The following alphabetical list represents papers published in 2021 with at least one Whitehead author (in red). Not all of this work was done at the Whitehead Institute. Some of these papers are collaborations with scientists elsewhere. The papers are gathered from PubMed and from Web of Science. Preceding the bibliography is an alphabetical list of the titles of the papers followed by the first author.

P.S. The journal links only work if you have a license to those respective online journals.

2021 Titles :

Adane, B., Alexe, G., Seong, B.K.A., Lu, D., Hwang, E.E., Hnisz, D., Lareau, C.A., Ross, L., Lin, S., Dela Cruz, F.S., Weintrub, A.S.,Young, R.A., et al. (2021). STAG2 loss rewires oncogenic and developmental programs to promote metastasis in Ewing sarcoma. Cancer Cell 39 : 827-844. The core cohesin subunit STAG2 is recurrently mutated in Ewing sarcoma but its biological role is less clear. Here, we demonstrate that cohesin complexes containing STAG2 occupy enhancer and polycomb repressive complex (PRC2)-marked regulatory regions. Genetic suppression of STAG2 leads to a compensatory increase in cohesin-STAG1 complexes, but not in enhancer-rich regions, and results in reprogramming of cis-chromatin interactions. Strikingly, in STAG2 knockout cells the oncogenic genetic program driven by the fusion transcription factor EWS/FLI1 was highly perturbed, in part due to altered enhancer-promoter contacts. Moreover, loss of STAG2 also disrupted PRC2-mediated regulation of gene expression. Combined, these transcriptional changes converged to modulate EWS/FLI1, migratory, and neurodevelopmental programs. Finally, consistent with clinical observations, functional studies revealed that loss of STAG2 enhances the metastatic potential of Ewing sarcoma xenografts. Our findings demonstrate that STAG2 mutations can alter chromatin architecture and transcriptional programs to promote an aggressive cancer phenotype. Full Text

Avagyan, S., Henninger, J.E. , Mannherz, W.P., Mistry, M., Yoon, J., Yang, S., Weber, M.C., Moore, J.L., and Zon, L.I. (2021). Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis. Science 374 (6568): 768-772. Clonal hematopoiesis results from enhanced fitness of a mutant hematopoietic stem and progenitor cell (HSPC), but how such clones expand is unclear. We developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in clonal hematopoiesis–associated genes such as asxl1 promoted clonal dominance. Single-cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti-inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator, nr4a1, abrogated the ability of asxl1-mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. Full Text

Avilés-Pagán, E.E., Hara, M., and Orr-Weaver, T.L. (2021). The GNU subunit of PNG kinase, the developmental regulator of mRNA translation, binds BIC-C to localize to RNP granules. eLife 10 : e67294. Control of mRNA translation is a key mechanism by which the differentiated oocyte transitions to a totipotent embryo. In Drosophila, the PNG kinase complex regulates maternal mRNA translation at the oocyte-to-embryo transition. We previously showed the GNU activating subunit is crucial in regulating PNG and timing its activity to the window between egg activation and early embryogenesis (Hara et al., 2017). In this study, we find associations between GNU and proteins of RNP granules and demonstrate that GNU localizes to cytoplasmic RNP granules in the mature oocyte, identifying GNU as a new component of a subset of RNP granules. Furthermore, we define roles for the domains of GNU. Interactions between GNU and the granule component BIC-C reveal potential conserved functions for translational regulation in metazoan development. We propose that by binding to BIC-C, upon egg activation GNU brings PNG to its initial targets, translational repressors in RNP granules. Full Text

Bellen, H.J., Hubbard, E.J.A., Lehmann, R., Madhani, H.D., Solnica-Krezel, L., and Southard-Smith, E.M. (2021). Model organism databases are in jeopardy. Development (2021) 148 (19): dev200193. Model organisms (MOs), including yeast, worm (C. elegans), fruit fly (Drosophila), zebrafish, frog (Xenopus), mouse and rat, contribute greatly to our understanding of human development and disease. To be successful, MO research critically depends on many shared resources. Particularly important are MO stock centers and MO databases (MODs), without which most MO work would not be possible. This article focuses on MODs, which are mostly supported by grants from the National Institutes of Health (NIH), especially the National Human Genome Research Institute (NHGRI). We are deeply concerned that the support for these vital databases is in jeopardy due to large cuts in their grant budgets. We fear these budget cuts will slow biomedical research worldwide and create increased waste of resources due to duplication of efforts. Indeed, the cuts threaten to erode access to reliable, expertly fact-checked data and cause an increase in mis-information due to the degraded organization of knowledge and information. Full Text

Bellott, D.W., and Page, D.C. (2021). Dosage-sensitive functions in embryonic development drove the survival of genes on sex-specific chromosomes in snakes, birds, and mammals. Genome Research. Online ahead of print. Different ancestral autosomes independently evolved into sex chromosomes in snakes, birds, and mammals. In snakes and birds, females are ZW and males are ZZ; in mammals, females are XX and males are XY. Although X and Z Chromosomes retain nearly all ancestral genes, sex-specific W and Y Chromosomes suffered extensive genetic decay. In both birds and mammals, the genes that survived on sex-specific chromosomes are enriched for broadly expressed, dosage-sensitive regulators of gene expression, subject to strong purifying selection. To gain deeper insight into the processes that govern survival on sex-specific chromosomes, we carried out a meta-analysis of survival across 41 species-three snakes, 24 birds, and 14 mammals-doubling the number of ancestral genes under investigation and increasing our power to detect enrichments among survivors relative to nonsurvivors. Of 2564 ancestral genes, representing an eighth of the ancestral amniote genome, only 324 survive on present-day sex-specific chromosomes. Survivors are enriched for dosage-sensitive developmental processes, particularly development of neural crest-derived structures, such as the face. However, there was no enrichment for expression in sex-specific tissues, involvement in sex determination or gonadogenesis pathways, or conserved sex-biased expression. Broad expression and dosage sensitivity contributed independently to gene survival, suggesting that pleiotropy imposes additional constraints on the evolution of dosage compensation. We propose that maintaining the viability of the heterogametic sex drove gene survival on amniote sex-specific chromosomes, and that subtle modulation of the expression of survivor genes and their autosomal orthologs has disproportionately large effects on development and disease. Full Text

Blobel, G.A., Higgs, D.R., Mitchell, J.A., Notani, D., and Young, R.A. (2021). Testing the super-enhancer concept. Nature Reviews Genetics. Many enhancers exist as clusters in the genome, which has led to the coining of the term ‘super-enhancer’. In this Viewpoint, five experts discuss our biological understanding of enhancer clusters, how we can responsibly study their functions, and their opinions on whether names for enhancer clusters are an informative reflection of their functional properties. They also provide their thoughts on key unanswered questions and future directions in the field. Full Text

Boija, A., Klein, I.A., and Young, R.A. (2021). Biomolecular Condensates and Cancer. Cancer Cell 39, 174-192. Malignant transformation is characterized by dysregulation of diverse cellular processes that have been the subject of detailed genetic, biochemical, and structural studies, but only recently has evidence emerged that many of these processes occur in the context of biomolecular condensates. Condensates are membrane-less bodies, often formed by liquid-liquid phase separation, that compartmentalize protein and RNA molecules with related functions. New insights from condensate studies portend a profound transformation in our understanding of cellular dysregulation in cancer. Here we summarize key features of biomolecular condensates, note where they have been implicated-or will likely be implicated-in oncogenesis, describe evidence that the pharmacodynamics of cancer therapeutics can be greatly influenced by condensates, and discuss some of the questions that must be addressed to further advance our understanding and treatment of cancer.Full Text

Bui, A.N., Tyan, K., Giobbie-Hurder, A., Klein, I.A. , Manos, M.P., Zubiri, L., Reynolds, K., Grover, S., Weinhouse, G.L., Ott, P.A., et al. (2021). Impact of COVID-19 on Patients with Cancer Receiving Immune Checkpoint Inhibitors. Journal of Immunotherapy and Precision Oncology 4: 35-44. To evaluate the impact of Sars-Cov-2 infection on mortality and immune checkpoint inhibitor (ICI) toxicity in patients with cancer receiving ICIs compared to those not receiving ICIs. METHODS: We conducted a retrospective matched cohort study of 25 patients receiving ICIs within 1 year of coronavirus disease 2019 (COVID-19) diagnosis between March 20, 2020, and June 3, 2020, at the Dana-Farber Cancer Institute/Mass General Brigham. Cases were matched 1:1 with controls based on age, sex, and anticancer therapy within the prior 6 months. RESULTS: Seven of 25 (28%) patients receiving ICIs died from COVID-19 as compared with nine of 25 (36%) controls. Through multivariable analysis adjusting for age, sex, and anticancer therapy, ICI use was not associated with increased risk for COVID-19 death (OR [odds ratio] 0.36, 95% CI 0.07-1.87). Determinants of mortality included age (OR 1.14, 95% CI 1.03-1.27) and chronic obstructive pulmonary disease (OR 12.26, 95% CI 1.76-85.14). Statin use was protective against mortality (OR 0.08, 95% CI 0.01-0.63). Two patients experienced persistent immune-related adverse events (irAEs) (hypophysitis); one had new-onset irAE (hypothyroidism) during their COVID-19 course. Patients with ICIs had significantly higher platelet (p = 0.017) and D-dimer (p = 0.037) levels. Elevated troponin levels (p = 0.01) were associated with COVID-19 death in patients using ICI. CONCLUSION: There is insufficient evidence to conclude COVID-19-related outcomes are associated with ICIs, and we did not observe an increased risk of COVID-19-related death associated with ICIs. The potential protective effect of statin therapy and role of laboratory biomarkers warrant further investigation. Full Text

Cable, J., Heard, E., Hirose, T., Prasanth, K.V., Chen, L.L., Henninger, J.E., Quinodoz, S.A., Spector, D.L., Diermeier, S.D., Porman, A.M., et al. Noncoding RNAs: biology and applications-a Keystone Symposia report. Annals of the New York Academy of Sciences. Online Ahead of Print. The human transcriptome contains many types of noncoding RNAs, which rival the number of protein-coding species. From long noncoding RNAs (lncRNAs) that are over 200 nucleotides long to piwi-interacting RNAs (piRNAs) of only 20 nucleotides, noncoding RNAs play important roles in regulating transcription, epigenetic modifications, translation, and cell signaling. Roles for noncoding RNAs in disease mechanisms are also being uncovered, and several species have been identified as potential drug targets. On May 11-14, 2021, the Keystone eSymposium "Noncoding RNAs: Biology and Applications" brought together researchers working in RNA biology, structure, and technologies to accelerate both the understanding of RNA basic biology and the translation of those findings into clinical applications. Full Text

Carlone, D.L., Riba-Wolman, R.D., Deary, L.T., Tovaglieri, A., Jiang, L., Ambruzs, D.M., Mead, B.E., Shah, M.S., Lengner, C.J., Jaenisch, R., et al. (2021). Telomerase expression marks transitional growth-associated skeletal progenitor/stem cells. Stem cells. Online ahead of print. Skeletal progenitor/stem cells (SSCs) play a critical role in postnatal bone growth and maintenance. Telomerase (Tert) activity prevents cellular senescence and is required for maintenance of stem cells in self-renewing tissues. Here we investigated the role of mTert-expressing cells in postnatal mouse long bone and found that mTert expression is enriched at the time of adolescent bone growth. mTert-GFP(+) cells were identified in regions known to house SSCs, including the metaphyseal stroma, growth plate, and the bone marrow. We also show that mTert-expressing cells are a distinct SSC population with enriched colony-forming capacity and contribute to multiple mesenchymal lineages, in vitro. In contrast, in vivo lineage-tracing studies identified mTert(+) cells as osteochondral progenitors and contribute to the bone-forming cell pool during endochondral bone growth with a subset persisting into adulthood. Taken together, our results show that mTert expression is temporally regulated and marks SSCs during a discrete phase of transitional growth between rapid bone growth and maintenance. Full Text

Cejas, P., Xie, Y., Font-Tello, A., Lim, K., Syamala, S., Qiu, X., Tewari, A.K., Shah, N., Nguyen, H.M., Patel, R.A., Alessandra Dall'Agnese, et al. (2021). Subtype heterogeneity and epigenetic convergence in neuroendocrine prostate cancer. Nature Communications 12(1): 5775. Neuroendocrine carcinomas (NEC) are tumors expressing markers of neuronal differentiation that can arise at different anatomic sites but have strong histological and clinical similarities. Here we report the chromatin landscapes of a range of human NECs and show convergence to the activation of a common epigenetic program. With a particular focus on treatment emergent neuroendocrine prostate cancer (NEPC), we analyze cell lines, patient-derived xenograft (PDX) models and human clinical samples to show the existence of two distinct NEPC subtypes based on the expression of the neuronal transcription factors ASCL1 and NEUROD1. While in cell lines and PDX models these subtypes are mutually exclusive, single-cell analysis of human clinical samples exhibits a more complex tumor structure with subtypes coexisting as separate sub-populations within the same tumor. These tumor sub-populations differ genetically and epigenetically contributing to intra- and inter-tumoral heterogeneity in human metastases. Overall, our results provide a deeper understanding of the shared clinicopathological characteristics shown by NECs. Furthermore, the intratumoral heterogeneity of human NEPCs suggests the requirement of simultaneous targeting of coexisting tumor populations as a therapeutic strategy. Full Text

Chen, C., and Yamashita, Y.M. (2021). Centrosome-centric view of asymmetric stem cell division. Open Biology 11(1):200314.Epub. The centrosome is a unique organelle: the semi-conservative nature of its duplication generates an inherent asymmetry between 'mother' and 'daughter' centrosomes, which differ in their age. This asymmetry has captivated many cell biologists, but its meaning has remained enigmatic. In the last two decades, many stem cell types have been shown to display stereotypical inheritance of either the mother or daughter centrosome. These observations have led to speculation that the mother and daughter centrosomes bear distinct information, contributing to differential cell fates during asymmetric cell divisions. This review summarizes recent progress and discusses how centrosome asymmetry may promote asymmetric fates during stem cell divisions. Full Text

Chen, P.J., Hussmann, J.A., Yan, J., Knipping, F., Ravisankar, P., Chen, P.F., Chen, C., Nelson, J.W., Newby, G.A., Sahin, M., Jonathan S. Weissman, et al. (2021). Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell. Online Ahead of Print. While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types. Full Text

Chiesa, I., Di Gesù, R., Overholt, K.J., and Gottardi, R. (2022). A Mesoscale 3D Culture System for Native and Engineered Biphasic Tissues: Application to the Osteochondral Unit. Methods in Molecular Biology 2022:2373:267-281. Interface tissues are functionally graded tissues characterized by a complex layered structure, which therefore present a great challenge to be reproduced and cultured in vitro. Here, we describe the design and operation of a 3D printed dual-chamber bioreactor as a culturing system for biphasic native or engineered osteochondral tissues. The bioreactor is designed to potentially accommodate a variety of interface tissues and enables the precise study of tissue crosstalk by creating two separate microenvironments while maintaining the tissue compartments in direct contact. Full Text

Choi, J.E., Sebastian, C., Ferrer, C.M., Lewis, C.A., Sade-Feldman, M., LaSalle, T., Gonye, A., Lopez, B.G.C., Abdelmoula, W.M., Regan, M.S., et al. (2021). A unique subset of glycolytic tumour-propagating cells drives squamous cell carcinoma. Nature Metabolism 3(2):182-195. Head and neck squamous cell carcinoma (SCC) remains among the most aggressive human cancers. Tumour progression and aggressiveness in SCC are largely driven by tumour-propagating cells (TPCs). Aerobic glycolysis, also known as the Warburg effect, is a characteristic of many cancers; however, whether this adaptation is functionally important in SCC, and at which stage, remains poorly understood. Here, we show that the NAD(+)-dependent histone deacetylase sirtuin 6 is a robust tumour suppressor in SCC, acting as a modulator of glycolysis in these tumours. Remarkably, rather than a late adaptation, we find enhanced glycolysis specifically in TPCs. More importantly, using single-cell RNA sequencing of TPCs, we identify a subset of TPCs with higher glycolysis and enhanced pentose phosphate pathway and glutathione metabolism, characteristics that are strongly associated with a better antioxidant response. Together, our studies uncover enhanced glycolysis as a main driver in SCC, and, more importantly, identify a subset of TPCs as the cell of origin for the Warburg effect, defining metabolism as a key feature of intra-tumour heterogeneity.Full Text

Cloutier, J.K., McMann, C.L., Oderberg, I.M., and Reddien, P.W. Activin-2 is required for regeneration of polarity on the planarian anterior-posterior axis. (2021). PLoS Genetics 17, e1009466. Planarians are flatworms and can perform whole-body regeneration. This ability involves a mechanism to distinguish between anterior-facing wounds that require head regeneration and posterior-facing wounds that require tail regeneration. How this head-tail regeneration polarity decision is made is studied to identify principles underlying tissue-identity specification in regeneration. We report that inhibition of activin-2, which encodes an Activin-like signaling ligand, resulted in the regeneration of ectopic posterior-facing heads following amputation. During tissue turnover in uninjured planarians, positional information is constitutively expressed in muscle to maintain proper patterning. Positional information includes Wnts expressed in the posterior and Wnt antagonists expressed in the anterior. Upon amputation, several wound-induced genes promote re-establishment of positional information. The head-versus-tail regeneration decision involves preferential wound induction of the Wnt antagonist notum at anterior-facing over posterior-facing wounds. Asymmetric activation of notum represents the earliest known molecular distinction between head and tail regeneration, yet how it occurs is unknown. activin-2 RNAi animals displayed symmetric wound-induced activation of notum at anterior- and posterior-facing wounds, providing a molecular explanation for their ectopic posterior-head phenotype. activin-2 RNAi animals also displayed anterior-posterior (AP) axis splitting, with two heads appearing in anterior blastemas, and various combinations of heads and tails appearing in posterior blastemas. This was associated with ectopic nucleation of anterior poles, which are head-tip muscle cells that facilitate AP and medial-lateral (ML) pattern, at posterior-facing wounds. These findings reveal a role for Activin signaling in determining the outcome of AP-axis-patterning events that are specific to regeneration. Full Text

Condon, K.J., Orozco, J.M., Adelmann, C.H., Spinelli, J.B., van der Helm, P.W., Roberts, J.M., Kunchok, T., and Sabatini, D.M. (2021). Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction. PNAS 118(4): e2022120118. In mammalian cells, nutrients and growth factors signal through an array of upstream proteins to regulate the mTORC1 growth control pathway. Because the full complement of these proteins has not been systematically identified, we developed a FACS-based CRISPR-Cas9 genetic screening strategy to pinpoint genes that regulate mTORC1 activity. Along with almost all known positive components of the mTORC1 pathway, we identified many genes that impact mTORC1 activity, including DCAF7, CSNK2B, SRSF2, IRS4, CCDC43, and HSD17B10 Using the genome-wide screening data, we generated a focused sublibrary containing single guide RNAs (sgRNAs) targeting hundreds of genes and carried out epistasis screens in cells lacking nutrient- and stress-responsive mTORC1 modulators, including GATOR1, AMPK, GCN2, and ATF4. From these data, we pinpointed mitochondrial function as a particularly important input into mTORC1 signaling. While it is well appreciated that mitochondria signal to mTORC1, the mechanisms are not completely clear. We find that the kinases AMPK and HRI signal, with varying kinetics, mitochondrial distress to mTORC1, and that HRI acts through the ATF4-dependent up-regulation of both Sestrin2 and Redd1. Loss of both AMPK and HRI is sufficient to render mTORC1 signaling largely resistant to mitochondrial dysfunction induced by the ATP synthase inhibitor oligomycin as well as the electron transport chain inhibitors piericidin and antimycin. Taken together, our data reveal a catalog of genes that impact the mTORC1 pathway and clarify the multifaceted ways in which mTORC1 senses mitochondrial dysfunction. Full Text

Coukos, R.W., Yao, D., Sanchez, M.L., Strand, E.T., Olive, M.E., Udeshi, N.D., Weissman, J.S., Carr, S.A., Bassik, M.C., and Ting, A.Y. (2021). An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens. eLife 10:e69142. The trafficking of specific protein cohorts to correct subcellular locations at correct times is essential for every signaling and regulatory process in biology. Gene perturbation screens could provide a powerful approach to probe the molecular mechanisms of protein trafficking, but only if protein localization or mislocalization can be tied to a simple and robust phenotype for cell selection, such as cell proliferation or fluorescence-activated cell sorting (FACS). To empower the study of protein trafficking processes with gene perturbation, we developed a genetically-encoded molecular tool named HiLITR. HiLITR converts protein colocalization into proteolytic release of a membrane-anchored transcription factor, which drives the expression of a chosen reporter gene. Using HiLITR in combination with FACS-based CRISPRi screening in human cell lines, we identified genes that influence the trafficking of mitochondrial and ER tail-anchored proteins. We show that loss of the SUMO E1 component SAE1 results in mislocalization and destabilization of many mitochondrial tail-anchored proteins. We also demonstrate a distinct regulatory role for EMC10 in the ER membrane complex, opposing the transmembrane-domain insertion activity of the complex. Through transcriptional integration of complex cellular functions, HiLITR expands the scope of biological processes that can be studied by genetic perturbation screening technologies. Full Text

Das, S., and Chadwick, B.P. (2021). CRISPR mediated targeting of DUX4 distal regulatory element represses DUX4 target genes dysregulated in Facioscapulohumeral muscular dystrophy. Scientific Reports 11(1) : 12598. Facioscapulohumeral muscular dystrophy (FSHD) is a debilitating muscle disease that currently does not have an effective cure or therapy. The abnormal reactivation of DUX4, an embryonic gene that is epigenetically silenced in somatic tissues, is causal to FSHD. Disease-specific reactivation of DUX4 has two common characteristics, the presence of a non-canonical polyadenylation sequence within exon 3 of DUX4 that stabilizes pathogenic transcripts, and the loss of repressive chromatin modifications at D4Z4, the macrosatellite repeat which encodes DUX4. We used CRISPR/Cas9 to silence DUX4 using two independent approaches. We deleted the DUX4 pathogenic polyadenylation signal, which resulted in downregulation of pathogenic DUX4-fl transcripts. In another approach, we transcriptionally repressed DUX4 by seeding heterochromatin using the dCas9-KRAB platform within exon 3. These feasibility of targeting DUX4 experiments were initially tested in a non-myogenic carcinoma cell line that we have previously characterized. Subsequently, in an immortalized patient myoblast cell line, we demonstrated that targeting DUX4 by either approach led to substantial downregulation of not only pathogenic DUX4 transcripts, but also a subset of its target genes that are known biomarkers of FSHD. These findings offer proof-of-concept of the effect of silencing the polyadenylation sequence on pathogenic DUX4 expression. Full Text

de la Calle Arregui, C., Plata-Gómez, A.B., Deleyto-Seldas, N., García, F., Ortega-Molina, A., Abril-Garrido, J., Rodriguez, E., Nemazanyy, I., Tribouillard, L., de Martino, A., David M. Sabatini & Alejo Efeyan, et al. (2021). Limited survival and impaired hepatic fasting metabolism in mice with constitutive Rag GTPase signaling. Nature Communications 12(1): 3660. The mechanistic target of rapamycin complex 1 (mTORC1) integrates cellular nutrient signaling and hormonal cues to control metabolism. We have previously shown that constitutive nutrient signaling to mTORC1 by means of genetic activation of RagA (expression of GTP-locked RagA, or RagA(GTP)) in mice resulted in a fatal energetic crisis at birth. Herein, we rescue neonatal lethality in RagA(GTP) mice and find morphometric and metabolic alterations that span glucose, lipid, ketone, bile acid and amino acid homeostasis in adults, and a median lifespan of nine months. Proteomic and metabolomic analyses of livers from RagA(GTP) mice reveal a failed metabolic adaptation to fasting due to a global impairment in PPARα transcriptional program. These metabolic defects are partially recapitulated by restricting activation of RagA to hepatocytes, and revert by pharmacological inhibition of mTORC1. Constitutive hepatic nutrient signaling does not cause hepatocellular damage and carcinomas, unlike genetic activation of growth factor signaling upstream of mTORC1. In summary, RagA signaling dictates dynamic responses to feeding-fasting cycles to tune metabolism so as to match the nutritional state. Full Text

Deshycka, R., Sudaryo, V., Huang, N.J., Xie, Y., Smeding, L.Y., Choi, M.K., Ploegh, H.L., Lodish, H.F., and Pishesha, N. (2021). Engineered red blood cells carrying PCSK9 inhibitors persistently lower LDL and prevent obesity. PloS One 16(11):e0259353. Low plasma levels of Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) are associated with decreased low-density lipoprotein (LDL) cholesterol and a reduced risk of cardiovascular disease. PCSK9 binds to the epidermal growth factor-like repeat A (EGFA) domain of LDL receptors (LDLR), very low-density lipoprotein receptors (VLDLR), apolipoprotein E receptor 2 (ApoER2), and lipoprotein receptor-related protein 1 (LRP1) and accelerates their degradation, thus acting as a key regulator of lipid metabolism. Antibody and RNAi-based PCSK9 inhibitor treatments lower cholesterol and prevent cardiovascular incidents in patients, but their high-cost hampers market penetration. We sought to develop a safe, long-term and one-time solution to treat hyperlipidemia. We created a cDNA encoding a chimeric protein in which the extracellular N- terminus of red blood cells (RBCs) specific glycophorin A was fused to the LDLR EGFA domain and introduced this gene into mouse bone marrow hematopoietic stem and progenitor cells (HSPCs). Following transplantation into irradiated mice, the animals produced RBCs with the EGFA domain (EGFA-GPA RBCs) displayed on their surface. These animals showed significantly reduced plasma PCSK9 (66.5% decrease) and reduced LDL levels (40% decrease) for as long as 12 months post-transplantation. Furthermore, the EGFA- GPA mice remained lean for life and maintained normal body weight under a high-fat diet. Hematopoietic stem cell gene therapy can generate red blood cells expressing an EGFA-glycophorin A chimeric protein as a practical and long-term strategy for treating chronic hyperlipidemia and obesity. Full Text

Dongre, A., and Weinberg, R.A. (2021). Leveraging immunochemotherapy for treating pancreatic cancer. Cell Research. Online Ahead of Print. The refractory response of pancreatic ductal adenocarcinoma (PDAC) to multiple treatment regimens can be attributed to the presence of a desmoplastic stroma together with an immunosuppressive tumor microenvironment. In a recent study published in Cell , Koikawa et al. demonstrate that targeting the proline isomerase, Pin1, potentiates the efficacy of immunochemotherapy and enhances the susceptibility of highly resistant PDAC to the anti-tumor immune response. Full Text

Feder, Z.A., Ali, A., Singh, A., Krakowiak, J., Zheng, X., Bindokas, V.P., Wolfgeher, D., Kron, S.J., and Pincus, D. (2021). Subcellular localization of the J-protein Sis1 regulates the heat shock response. Journal of Cell Biology 220(1):e202005165. Cells exposed to heat shock induce a conserved gene expression program, the heat shock response (HSR), encoding protein homeostasis (proteostasis) factors. Heat shock also triggers proteostasis factors to form subcellular quality control bodies, but the relationship between these spatial structures and the HSR is unclear. Here we show that localization of the J-protein Sis1, a cofactor for the chaperone Hsp70, controls HSR activation in yeast. Under nonstress conditions, Sis1 is concentrated in the nucleoplasm, where it promotes Hsp70 binding to the transcription factor Hsf1, repressing the HSR. Upon heat shock, Sis1 forms an interconnected network with other proteostasis factors that spans the nucleolus and the surface of the endoplasmic reticulum. We propose that localization of Sis1 to this network directs Hsp70 activity away from Hsf1 in the nucleoplasm, leaving Hsf1 free to induce the HSR. In this manner, Sis1 couples HSR activation to the spatial organization of the proteostasis network.Full Text

Fujita, D., Suzuki, R., Fujii, Y., Yamada, M., Nakama, T., Matsugami, A., Hayashi, F., Weng, J.K., Yagi-Utsumi, M., and Fujita, M. (2021). Protein stabilization and refolding in a gigantic self-assembled cage. Chem 7 : 2672-2683. Spatial isolation of molecules is often a powerful strategy for regulating their molecular behavior. Biological systems employ such mechanisms well; however, scientists have yet to rival nature, particularly for macromolecular substrates. We demonstrated that the encapsulation of a protein in a molecular cage with an open framework stabilizes the tertiary structure of the protein and improves its enzymatic activity. Particularly, when the three-dimensionally confined enzyme was exposed to an organic solvent, its half-life was prolonged 1,000-fold. Kinetic and spectroscopic analysis of the enzymatic reaction revealed that the key to this stability is the isolated space; this is reminiscent of chaperonins, which use their large internal cavities to assist the folding of client proteins. The single-molecule protein caging affords a new type of protein-based nanobiotechnology that accelerates molecular biology research as well as industrial applications. Full Text

Gebert, D., Neubert, L.K., Lloyd, C., Gui, J.H., Lehmann, R., and Teixeira, F.K. (2021). Large Drosophila germline piRNA clusters are evolutionarily labile and dispensable for transposon regulation. Molecular Cell 81:3965-78. PIWI proteins and their guiding Piwi-interacting small RNAs (piRNAs) are crucial for fertility and transposon defense in the animal germline. In most species, the majority of piRNAs are produced from distinct large genomic loci, called piRNA clusters. It is assumed that germline-expressed piRNA clusters, particularly in Drosophila, act as principal regulators to control transposons dispersed across the genome. Here, using synteny analysis, we show that large clusters are evolutionarily labile, arise at loci characterized by recurrent chromosomal rearrangements, and are mostly species-specific across the Drosophila genus. By engineering chromosomal deletions in D. melanogaster, we demonstrate that the three largest germline clusters, which account for the accumulation of >40% of all transposon-targeting piRNAs in ovaries, are neither required for fertility nor for transposon regulation in trans. We provide further evidence that dispersed elements, rather than the regulatory action of large Drosophila germline clusters in trans, may be central for transposon defense. Full Text

Gebert, N., Rahman, S., Lewis, C.A., Ori, A., and Cheng, C.W. (2021). Identifying Cell-Type-Specific Metabolic Signatures Using Transcriptome and Proteome Analyses. Current Protocols 1(9):e245. Studies in various tissues have revealed a central role of metabolic pathways in regulating adult stem cell function in tissue regeneration and tumor initiation. The unique metabolic dependences or preferences of adult stem cells, therefore, are emerging as a new category of therapeutic target. Recently, advanced methods including high-resolution metabolomics, proteomics, and transcriptomics have been developed to address the growing interest in stem cell metabolism. A practical framework integrating the omics analyses is needed to systematically perform metabolic characterization in a cell-type-specific manner. Here, we leverage recent advances in transcriptomics and proteomics research to identify cell-type-specific metabolic features by reconstructing cell identity using genes and the encoded enzymes involved in major metabolic pathways. We provide protocols for cell isolation, transcriptome and proteome analyses, and metabolite profiling and measurement. The workflow for mapping cell-type-specific metabolic signatures presented here, although initially developed for intestinal crypt cells, can be easily implemented for cell populations in other tissues, and is highly compatible with most public datasets. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Intestinal crypt isolation and cell population purification Basic Protocol 2: Transcriptome analyses for cell-type-specific metabolic gene expression Basic Protocol 3: Proteome analyses for cell-type-specific metabolic enzyme levels Basic Protocol 4: Metabolite profiling and measurement. Full Text

Gupta, P., and Rouskin, S. (2022). In Vivo RNA Structure Probing with DMS-MaPseq. Methods in Molecular Biology 2404: 299-310. RNA has an extraordinary capacity to fold and form intrinsic secondary structures that play a central role in maintaining its functionality. It is crucial to have ways to study RNA structures and identify their functions in their biological environment. In the last few decades, a number of different chemical probing methods have been used to study RNA secondary structure. Here, we present a dimethyl sulfate-based (DMS) chemical probing method coupled with Next Generation sequencing (DMS-MaPseq) to study RNA secondary structure in vivo.DMS modifies unpaired adenine and cytosine bases which are then converted to mutations/mismatches using a thermostable group II intron reverse transcriptase (TGIRT) and further analyzed using sequencing. We validated the technique in model systems ranging from Drosophila to human cell lines, thus increasing the technique's broad range of applications. DMS-MaPseq provides high quality data and can be used for both gene-targeted as well as genome-wide analysis. Full Text

Hamza, B., Miller, A.B., Meier, L., Stockslager, M., Ng, S.R., King, E.M., Lin, L., DeGouveia, K.L., Mulugeta, N., Calistri, N.L., Robert A Weinberg, et al. (2021). Measuring kinetics and metastatic propensity of CTCs by blood exchange between mice. Nature Communications 12 (1):5680. Existing preclinical methods for acquiring dissemination kinetics of rare circulating tumor cells (CTCs) en route to forming metastases have not been capable of providing a direct measure of CTC intravasation rate and subsequent half-life in the circulation. Here, we demonstrate an approach for measuring endogenous CTC kinetics by continuously exchanging CTC-containing blood over several hours between un-anesthetized, tumor-bearing mice and healthy, tumor-free counterparts. By tracking CTC transfer rates, we extrapolated half-life times in the circulation of between 40 and 260 s and intravasation rates between 60 and 107,000 CTCs/hour in mouse models of small-cell lung cancer (SCLC), pancreatic ductal adenocarcinoma (PDAC), and non-small cell lung cancer (NSCLC). Additionally, direct transfer of only 1-2% of daily-shed CTCs using our blood-exchange technique from late-stage, SCLC-bearing mice generated macrometastases in healthy recipient mice. We envision that our technique will help further elucidate the role of CTCs and the rate-limiting steps in metastasis. Full Text

Hanna, R., Flamier, A. , Barabino, A., and Bernier, G. (2021). G-quadruplexes originating from evolutionary conserved L1 elements interfere with neuronal gene expression in Alzheimer's disease. Nature Communications 12(1):1828. DNA sequences containing consecutive guanines organized in 4-interspaced tandem repeats can form stable single-stranded secondary structures, called G-quadruplexes (G4). Herein, we report that the Polycomb group protein BMI1 is enriched at heterochromatin regions containing putative G4 DNA sequences, and that G4 structures accumulate in cells with reduced BMI1 expression and/or relaxed chromatin, including sporadic Alzheimer's disease (AD) neurons. In AD neurons, G4 structures preferentially accumulate in lamina-associated domains, and this is rescued by re-establishing chromatin compaction. ChIP-seq analyses reveal that G4 peaks correspond to evolutionary conserved Long Interspersed Element-1 (L1) sequences predicted to be transcriptionally active. Hence, G4 structures co-localize with RNAPII, and inhibition of transcription can reverse the G4 phenotype without affecting chromatin's state, thus uncoupling both components. Intragenic G4 structures affecting splicing events are furthermore associated with reduced neuronal gene expression in AD. Active L1 sequences are thus at the origin of most G4 structures observed in human neurons. Full Text

He, Y., Tang, X., Huang, J., Ren, J., Zhou, H., Chen, K., Liu, A., Shi, H., Lin, Z., Li, Q., Johain Ounadjela, Emanuelle I Grody, Jian Shu, et al. (2021). ClusterMap for multi-scale clustering analysis of spatial gene expression. Nature Communications 12 : 5909. Quantifying RNAs in their spatial context is crucial to understanding gene expression and regulation in complex tissues. In situ transcriptomic methods generate spatially resolved RNA profiles in intact tissues. However, there is a lack of a unified computational framework for integrative analysis of in situ transcriptomic data. Here, we introduce an unsupervised and annotation-free framework, termed ClusterMap, which incorporates the physical location and gene identity of RNAs, formulates the task as a point pattern analysis problem, and identifies biologically meaningful structures by density peak clustering (DPC). Specifically, ClusterMap precisely clusters RNAs into subcellular structures, cell bodies, and tissue regions in both two- and three-dimensional space, and performs consistently on diverse tissue types, including mouse brain, placenta, gut, and human cardiac organoids. We demonstrate ClusterMap to be broadly applicable to various in situ transcriptomic measurements to uncover gene expression patterns, cell niche, and tissue organization principles from images with high-dimensional transcriptomic profiles. Full Text

Hein, M.Y., and Weissman, J.S. (2021). Functional single-cell genomics of human cytomegalovirus infection. Nature Biotechnology. Online Ahead of Print. Understanding how viral and host factors interact and how perturbations impact infection is the basis for designing antiviral interventions. Here we define the functional contribution of each viral and host factor involved in human cytomegalovirus infection in primary human fibroblasts through pooled CRISPR interference and nuclease screening. To determine how genetic perturbation of critical host and viral factors alters the timing, course and progression of infection, we applied Perturb-seq to record the transcriptomes of tens of thousands of CRISPR-modified single cells and found that, normally, most cells follow a stereotypical transcriptional trajectory. Perturbing critical host factors does not change the stereotypical transcriptional trajectory per se but can stall, delay or accelerate progression along the trajectory, allowing one to pinpoint the stage of infection at which host factors act. Conversely, perturbation of viral factors can create distinct, abortive trajectories. Our results reveal the roles of host and viral factors and provide a roadmap for the dissection of host-pathogen interactions. Full Text

Henninger, J.E., Oksuz, O., Shrinivas, K., Sagi, I., LeRoy, G., Zheng, M.M., Andrews, J.O., Zamudio, A.V., Lazaris, C., Hannett, N.M., Lee, T.I., Young, R.A., et al. (2021). RNA-Mediated Feedback Control of Transcriptional Condensates. Cell 184(1):207-225. Regulation of biological processes typically incorporates mechanisms that initiate and terminate the process and, where understood, these mechanisms often involve feedback control. Regulation of transcription is a fundamental cellular process where the mechanisms involved in initiation have been studied extensively, but those involved in arresting the process are poorly understood. Modeling of the potential roles of RNA in transcriptional control suggested a non-equilibrium feedback control mechanism where low levels of RNA promote condensates formed by electrostatic interactions whereas relatively high levels promote dissolution of these condensates. Evidence from in vitro and in vivo experiments support a model where RNAs produced during early steps in transcription initiation stimulate condensate formation, whereas the burst of RNAs produced during elongation stimulate condensate dissolution. We propose that transcriptional regulation incorporates a feedback mechanism whereby transcribed RNAs initially stimulate but then ultimately arrest the process. Full Text

Herneisen, A.L., and Lourido, S. (2021). Thermal Proteome Profiling to Identify Protein-ligand Interactions in the Apicomplexan Parasite Toxoplasma gondii. Bio-Protocol 11(21):e4207. Toxoplasma gondii is a single-celled eukaryotic parasite that chronically infects a quarter of the global population. In recent years, phenotypic screens have identified compounds that block parasite replication. Unraveling the pathways and molecular mechanisms perturbed by such compounds requires target deconvolution. In parasites, such deconvolution has been achieved via chemogenomic approaches-for example, directed evolution followed by whole-genome sequencing or genome-wide knockout screens. As a proteomic alternative that directly probes the physical interaction between compound and protein, thermal proteome profiling (TPP), also known as the cellular thermal shift assay (CETSA), recently emerged as a method to identify small molecule-target interactions in living cells and cell extracts in a variety of organisms, including unicellular eukaryotic pathogens. Ligand binding induces a thermal stability shift-stabilizing or destabilizing proteins that change conformationally in response to the ligand-that can be measured by mass spectrometry (MS). Cells are incubated with different concentrations of ligand and heated, causing thermal denaturation of proteins. The soluble protein is extracted and quantified with multiplexed, quantitative MS, resulting in thousands of thermal denaturation profiles. Proteins engaging the ligand can be identified by their compound-dependent thermal shift. The protocol provided here can be used to identify ligand-target interactions and assess the impact of environmental or genetic perturbations on the thermal stability of the proteome in T gondii and other eukaryotic pathogens. Full Text

Huang X., Park, K.M., Gontarz, P., Zhang, B., Pan, J., McKenzie, Z., Fischer, L.A., Dong, C., Dietmann, S., Xing, X., Tenzin Lungjangwa , Maya Mitalipovaet, Sumeth Imsoonthornruksa, Rudolph Jaenisch, et al. (2021). OCT4 cooperates with distinct ATP-dependent chromatin remodelers in naïve and primed pluripotent states in human. Nature Communications 12 : 5123. Understanding the molecular underpinnings of pluripotency is a prerequisite for optimal maintenance and application of embryonic stem cells (ESCs). While the protein-protein interactions of core pluripotency factors have been identified in mouse ESCs, their interactome in human ESCs (hESCs) has not to date been explored. Here we mapped the OCT4 interactomes in naïve and primed hESCs, revealing extensive connections to mammalian ATP-dependent nucleosome remodeling complexes. In naïve hESCs, OCT4 is associated with both BRG1 and BRM, the two paralog ATPases of the BAF complex. Genome-wide location analyses and genetic studies reveal that these two enzymes cooperate in a functionally redundant manner in the transcriptional regulation of blastocyst-specific genes. In contrast, in primed hESCs, OCT4 cooperates with BRG1 and SOX2 to promote chromatin accessibility at ectodermal genes. This work reveals how a common transcription factor utilizes differential BAF complexes to control distinct transcriptional programs in naïve and primed hESCs. Full Text

Hussmann, J.A., Ling, J., Ravisankar, P., Yan, J., Cirincione, A., Xu, A., Simpson, D., Yang, D., Bothmer, A., Cotta-Ramusino, C., Jonathan S Weissman, et al. (2021). Mapping the genetic landscape of DNA double-strand break repair. Cell. Online Ahead of Print. Cells repair DNA double-strand breaks (DSBs) through a complex set of pathways critical for maintaining genomic integrity. To systematically map these pathways, we developed a high-throughput screening approach called Repair-seq that measures the effects of thousands of genetic perturbations on mutations introduced at targeted DNA lesions. Using Repair-seq, we profiled DSB repair products induced by two programmable nucleases (Cas9 and Cas12a) in the presence or absence of oligonucleotides for homology-directed repair (HDR) after knockdown of 476 genes involved in DSB repair or associated processes. The resulting data enabled principled, data-driven inference of DSB end joining and HDR pathways. Systematic interrogation of this data uncovered unexpected relationships among DSB repair genes and demonstrated that repair outcomes with superficially similar sequence architectures can have markedly different genetic dependencies. This work provides a foundation for mapping DNA repair pathways and for optimizing genome editing across diverse modalities. Full Text

Jackson, E.K., Bellott, D.W., Skaletsky, H., and Page, D.C. (2021). GC-biased gene conversion in X-chromosome palindromes conserved in human, chimpanzee, and rhesus macaque. G3 11(11):jkab224. Gene conversion is GC-biased across a wide range of taxa. Large palindromes on mammalian sex chromosomes undergo frequent gene conversion that maintains arm-to-arm sequence identity greater than 99%, which may increase their susceptibility to the effects of GC-biased gene conversion. Here, we demonstrate a striking history of GC-biased gene conversion in 12 palindromes conserved on the X chromosomes of human, chimpanzee, and rhesus macaque. Primate X-chromosome palindrome arms have significantly higher GC content than flanking single-copy sequences. Nucleotide replacements that occurred in human and chimpanzee palindrome arms over the past 7 million years are one-and-a-half times as GC-rich as the ancestral bases they replaced. Using simulations, we show that our observed pattern of nucleotide replacements is consistent with GC-biased gene conversion with a magnitude of 70%, similar to previously reported values based on analyses of human meioses. However, GC-biased gene conversion since the divergence of human and rhesus macaque explains only a fraction of the observed difference in GC content between palindrome arms and flanking sequence, suggesting that palindromes are older than 29 million years and/or had elevated GC content at the time of their formation. This work supports a greater than 2:1 preference for GC bases over AT bases during gene conversion and demonstrates that the evolution and composition of mammalian sex chromosome palindromes is strongly influenced by GC-biased gene conversion. Full Text

Jackson, E.K., Bellott, D.W., Ting-Jan Cho, Skaletsky, H., Jennifer F Hughes , Tatyana Pyntikova, and Page, D.C. (2021). Large palindromes on the primate X Chromosome are preserved by natural selection. Genome Research 31(8):1337-1352. Mammalian sex chromosomes carry large palindromes that harbor protein-coding gene families with testis-biased expression. However, there are few known examples of sex-chromosome palindromes conserved between species. We identified 26 palindromes on the human X Chromosome, constituting more than 2% of its sequence, and characterized orthologous palindromes in the chimpanzee and the rhesus macaque using a clone-based sequencing approach that incorporates full-length nanopore reads. Many of these palindromes are missing or misassembled in the current reference assemblies of these species' genomes. We find that 12 human X palindromes have been conserved for at least 25 million years, with orthologs in both chimpanzee and rhesus macaque. Insertions and deletions between species are significantly depleted within the X palindromes' protein-coding genes compared to their noncoding sequence, demonstrating that natural selection has preserved these gene families. The spacers that separate the left and right arms of palindromes are a site of localized structural instability, with seven of 12 conserved palindromes showing no spacer orthology between human and rhesus macaque. Analysis of the 1000 Genomes Project data set revealed that human X-palindrome spacers are enriched for deletions relative to arms and flanking sequence, including a common spacer deletion that affects 13% of human X Chromosomes. This work reveals an abundance of conserved palindromes on primate X Chromosomes and suggests that protein-coding gene families in palindromes (most of which remain poorly characterized) promote X-palindrome survival in the face of ongoing structural instability. Full Text

Jagannathan, M., and Yamashita, Y.M. (2021). Defective satellite DNA clustering into chromocenters underlies hybrid incompatibility in Drosophila. Molecular Biology and Evolution. Online Ahead of Print. Although rapid evolution of pericentromeric satellite DNA repeats is theorized to promote hybrid incompatibility (HI) (Yunis and Yasmineh 1971; Henikoff et al. 2001; Ferree and Barbash 2009; Sawamura 2012; Jagannathan and Yamashita 2017), how divergent repeats affect hybrid cells remains poorly understood. Recently, we demonstrated that sequence-specific DNA-binding proteins cluster satellite DNA from multiple chromosomes into 'chromocenters', thereby bundling chromosomes to maintain the entire genome in a single nucleus (Jagannathan et al. 2018; Jagannathan et al. 2019). Here we show that ineffective clustering of divergent satellite DNA in the cells of Drosophila hybrids results in chromocenter disruption, associated micronuclei formation and tissue atrophy. We further demonstrate that previously identified HI factors trigger chromocenter disruption and micronuclei in hybrids, linking their function to a conserved cellular process. Together, we propose a unifying framework that explains how the widely observed satellite DNA divergence between closely related species can cause reproductive isolation. Full Text

Jbara, M., Pomplun, S., Schissel, C.K., Hawken, S.W., Boija, A., Klein, I. , Rodriguez, J., Buchwald, S.L., and Pentelute, B.L. (2021). Engineering Bioactive Dimeric Transcription Factor Analogs via Palladium Rebound Reagents. Journal of the American Chemical Society. Online Ahead of Print. Transcription factors (TF), such as Myc, are proteins implicated in disease pathogenesis, with dysregulation of Myc expression in 50% of all human cancers. Still, targeting Myc remains a challenge due to the lack of small molecule binding pockets in the tertiary structure. Here, we report synthetic covalently linked TF mimetics that inhibit oncogenic Myc-driven transcription by antagonistic binding of the target DNA-binding site. We combined automated flow peptide chemistry with palladium(II) oxidative addition complexes (OACs) to engineer covalent protein dimers derived from the DNA-binding domains of Myc, Max, and Omomyc TF analogs. Palladium-mediated cross-coupling of synthesized protein monomers resulted in milligram quantities of seven different covalent homo- and heterodimers. The covalent helical dimers were found to bind DNA and exhibited improved thermal stability. Cell-based studies revealed the Max-Max covalent dimer is cell-penetrating and interfered with Myc-dependent gene transcription resulting in reduced cancer cell proliferation (EC(50) of 6 μM in HeLa). RNA sequencing and gene analysis of extracted RNA from treated cancer cells confirmed that the covalent Max-Max homodimer interferes with Myc-dependent transcription. Flow chemistry, combined with palladium(II) OACs, has enabled a practical strategy to generate new bioactive compounds to inhibit tumor cell proliferation. Full Text

Johnnidis, J.B., Muroyama, Y., Ngiow, S.F., Chen, Z., Manne, S., Cai, Z., Song, S., Platt, J.M. , Schenkel, J.M., Abdel-Hakeem, M., et al. (2021). Inhibitory signaling sustains a distinct early memory CD8(+) T cell precursor that is resistant to DNA damage. Science Immunology 6(55) : eabe3702. The developmental origins of memory T cells remain incompletely understood. During the expansion phase of acute viral infection, we identified a distinct subset of virus-specific CD8(+) T cells that possessed distinct characteristics including expression of CD62L, T cell factor 1 (TCF-1), and Eomesodermin; relative quiescence; expression of activation markers; and features of limited effector differentiation. These cells were a quantitatively minor subpopulation of the TCF-1(+) pool and exhibited self-renewal, heightened DNA damage surveillance activity, and preferential long-term recall capacity. Despite features of memory and somewhat restrained proliferation during the expansion phase, this subset displayed evidence of stronger TCR signaling than other responding CD8(+) T cells, coupled with elevated expression of multiple inhibitory receptors including programmed cell death 1 (PD-1), lymphocyte activating gene 3 (LAG-3), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), CD5, and CD160. Genetic ablation of PD-1 and LAG-3 compromised the formation of this CD62L(hi) TCF-1(+) subset and subsequent CD8(+) T cell memory. Although central memory phenotype CD8(+) T cells were formed in the absence of these cells, subsequent memory CD8(+) T cell recall responses were compromised. Together, these results identify an important link between genome integrity maintenance and CD8(+) T cell memory. Moreover, the data indicate a role for inhibitory receptors in preserving key memory CD8(+) T cell precursors during initial activation and differentiation. Identification of this rare subpopulation within the memory CD8(+) T cell precursor pool may help reconcile models of the developmental origin of long-term CD8(+) T cell memory. Full Text

Jost, M., Jacobson, A.N., Hussmann, J.A., Cirolia, G., Fischbach, M.A., and Weissman, J.S. (2021). CRISPR-based functional genomics in human dendritic cells. eLife 10:e65856. Dendritic cells (DCs) regulate processes ranging from antitumor and antiviral immunity to host-microbe communication at mucosal surfaces. It remains difficult, however, to genetically manipulate human DCs, limiting our ability to probe how DCs elicit specific immune responses. Here, we develop a CRISPR-Cas9 genome editing method for human monocyte-derived DCs (moDCs) that mediates knockouts with a median efficiency of >94% across >300 genes. Using this method, we perform genetic screens in moDCs, identifying mechanisms by which DCs tune responses to lipopolysaccharides from the human microbiome. In addition, we reveal donor-specific responses to lipopolysaccharides, underscoring the importance of assessing immune phenotypes in donor-derived cells, and identify candidate genes that control this specificity, highlighting the potential of our method to pinpoint determinants of inter-individual variation in immunity. Our work sets the stage for a systematic dissection of the immune signaling at the host-microbiome interface and for targeted engineering of DCs for neoantigen vaccination. Full Text

Joyner, A., Lehmann, R., and Niswander, L. (2021). In Memoriam: Kathryn V. Anderson (1952-2020). Developmental Biology 472, 125-126.[Epub ahead of print]. It is with deepest respect that we write to honor the memory and accomplishments of Dr. Kathryn V. Anderson, who died peacefully on November 30, 2020. Kathryn was a world leader in the developmental biology community who made ground-breaking contributions throughout her career. She built a highly respected Program in Developmental Biology at Memorial Sloan Kettering Cancer Center, she fought against sexism and for inclusion, and was a role model for young scientists and an unselfish mentor and colleague. Until recently, Kathryn could be seen in the mouse room attending to her precious mouse lines. Full Text

Kamal, Y., Dwan, D., Hoehn, H.J., Sanz-Pamplona, R., Alonso, M.H., Moreno, V., Cheng, C., Schell, M.J., Kim, Y., Felder, S.I., Lazaris, C., et al. (2021). Tumor immune infiltration estimated from gene expression profiles predicts colorectal cancer relapse. Oncoimmunology 10(1):1862529. A substantial fraction of patients with stage I-III colorectal adenocarcinoma (CRC) experience disease relapse after surgery with curative intent. However, biomarkers for predicting the likelihood of CRC relapse have not been fully explored. Therefore, we assessed the association between tumor infiltration by a broad array of innate and adaptive immune cell types and CRC relapse risk. We implemented a discovery-validation design including a discovery dataset from Moffitt Cancer Center (MCC; Tampa, FL) and three independent validation datasets: (1) GSE41258 (2) the Molecular Epidemiology of Colorectal Cancer (MECC) study, and (3) GSE39582. Infiltration by 22 immune cell types was inferred from tumor gene expression data, and the association between immune infiltration by each cell type and relapse-free survival was assessed using Cox proportional hazards regression. Within each of the four independent cohorts, CD4+ memory activated T cell (HR: 0.93, 95% CI: 0.90-0.96; FDR = 0.0001) infiltration was associated with longer time to disease relapse, independent of stage, microsatellite instability, and adjuvant therapy. Based on our meta-analysis across the four datasets, 10 innate and adaptive immune cell types associated with disease relapse of which 2 were internally validated using multiplex immunofluorescence. Moreover, immune cell type infiltration was a better predictors of disease relapse than Consensus Molecular Subtype (CMS) and other expression-based biomarkers (Immune-AIC(MCC):238.1-238.9; CMS-AIC(MCC): 241.0). These data suggest that transcriptome-derived immune profiles are prognostic indicators of CRC relapse and quantification of both innate and adaptive immune cell types may serve as candidate biomarkers for predicting prognosis and guiding frequency and modality of disease surveillance.Full Text

Katz, D.H., Tahir, U.A., Ngo, D., Benson, M.D., Gao, Y., Shi, X., Nayor, M., Keyes, M.J., Larson, M.G., Hall, M.E., Sinha, S., et al. (2021). Multi-Omic Profiling in Black and White Populations Reveals Novel Candidate Pathways in Left Ventricular Hypertrophy and Incident Heart Failure Specific to Black Adults. Circulation.Genomic and Precision Medicine. Online Ahead of Print. Increased left ventricular (LV) mass is associated with adverse cardiovascular events including heart failure (HF). Both increased LV mass and HF disproportionately affect Black individuals. To understand underlying mechanisms, we undertook a proteomic screen in a Black cohort and compared the findings to results from a white cohort. Methods - We measured 1305 plasma proteins using the SomaScan® platform in 1772 Black participants (mean age 56 years, 62% women) in the Jackson Heart Study (JHS) with LV mass assessed by 2D echocardiography. Incident HF was assessed in 1600 participants. We then compared protein associations in JHS to those observed in white participants from the Framingham Heart Study (FHS, mean age 54 years, 56% women). Results - In JHS, there were 110 proteins associated with LV mass and 13 proteins associated with incident HF hospitalization with false discovery rate <5% after multivariable adjustment. Several proteins showed expected associations with both LV mass and HF, including N-terminal pro-BNP (β = 0.04, p = 2 × 10(-8); HR = 1.48, p = 0.0001). The strongest association with LV mass was novel: Leukotriene A-4 hydrolase (LKHA4) (β = 0.05, p = 5 × 10(-15)). This association was confirmed on an alternate proteomics platform and further supported by related metabolomic data. Fractalkine/CX3CL1 showed a novel association with incident HF (HR = 1.32, p = 0.0002). While established biomarkers such as cystatin C and N-terminal pro-BNP showed consistent associations in Black and white individuals, LKHA4 and fractalkine were significantly different between the two groups. Conclusions - We identified several novel biological pathways specific to Black adults hypothesized to contribute to the pathophysiologic cascade of LV hypertrophy and incident HF including LKHA4 and fractalkine. Full Text

Khan, S.A., Park, K.M., Fischer, L.A., Dong, C., Lungjangwa, T., Jimenez, M., Casalena, D., Chew, B., Dietmann, S., Auld, D.S., Rudolph Jaenisch, et al. (2021). Probing the signaling requirements for naive human pluripotency by high-throughput chemical screening. Cell Reports 35 (11) : 109233. Naive human embryonic stem cells (hESCs) have been isolated that more closely resemble the pre-implantation epiblast compared to conventional "primed" hESCs, but the signaling principles underlying these discrete stem cell states remain incompletely understood. Here, we describe the results from a high-throughput screen using ∼3,000 well-annotated compounds to identify essential signaling requirements for naive human pluripotency. We report that MEK1/2 inhibitors can be replaced during maintenance of naive human pluripotency by inhibitors targeting either upstream (FGFR, RAF) or downstream (ERK1/2) kinases. Naive hESCs maintained under these alternative conditions display elevated levels of ERK phosphorylation but retain genome-wide DNA hypomethylation and a transcriptional identity of the pre-implantation epiblast. In contrast, dual inhibition of MEK and ERK promotes efficient primed-to-naive resetting in combination with PKC, ROCK, and TNKS inhibitors and activin A. This work demonstrates that induction and maintenance of naive human pluripotency are governed by distinct signaling requirements. Full Text

Kim, J.S., Pineda, M., and Li, P. (2021). Reconstitution of Morphogen Signaling Gradients in Cultured Cells. Methods in Molecular Biology 2258, 43-56. Development of multicellular organisms depends on the proper establishment of signaling information in space and time. Secreted molecules called morphogens form concentration gradients in space and provide positional information to differentiating cells within the organism. Although the key molecular components of morphogen pathways have been identified, how the architectures and key parameters of morphogen pathways control the properties of signaling gradients, such as their size, speed, and robustness to perturbations, remains challenging to study in developing embryos. Reconstituting morphogen gradients in cell culture provides an alternative approach to address this question. Here we describe the methodology for reconstituting Sonic Hedgehog (SHH) signaling gradients in mouse fibroblast cells. The protocol includes the design of morphogen sending and receiving cell lines, the setup of radial and linear gradients, the quantitative time-lapse imaging, and the data analysis. Similar approaches could potentially be applied to other cell-cell communication pathways.Full Text

Kingston, E.R., and Bartel, D.P. (2021). Ago2 protects Drosophila siRNAs and microRNAs from target-directed degradation, even in the absence of 2'-O-methylation. RNA. Online Ahead of Print. Target-directed microRNA (miRNA) degradation (TDMD), which is mediated by the protein ZSWIM8, plays a widespread role in shaping miRNA abundances across bilateria. Some endogenous small interfering RNAs (siRNAs) of Drosophila cells have target sites resembling those that trigger TDMD, raising the question as to whether they too might undergo such regulation by Dora, the Drosophila ZSWIM8 homolog. Here, we find that some of these siRNAs are indeed sensitive to Dora when loaded into Ago1, the Argonaute paralog that preferentially associates with miRNAs. Despite this sensitivity when loaded into Ago1, these siRNAs are not detectably regulated by target-directed degradation because most molecules are loaded into Ago2, the Argonaute paralog that preferentially associates with siRNAs, and we find that siRNAs and miRNAs loaded into Ago2 are insensitive to Dora. One explanation for the protection of these small RNAs loaded into Ago2 is that these small RNAs are 2'-O-methylated at their 3' termini. However, 2'-O-methylation does not protect these RNAs from Dora-mediated target-directed degradation, which indicates that their protection is instead conferred by features of the Ago2 protein itself. Together, these observations clarify the requirements for regulation by target-directed degradation and expand our understanding of the role of 2'-O-methylation in small-RNA biology. Full Text

Klein, K., Hölzemer, A., Wang, T., Kim, T.E., Dugan, H.L., Jost, S., Altfeld, M., and Garcia-Beltran, W.F. (2021). A Genome-Wide CRISPR/Cas9-Based Screen Identifies Heparan Sulfate Proteoglycans as Ligands of Killer-Cell Immunoglobulin-Like Receptors. Frontiers in Immunology 12 : 798235. While human leukocyte antigen (HLA) and HLA-like proteins comprise an overwhelming majority of known ligands for NK-cell receptors, the interactions of NK-cell receptors with non-conventional ligands, particularly carbohydrate antigens, is less well described. We previously found through a bead-based HLA screen that KIR3DS1, a formerly orphan member of the killer-cell immunoglobulin-like receptor (KIR) family, binds to HLA-F. In this study, we assessed the ligand binding profile of KIR3DS1 to cell lines using Fc fusion constructs, and discovered that KIR3DS1-Fc exhibited binding to several human cell lines including ones devoid of HLA. To identify these non-HLA ligands, we developed a magnetic enrichment-based genome-wide CRISPR/Cas9 knock-out screen approach, and identified enzymes involved in the biosynthesis of heparan sulfate as crucial for the binding of KIR3DS1-Fc to K562 cells. This interaction between KIR3DS1 and heparan sulfate was confirmed via surface plasmon resonance, and removal of heparan sulfate proteoglycans from cell surfaces abolished KIR3DS1-Fc binding. Testing of additional KIR-Fc constructs demonstrated that KIR family members containing a D0 domain (KIR3DS1, KIR3DL1, KIR3DL2, KIR2DL4, and KIR2DL5) bound to heparan sulfate, while those without a D0 domain (KIR2DL1, KIR2DL2, KIR2DL3, and KIR2DS4) did not. Overall, this study demonstrates the use of a genome-wide CRISPR/Cas9 knock-out strategy to unbiasedly identify unconventional ligands of NK-cell receptors. Furthermore, we uncover a previously underrecognized binding of various activating and inhibitory KIRs to heparan sulfate proteoglycans that may play a role in NK-cell receptor signaling and target-cell recognition. Full Text

Koblan, L.W., Arbab, M., Shen, M.W., Hussmann, J.A., Anzalone, A.V., Doman, J.L., Newby, G.A., Yang, D., Mok, B., Replogle, J.M., Weissman J.S., et al. (2021). Efficient C•G-to-G•C base editors developed using CRISPRi screens, target-library analysis, and machine learning. Nature Biotechnology. Online Ahead of Print. Programmable C•G-to-G•C base editors (CGBEs) have broad scientific and therapeutic potential, but their editing outcomes have proved difficult to predict and their editing efficiency and product purity are often low. We describe a suite of engineered CGBEs paired with machine learning models to enable efficient, high-purity C•G-to-G•C base editing. We performed a CRISPR interference (CRISPRi) screen targeting DNA repair genes to identify factors that affect C•G-to-G•C editing outcomes and used these insights to develop CGBEs with diverse editing profiles. We characterized ten promising CGBEs on a library of 10,638 genomically integrated target sites in mammalian cells and trained machine learning models that accurately predict the purity and yield of editing outcomes (R = 0.90) using these data. These CGBEs enable correction to the wild-type coding sequence of 546 disease-related transversion single-nucleotide variants (SNVs) with >90% precision (mean 96%) and up to 70% efficiency (mean 14%). Computational prediction of optimal CGBE-single-guide RNA pairs enables high-purity transversion base editing at over fourfold more target sites than achieved using any single CGBE variant. Full Text

Lam, F.H., Turanlı-Yıldız, B., Liu, D., Resch, M.G., Fink, G.R., and Stephanopoulos, G. (2021). Engineered yeast tolerance enables efficient production from toxified lignocellulosic feedstocks. Science Advances Vol. 7, no. 26 : eabf7613. Lignocellulosic biomass remains unharnessed for the production of renewable fuels and chemicals due to challenges in deconstruction and the toxicity its hydrolysates pose to fermentation microorganisms. Here, we show in Saccharomyces cerevisiae that engineered aldehyde reduction and elevated extracellular potassium and pH are sufficient to enable near-parity production between inhibitor-laden and inhibitor-free feedstocks. By specifically targeting the universal hydrolysate inhibitors, a single strain is enhanced to tolerate a broad diversity of highly toxified genuine feedstocks and consistently achieve industrial-scale titers (cellulosic ethanol of >100 grams per liter when toxified). Furthermore, a functionally orthogonal, lightweight design enables seamless transferability to existing metabolically engineered chassis strains: We endow full, multifeedstock tolerance on a xylose-consuming strain and one producing the biodegradable plastics precursor lactic acid. The demonstration of "drop-in" hydrolysate competence enables the potential of cost-effective, at-scale biomass utilization for cellulosic fuel and nonfuel products alike. Full Text

Lambert, A.W., and Weinberg, R.A. (2021). Linking EMT programmes to normal and neoplastic epithelial stem cells.Nature Reviews Cancer. Online Ahead of Print. Epithelial stem cells serve critical physiological functions in the generation, maintenance and repair of diverse tissues through their ability to self-renew and spawn more specialized, differentiated cell types. In an analogous fashion, cancer stem cells have been proposed to fuel the growth, progression and recurrence of many carcinomas. Activation of an epithelial-mesenchymal transition (EMT), a latent cell-biological programme involved in development and wound healing, has been linked to the formation of both normal and neoplastic stem cells, but the mechanistic basis underlying this connection remains unclear. In this Perspective, we outline the instances where aspects of an EMT have been implicated in normal and neoplastic epithelial stem cells and consider the involvement of this programme during tissue regeneration and repair. We also discuss emerging concepts and evidence related to the heterogeneous and plastic cell states generated by EMT programmes and how these bear on our understanding of cancer stem cell biology and cancer metastasis. A more comprehensive accounting of the still-elusive links between EMT programmes and the stem cell state will surely advance our understanding of both normal stem cell biology and cancer pathogenesis. Full Text

Lehmann, R., and Peters, J.M. (2021). Angelika Amon (1967-2020). Cell 184, 10-14. A trailblazer of cell biology and genetics, Angelika Amon, died at the age of 53 on October 29, 2020, after a two-and-half year battle with cancer. The scientific community has lost a fearless leader and a truly exceptional colleague, and those who knew her lost a passionate, spirited mentor and friend with a bottom- less heart. Full Text

Le Vasseur, M., Friedman, J., Jost, M., Xu, J., Yamada, J., Kampmann, M., Horlbeck, M.A., Salemi, M.R., Phinney, B.S., Weissman, J.S., et al. (2021). Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells. Elife 10:e67624. Dysfunction of the mitochondrial electron transport chain (mETC) is a major cause of human mitochondrial diseases. To identify determinants of mETC function, we screened a genome-wide human CRISPRi library under oxidative metabolic conditions with selective inhibition of mitochondrial Complex III and identified ovarian carcinoma immunoreactive antigen (OCIA) domain-containing protein 1 (OCIAD1) as a Complex III assembly factor. We find that OCIAD1 is an inner mitochondrial membrane protein that forms a complex with supramolecular prohibitin assemblies. Our data indicate that OCIAD1 is required for maintenance of normal steady-state levels of Complex III and the proteolytic processing of the catalytic subunit cytochrome c(1) (CYC1). In OCIAD1 depleted mitochondria, unprocessed CYC1 is hemylated and incorporated into Complex III. We propose that OCIAD1 acts as an adaptor within prohibitin assemblies to stabilize and/or chaperone CYC1 and to facilitate its proteolytic processing by the IMMP2L protease. Full Text

Luengo, A., Li, Z., Gui, D.Y., Sullivan, L.B., Zagorulya, M., Do, B.T., Ferreira, R., Naamati, A., Ali, A., Lewis, C.A.,, et al.(2021). Increased demand for NAD(+) relative to ATP drives aerobic glycolysis. Molecular Cell 81(4):691-707. Increased demand for NAD(+) relative to ATP drives aerobic glycolysis. Increased demand for NAD(+) relative to ATP drives aerobic glycolysis. Increased demand for NAD(+) relative to ATP drives aerobic glycolysis. Aerobic glycolysis, or preferential fermentation of glucose-derived pyruvate to lactate despite available oxygen, is associated with proliferation across many organisms and conditions. To better understand that association, we examined the metabolic consequence of activating the pyruvate dehydrogenase complex (PDH) to increase pyruvate oxidation at the expense of fermentation. We find that increasing PDH activity impairs cell proliferation by reducing the NAD(+)/NADH ratio. This change in NAD(+)/NADH is caused by increased mitochondrial membrane potential that impairs mitochondrial electron transport and NAD(+) regeneration. Uncoupling respiration from ATP synthesis or increasing ATP hydrolysis restores NAD(+)/NADH homeostasis and proliferation even when glucose oxidation is increased. These data suggest that when demand for NAD(+) to support oxidation reactions exceeds the rate of ATP turnover in cells, NAD(+) regeneration by mitochondrial respiration becomes constrained, promoting fermentation, despite available oxygen. This argues that cells engage in aerobic glycolysis when the demand for NAD(+) is in excess of the demand for ATP. Full Text

Luo, L., Chiu, L.Y., Sugarman, A., Gupta, P., Rouskin, S. , and Tolbert, B.S. (2021). HnRNP A1/A2 Proteins Assemble onto 7SK snRNA via Context Dependent Interactions. Journal of Molecular Biology : 166885. Online Ahead of Print. 7SK small nuclear RNA (snRNA) is an abundant and ubiquitously expressed noncoding RNA that functions to modulate the activity of RNA Polymerase II (RNAPII) in part by stabilizing distinct pools of 7SK-protein complexes. Prevailing models suggest that the secondary structure of 7SK is dynamically remodeled within its alternative RNA-protein pools such that its architecture differentially regulates the exchange of cognate binding partners. The nuclear hnRNP A1/A2 proteins influence the biology of 7SK snRNA via processes that require an intact stem loop (SL) 3 domain; however, the molecular details by which hnRNPs assemble onto 7SK snRNA are yet to be described. Here, we have taken an integrated approach to present a detailed description of the 7SK-hnRNP A1 complex. We show that unbound 7SK snRNA adopts at least two major conformations in solution, with significant structural differences localizing to the SL2-3 linker and the base of SL3. Phylogenetic analysis indicates that this same region is the least genetically conserved feature of 7SK snRNA. By performing DMS modifications with the presence of excess protein, we reveal that hnRNP A1 binds with selectivity to SL3 through mechanisms that increase the flexibility of the RNA adjacent to putative binding sites. Calorimetric titrations further validate that hnRNP A1-SL3 assembly is complex with the affinity of discrete binding events modulated by the surrounding RNA structure. To interpret this context-dependent binding phenomenon, we determined a 3D model of SL3 to show that it folds to position minimal hnRNP A1/A2 binding sites (5'-Y/RAG-3') within different local environments. SL3-protein complexes resolved by SEC-MALS-SAXS confirm that up to four hnRNP A1 proteins bind along the entire surface of SL3 via interactions that preserve the overall structural integrity of this domain. In sum, the collective results presented here reveal a specific role for a folded SL3 domain to scaffold hnRNP A1/A2-7SK assembly via mechanisms modulated by the surrounding RNA structure. Full Text

Maier, N.K., Ma, J., Lampson, M.A., and Cheeseman, I.M. (2021). Separase cleaves the kinetochore protein Meikin at the meiosis I/II transition. Developmental Cell. Online Ahead of Print. To generate haploid gametes, germ cells undergo two consecutive meiotic divisions requiring key changes to the cell division machinery. Here, we demonstrate that the protease separase rewires key cell division processes at the meiosis I/II transition by cleaving the meiosis-specific protein Meikin. Separase proteolysis does not inactivate Meikin but instead alters its function to create a distinct activity state. Full-length Meikin and the C-terminal Meikin separase cleavage product both localize to kinetochores, bind to Plk1 kinase, and promote Rec8 cleavage, but our results reveal distinct roles for these proteins in controlling meiosis. Mutations that prevent Meikin cleavage or that conditionally inactivate Meikin at anaphase I result in defective meiosis II chromosome alignment in mouse oocytes. Finally, as oocytes exit meiosis, C-Meikin is eliminated by APC/C-mediated degradation prior to the first mitotic division. Thus, multiple regulatory events irreversibly modulate Meikin activity during successive meiotic divisions to rewire the cell division machinery at two distinct transitions. Full Text

Mana, M.D., Hussey, A.M., Tzouanas, C.N., Imada, S., Barrera Millan, Y., Bahceci, D., Saiz, D.R., Webb, A.T., Lewis, C.A., Carmeliet, P., et al. (2021). High-fat diet-activated fatty acid oxidation mediates intestinal stemness and tumorigenicity. Cell Reports 35(10): 109212. Obesity is an established risk factor for cancer in many tissues. In the mammalian intestine, a pro-obesity high-fat diet (HFD) promotes regeneration and tumorigenesis by enhancing intestinal stem cell (ISC) numbers, proliferation, and function. Although PPAR (peroxisome proliferator-activated receptor) nuclear receptor activity has been proposed to facilitate these effects, their exact role is unclear. Here we find that, in loss-of-function in vivo models, PPARα and PPARδ contribute to the HFD response in ISCs. Mechanistically, both PPARs do so by robustly inducing a downstream fatty acid oxidation (FAO) metabolic program. Pharmacologic and genetic disruption of CPT1A (the rate-controlling enzyme of mitochondrial FAO) blunts the HFD phenotype in ISCs. Furthermore, inhibition of CPT1A dampens the pro-tumorigenic consequences of a HFD on early tumor incidence and progression. These findings demonstrate that inhibition of a HFD-activated FAO program creates a therapeutic opportunity to counter the effects of a HFD on ISCs and intestinal tumorigenesis. Full Text

Markus, B.M., Boydston, E.A., and Lourido, S. (2021). CRISPR-Mediated Transcriptional Repression in Toxoplasma gondii. mSphere : e0047421. Online ahead of print. Tools for tuning endogenous gene expression are key to determining the genetic basis of diverse cellular phenotypes. Although synthetic regulatable promoters are available in Toxoplasma, scalable methods for targeted and combinatorial downregulation of gene expression-like RNA interference-have yet to be developed. To investigate the feasibility of CRISPR-mediated transcriptional regulation, we examined the function of two catalytically inactive Cas9 (dCas9) orthologs, from Streptococcus pyogenes and Streptococcus thermophilus, in Toxoplasma. Following the addition of single-guide RNAs (sgRNAs) targeting the promoter and 5' untranslated region (UTR) of the surface antigen gene SAG1, we profiled changes in protein abundance of targeted genes by flow cytometry for transcriptional reporters and immunoblotting. We found that the dCas9 orthologs generated a range of target gene expression levels, and the degree of repression was durable and stably inherited. Therefore, S. pyogenes and S. thermophilus dCas9 can effectively produce intermediate levels of gene expression in Toxoplasma. The distinct sgRNA scaffold requirements of the two dCas9s permit their orthogonal use for simultaneous examination of two distinct loci through transcriptional modulation, labeling for microscopy-based studies, or other dCas9-based approaches. Taking advantage of newly available genomic transcription start site data, these tools will aid in the development of new loss-of-function screening approaches in Toxoplasma. IMPORTANCE Toxoplasma gondii is a ubiquitous intracellular parasite of humans and animals that causes life-threatening disease in immunocompromised patients, fetal abnormalities when contracted during gestation, and recurrent eye lesions in some patients. Despite its health implications, about half of the Toxoplasma genome still lacks functional annotation. A particularly powerful tool for the investigation of an organism's cell biology is the modulation of gene expression, which can produce the subtle phenotypes often required for informing gene function. In Toxoplasma, such tools have limited throughput and versatility. Here, we detail the adaptation of a new set of tools based on CRISPR-Cas9, which allows the targeted downregulation of gene expression in Toxoplasma. With its scalability and adaptability to diverse genomic loci, this approach has the potential to greatly accelerate the functional characterization of the Toxoplasma genome. Full Text

McCormick, D.M., Lalanne, J.B., Lan, T.C.T., Rouskin, S., and Li, G.W. (2021). Sigma factor dependent translational activation in Bacillus subtilis. RNA. 078747.121. Sigma factors are an important class of bacterial transcription factors that lend specificity to RNA polymerases by binding to distinct promoter elements for genes in their regulons. Here we show that activation of the general stress sigma factor, σ(B), in Bacillus subtilis paradoxically leads to dramatic induction of translation for a subset of its regulon genes. These genes are translationally repressed when transcribed by the housekeeping sigma factor, σ(A), owing to extended RNA secondary structures as determined in vivo using DMS-MaPseq. Transcription from σ(B)-dependent promoters ablates the secondary structures and activates translation, leading to dual induction. Translation efficiencies between σ(B)- and σ(A)-dependent RNA isoforms can vary by up to 100-fold, which in multiple cases exceeds the magnitude of transcriptional induction. These results highlight the role of long-range RNA folding in modulating translation and demonstrate that a transcription factor can regulate protein synthesis beyond its effects on transcript levels. Full Text

Montalban-Lopez, M., Scott, T.A., Ramesh, S., Rahman, I.R., van Heel, A.J., Viel, J.H., Bandarian, V., Dittmann, E., Genilloud, O., Goto, Y., Weng, J.K., et al. (2021). New developments in RiPP discovery, enzymology and engineering. Natural Product Reports 38:130-239. Covering: up to June 2020 Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research. Full Text

Muus, C., Luecken, M.D., Eraslan, G., Sikkema, L., Waghray, A., Heimberg, G., Kobayashi, Y., Vaishnav, E.D., Subramanian, A., Smillie, C., Shu, J., et al. (2021). Single-cell meta-analysis of SARS-CoV-2 entry genes across tissues and demographics. Nature Medicine. Online Ahead of Print. Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2(+)TMPRSS2(+) cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention. Full Text

Nardini, E., Hogan, R., Flamier, A., and Bernier, G. (2021). Alzheimer's disease: a tale of two diseases? Neural Regeneration Research 16(10): 1958-1964. Sporadic late-onset Alzheimer's disease (SLOAD) and familial early-onset Alzheimer's disease (FEOAD) associated with dominant mutations in APP, PSEN1 and PSEN2, are thought to represent a spectrum of the same disorder based on near identical behavioral and histopathological features. Hence, FEOAD transgenic mouse models have been used in past decades as a surrogate to study SLOAD pathogenic mechanisms and as the gold standard to validate drugs used in clinical trials. Unfortunately, such research has yielded little output in terms of therapeutics targeting the disease's development and progression. In this short review, we interrogate the widely accepted view of one, dimorphic disease through the prism of the Bmi1(+/-) mouse model and the distinct chromatin signatures observed between SLOAD and FEOAD brains. Full Text

Navarro, A.P., and Cheeseman, I.M. (2021). Kinetochore assembly throughout the cell cycle. Seminars in Cell & Developmental Biology. Online Ahead of Print. The kinetochore plays an essential role in facilitating chromosome segregation during cell division. This massive protein complex assembles onto the centromere of chromosomes and enables their attachment to spindle microtubules during mitosis. The kinetochore also functions as a signaling hub to regulate cell cycle progression, and is crucial to ensuring the fidelity of chromosome segregation. Despite the fact that kinetochores are large and robust molecular assemblies, they are also highly dynamic structures that undergo structural and organizational changes throughout the cell cycle. This review will highlight our current understanding of kinetochore structure and function, focusing on the dynamic processes that underlie kinetochore assembly. Full Text

Ngo, B., Kim, E., Osorio-Vasquez, V., Doll, S., Bustraan, S., Liang, R.J., Luengo, A., Davidson, S.M., Ali, A., Ferraro, G.B., David M. Sabatini, et al. (2020). Limited Environmental Serine and Glycine Confer Brain Metastasis Sensitivity to PHGDH Inhibition. Cancer Discovery 10 : 1352-1373. A hallmark of metastasis is the adaptation of tumor cells to new environments. Metabolic constraints imposed by the serine and glycine-limited brain environment restrict metastatic tumor growth. How brain metastases overcome these growth-prohibitive conditions is poorly understood. Here, we demonstrate that 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of glucose-derived serine synthesis, is a major determinant of brain metastasis in multiple human cancer types and preclinical models. Enhanced serine synthesis proved important for nucleotide production and cell proliferation in highly aggressive brain metastatic cells. In vivo, genetic suppression and pharmacologic inhibition of PHGDH attenuated brain metastasis, but not extracranial tumor growth, and improved overall survival in mice. These results reveal that extracellular amino acid availability determines serine synthesis pathway dependence, and suggest that PHGDH inhibitors may be useful in the treatment of brain metastasis. SIGNIFICANCE: Using proteomics, metabolomics, and multiple brain metastasis models, we demonstrate that the nutrient-limited environment of the brain potentiates brain metastasis susceptibility to serine synthesis inhibition. These findings underscore the importance of studying cancer metabolism in physiologically relevant contexts, and provide a rationale for using PHGDH inhibitors to treat brain metastasis. Full Text

Nguyen, A.L., Fadel, M.D., and Cheeseman, I.M. (2021). Differential Requirements for the CENP-O Complex Reveal Parallel PLK1 Kinetochore Recruitment Pathways. Molecular Biology of the Cell:mbcE20110751. Online Ahead of Print. Similar to other core biological processes, the vast majority of cell division components are essential for viability across human cell lines. However, recent genome-wide screens have identified a number of proteins that exhibit cell line-specific essentiality. Defining the behaviors of these proteins is critical to our understanding of complex biological processes. Here, we harness differential essentiality to reveal the contributions of the 4-subunit centromere-localized CENP-O complex, whose precise function has been difficult to define. Our results support a model in which the CENP-O complex and BUB1 act in parallel pathways to recruit a threshold level of PLK1 to mitotic kinetochores, ensuring accurate chromosome segregation. We demonstrate that targeted changes to either pathway sensitizes cells to the loss of the other component, resulting in cell-state dependent requirements. This approach also highlights the advantage of comparing phenotypes across diverse cell lines to define critical functional contributions and behaviors that could be exploited for the targeted treatment of disease. Full Text

Nicholls, P.K., and Page, D.C. (2021). Germ cell determination and the developmental origin of germ cell tumors. Development 148(8):dev198150. In each generation, the germline is tasked with producing somatic lineages that form the body, and segregating a population of cells for gametogenesis. During animal development, when do cells of the germline irreversibly commit to producing gametes? Integrating findings from diverse species, we conclude that the final commitment of the germline to gametogenesis - the process of germ cell determination - occurs after primordial germ cells (PGCs) colonize the gonads. Combining this understanding with medical findings, we present a model whereby germ cell tumors arise from cells that failed to undertake germ cell determination, regardless of their having colonized the gonads. We propose that the diversity of cell types present in these tumors reflects the broad developmental potential of migratory PGCs. Full Text

Nishida, Y., Zhao, R., Heese, L.E., Akiyama, H., Patel, S., Jaeger, A.M., Jacamo, R.O., Kojima, K., Ma, M.C.J., Ruvolo, V.R., Lindquist, Susan, Whitesell, Luke, et al. (2021). Inhibition of translation initiation factor eIF4a inactivates heat shock factor 1 (HSF1) and exerts anti-leukemia activity in AML. Leukemia. Online Ahead of Print. Eukaryotic initiation factor 4A (eIF4A), the enzymatic core of the eIF4F complex essential for translation initiation, plays a key role in the oncogenic reprogramming of protein synthesis, and thus is a putative therapeutic target in cancer. As important component of its anticancer activity, inhibition of translation initiation can alleviate oncogenic activation of HSF1, a stress-inducible transcription factor that enables cancer cell growth and survival. Here, we show that primary acute myeloid leukemia (AML) cells exhibit the highest transcript levels of eIF4A1 compared to other cancer types. eIF4A inhibition by the potent and specific compound rohinitib (RHT) inactivated HSF1 in these cells, and exerted pronounced in vitro and in vivo anti-leukemia effects against progenitor and leukemia-initiating cells, especially those with FLT3-internal tandem duplication (ITD). In addition to its own anti-leukemic activity, genetic knockdown of HSF1 also sensitized FLT3-mutant AML cells to clinical FLT3 inhibitors, and this synergy was conserved in FLT3 double-mutant cells carrying both ITD and tyrosine kinase domain mutations. Consistently, the combination of RHT and FLT3 inhibitors was highly synergistic in primary FLT3-mutated AML cells. Our results provide a novel therapeutic rationale for co-targeting eIF4A and FLT3 to address the clinical challenge of treating FLT3-mutant AML. Full Text

Nofal, M., Wang, T., Yang, L., Jankowski, C.S.R., Hsin-Jung Li, S., Han, S., Parsons, L., Frese, A.N., Gitai, Z., Anthony, T.G., David M. Sabatini, et al. (2021). GCN2 adapts protein synthesis to scavenging-dependent growth. Cell Systems In Press. Pancreatic cancer cells with limited access to free amino acids can grow by scavenging extracellular protein. In a murine model of pancreatic cancer, we performed a genome-wide CRISPR screen for genes required for scavenging-dependent growth. The screen identified key mediators of macropinocytosis, peripheral lysosome positioning, endosome-lysosome fusion, lysosomal protein catabolism, and translational control. The top hit was GCN2, a kinase that suppresses translation initiation upon amino acid depletion. Using isotope tracers, we show that GCN2 is not required for protein scavenging. Instead, GCN2 prevents ribosome stalling but without slowing protein synthesis; cells still use all of the limiting amino acids as they emerge from lysosomes. GCN2 also adapts gene expression to the nutrient-poor environment, reorienting protein synthesis away from ribosomes and toward lysosomal hydrolases, such as cathepsin L. GCN2, cathepsin L, and the other genes identified in the screen are potential therapeutic targets in pancreatic cancer. Full Text

Nuñez, J.K., Chen, J., Pommier, G.C., Cogan, J.Z., Replogle, J.M., Adriaens, C., Ramadoss, G.N., Shi, Q., Hung, K.L., Samelson, A.J., Pogson, A.N., Weissman, J.S., et al. (2021). Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing.Cell. Online ahead of print. A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance. Full Text

Papathanasiou, S., Markoulaki, S., Blaine, L.J., Leibowitz, M.L., Zhang, C.Z., Jaenisch, R., and Pellman, D. (2021). Whole chromosome loss and genomic instability in mouse embryos after CRISPR-Cas9 genome editing. Nature Communications 12(1):5855. Karyotype alterations have emerged as on-target complications from CRISPR-Cas9 genome editing. However, the events that lead to these karyotypic changes in embryos after Cas9-treatment remain unknown. Here, using imaging and single-cell genome sequencing of 8-cell stage embryos, we track both spontaneous and Cas9-induced karyotype aberrations through the first three divisions of embryonic development. We observe the generation of abnormal structures of the nucleus that arise as a consequence of errors in mitosis, including micronuclei and chromosome bridges, and determine their contribution to common karyotype aberrations including whole chromosome loss that has been recently reported after editing in embryos. Together, these data demonstrate that Cas9-mediated germline genome editing can lead to unwanted on-target side effects, including major chromosome structural alterations that can be propagated over several divisions of embryonic development. Full Text

Payne, A.C., Chiang, Z.D., Reginato, P.L., Mangiameli, S.M., Murray, E.M., Yao, C.C., Markoulaki, S., Earl, A.S., Labade, A.S., Jaenisch, R. , et al. (2021). In situ genome sequencing resolves DNA sequence and structure in intact biological samples. Science 371(6532):eaay3446. Understanding genome organization requires integration of DNA sequence and three-dimensional spatial context; however, existing genome-wide methods lack either base pair sequence resolution or direct spatial localization. Here, we describe in situ genome sequencing (IGS), a method for simultaneously sequencing and imaging genomes within intact biological samples. We applied IGS to human fibroblasts and early mouse embryos, spatially localizing thousands of genomic loci in individual nuclei. Using these data, we characterized parent-specific changes in genome structure across embryonic stages, revealed single-cell chromatin domains in zygotes, and uncovered epigenetic memory of global chromosome positioning within individual embryos. These results demonstrate how IGS can directly connect sequence and structure across length scales from single base pairs to whole organisms. Full Text

Perillo, M., Swartz, S.Z., and Wessel, G.M. (2021). A conserved node in the regulation of Vasa between an induced and an inherited program of primordial germ cell specification. Developmental Biology.Online Ahead of Print. Primordial germ cells (PGCs) are specified by diverse mechanisms in early development. In some animals, PGCs are specified via inheritance of maternal determinants, while in others, in a process thought to represent the ancestral mode, PGC fate is induced by cell interactions. Although the terminal factors expressed in specified germ cells are widely conserved, the mechanisms by which these factors are regulated can be widely diverse. Here we show that a post-translational mechanism of germ cell specification is conserved between two echinoderm species thought to employ divergent germ line segregation strategies. Sea urchins segregate their germ line early by an inherited mechanism. The DEAD-box RNA - helicase Vasa, a conserved germline factor, becomes enriched in the PGCs by degradation in future somatic cells by the E3-ubiquitin-ligase Gustavus (Gustafson et al., 2011). This post-translational activity occurs early in development, substantially prior to gastrulation. Here we test this process in germ cell specification of sea star embryos, which use inductive signaling mechanisms after gastrulation for PGC fate determination. We find that Vasa-GFP protein becomes restricted to the PGCs in the sea star even though the injected mRNA is present throughout the embryo. Gustavus depletion, however, results in uniform accumulation of the protein. These data demonstrate that Gustavus-mediated Vasa turnover in somatic cells is conserved between species with otherwise divergent PGC specification mechanisms. Since Gustavus was originally identified in Drosophila melanogaster to have similar functions in Vasa regulation (Kugler et al., 2010), we conclude that this node of Vasa regulation in PGC formation is ancestral and evolutionarily transposable from the ancestral, induced PGC specification program to an inherited PGC specification mechanism. Full Text

Picard, C.L., Povilus, R.A., Williams, B.P., and Gehring, M. (2021). Transcriptional and imprinting complexity in Arabidopsis seeds at single-nucleus resolution. Nature Plants. Online Ahead of Print. Seeds are a key life cycle stage for many plants. Seeds are also the basis of agriculture and the primary source of calories consumed by humans(1). Here, we employ single-nucleus RNA-sequencing to generate a transcriptional atlas of developing Arabidopsis thaliana seeds, with a focus on endosperm. Endosperm, the primary site of gene imprinting in flowering plants, mediates the relationship between the maternal parent and the embryo(2). We identify transcriptionally uncharacterized nuclei types in the chalazal endosperm, which interfaces with maternal tissue for nutrient unloading(3,4). We demonstrate that the extent of parental bias of maternally expressed imprinted genes varies with cell-cycle phase, and that imprinting of paternally expressed imprinted genes is strongest in chalazal endosperm. Thus, imprinting is spatially and temporally heterogeneous. Increased paternal expression in the chalazal region suggests that parental conflict, which is proposed to drive imprinting evolution, is fiercest at the boundary between filial and maternal tissues. Full Text

Pikman, Y., Ocasio-Martinez, N., Alexe, G., Dimitrov, B., Kitara, S., Diehl, F.F., Robichaud, A.L., Conway, A.S., Ross, L., Su, A., Caroline A Lewis, et al. (2021). Targeting serine hydroxymethyltransferases 1 and 2 for T-cell acute lymphoblastic leukemia therapy. Leukemia. Online Ahead of Print. Despite progress in the treatment of acute lymphoblastic leukemia (ALL), T-cell ALL (T-ALL) has limited treatment options, particularly in the setting of relapsed/refractory disease. Using an unbiased genome-scale CRISPR-Cas9 screen we sought to identify pathway dependencies for T-ALL which could be harnessed for therapy development. Disruption of the one-carbon folate, purine and pyrimidine pathways scored as the top metabolic pathways required for T-ALL proliferation. We used a recently developed inhibitor of SHMT1 and SHMT2, RZ-2994, to characterize the effect of inhibiting these enzymes of the one-carbon folate pathway in T-ALL and found that T-ALL cell lines were differentially sensitive to RZ-2994, with the drug inducing a S/G2 cell cycle arrest. The effects of SHMT1/2 inhibition were rescued by formate supplementation. Loss of both SHMT1 and SHMT2 was necessary for impaired growth and cell cycle arrest, with suppression of both SHMT1 and SHMT2 inhibiting leukemia progression in vivo. RZ-2994 also decreased leukemia burden in vivo and remained effective in the setting of methotrexate resistance in vitro. This study highlights the significance of the one-carbon folate pathway in T-ALL and supports further development of SHMT inhibitors for treatment of T-ALL and other cancers. Full Text

Pomplun, S., Jbara, M., Schissel, C.K., Hawken, S.W., Boija, A., Li, C., Klein, I., and Pentelute, B.L. (2021). Parallel Automated Flow Synthesis of Covalent Protein Complexes That Can Inhibit MYC-Driven Transcription. ACS Central Science 7, 8 : 1408-1418. Dysregulation of the transcription factor MYC is involved in many human cancers. The dimeric transcription factor complexes of MYC/MAX and MAX/MAX activate or inhibit, respectively, gene transcription upon binding to the same enhancer box DNA. Targeting these complexes in cancer is a long-standing challenge. Inspired by the inhibitory activity of the MAX/MAX dimer, we engineered covalently linked, synthetic homo- and heterodimeric protein complexes to attenuate oncogenic MYC-driven transcription. We prepared the covalent protein complexes (∼20 kDa, 167-231 residues) in a single shot via parallel automated flow synthesis in hours. The stabilized covalent dimers display DNA binding activity, are intrinsically cell-penetrant, and inhibit cancer cell proliferation in different cell lines. RNA sequencing and gene set enrichment analysis in A549 cancer cells confirmed that the synthetic dimers interfere with MYC-driven transcription. Our results demonstrate the potential of automated flow technology to rapidly deliver engineered synthetic protein complex mimetics that can serve as a starting point in developing inhibitors of MYC-driven cancer cell growth. Full Text

Povilus, R.A., and Gehring, M. (2021). Maternal-filial transfer structures in endosperm: A nexus of nutritional dynamics and seed development. Current Opinion in Plant Biology 65 : 102121. Although the ultimate purpose of a seed is the successful establishment of the next generation, seed development involves more than embryo growth. In angiosperms, seed development requires the intimate coordination of three distinct entities - maternal tissue and two offspring, embryo and embryo-nourishing endosperm. Although seeds are cornerstones of many terrestrial ecosystems and human diets, we are only beginning to understand the interactions among seed tissues and the molecular processes and genes that determine them. Recent studies of gene expression and function in distantly related angiosperms, combined with over 100 years of embryological research, have repeatedly highlighted the endosperm associated with maternal-filial boundaries as a central point in seed developmental dynamics. In this review, we highlight evidence that links this zone with nutritional dynamics, developmental signaling, and imprinted gene expression. We suggest that the underappreciated diversity of this specialized endosperm across angiosperms deserves further study from developmental, molecular, and genetic perspectives. Full Text

Qi, Y., and Weng, J.K. (2021). Editorial overview: Advancing basic plant research and crop improvement through cutting-edge biotechnologies.Current Opinion in Plant Biology 60 : 102069. To feed and nourish the growing human population with a healthy and diverse diet, innovative agricultural solutions are needed. Right now, this quest is of particular urgency in light of the looming climate change. One of the central missions of plant sciences is to provide such solutions by developing crop plants with better yield, nutritional value, and resistance to various adversary biotic and abiotic environments. Biotechnologies have been a driving force for both fundamental discovery and translational applications in plant sciences. Full Text

Quinn, J.J., Jones, M.G., Okimoto, R.A., Nanjo, S., Chan, M.M., Yosef, N., Bivona, T.G., and Weissman, J.S. (2021). Single-cell lineages reveal the rates, routes, and drivers of metastasis in cancer xenografts. Science. Online ahead of print. . Detailed phylogenies of tumor populations can recount the history and chronology of critical events during cancer progression, such as metastatic dissemination. We applied a Cas9-based, single-cell lineage tracer to study the rates, routes, and drivers of metastasis in a lung cancer xenograft mouse model. We report deeply resolved phylogenies for tens of thousands of cancer cells traced over months of growth and dissemination. This revealed stark heterogeneity in metastatic capacity, arising from pre-existing and heritable differences in gene expression. We demonstrate that these identified genes can drive invasiveness, and uncovered an unanticipated suppressive role for KRT17 We also show that metastases disseminated via multidirectional tissue routes and complex seeding topologies. Overall, we demonstrate the power of tracing cancer progression at subclonal resolution and vast scale. Full Text

Rauner, G., Jin, D.X., Miller, D.H., Gierahn, T.M., Li, C.M., Sokol, E.S., Feng, Y.X., Mathis, R.A., Love, J.C., Gupta, P.B., et al. (2021). Breast tissue regeneration is driven by cell-matrix interactions coordinating multi-lineage stem cell differentiation through DDR1. Nature Communications 12(1): 7116. Mammary morphogenesis is an orchestrated process involving differentiation, proliferation and organization of cells to form a bi-layered epithelial network of ducts and lobules embedded in stromal tissue. We have engineered a 3D biomimetic human breast that makes it possible to study how stem cell fate decisions translate to tissue-level structure and function. Using this advancement, we describe the mechanism by which breast epithelial cells build a complex three-dimensional, multi-lineage tissue by signaling through a collagen receptor. Discoidin domain receptor tyrosine kinase 1 induces stem cells to differentiate into basal cells, which in turn stimulate luminal progenitor cells via Notch signaling to differentiate and form lobules. These findings demonstrate how human breast tissue regeneration is triggered by transmission of signals from the extracellular matrix through an epithelial bilayer to coordinate structural changes that lead to formation of a complex ductal-lobular network. Full Text

Raz, A.A., and Yamashita, Y.M. (2021). Stem cell niche signaling goes both ways. Developmental Cell 56:2267-2268. Stem cell niches are well-characterized factories of signaling information, but niche cells themselves also rely on their neighbors for fate maintenance. In this issue of Developmental Cell, Herrera et al. reveal bi-directional communication between Drosophila testis niche "hub" cells and somatic cyst stem cells. Full Text

Raz, A.A., and Yamashita, Y.M. (2021). Molding immortality from a plastic germline. Current Opinion in Cell Biology 73:1-8 Online Ahead of Print. Germ cells are uniquely capable of maintaining cellular immortality, allowing them to give rise to new individuals in generation after generation. Recent studies have identified that the germline state is plastic, with frequent interconversion between germline differentiation states and across the germline/soma border. Therefore, features that grant germline immortality must be inducible, with other cells undergoing some form of rejuvenation to a germline state. In this review, we summarize the breadth of our current interpretations of germline plasticity and the ways in which these fate conversion events can aid our understanding of the underlying hallmarks of germline immortality. Full Text

Raz, A.A., Wurtzel, O., and Reddien, P.W. (2021). Planarian stem cells specify fate yet retain potency during the cell cycle. Cell Stem Cell.Online Ahead of Print. Planarian whole-body regeneration is enabled by stem cells called neoblasts. At least some neoblasts are individually pluripotent. Neoblasts are also heterogeneous, with subpopulations of specialized neoblasts having different specified fates. Fate specification in neoblasts is regulated by fate-specific transcription factor (FSTF) expression. Here, we find that FSTF expression is common in neoblast S/G2/M cell-cycle phases but less common in G1. We find that specialized neoblasts can divide to produce progeny with asymmetric cell fates, suggesting that they could retain pluripotency. Furthermore, no known neoblast class was present in all neoblast colonies, suggesting that pluripotency is not the exclusive property of any known class. We tested this possibility with single-cell transplantations, which indicate that at least some specialized neoblasts are likely clonogenic. On the basis of these findings, we propose a model for neoblast pluripotency in which neoblasts can undergo specialization during the cell cycle without loss of potency. Full Text

Reddien, P.W. (2021). Positional Information and Stem Cells Combine to Result in Planarian Regeneration. Cold Spring Harbor Perspectives in Biology. Online ahead of Print. The capacity for regeneration is broad in the animal kingdom. Planarians are flatworms that can regenerate any missing body part and their regenerative powers have combined with ease of experimentation to make them a classic regeneration model for more than a century. Pluripotent stem cells called neoblasts generate missing planarian tissues. Fate specification happens in the neoblasts, and this can occur in response to regeneration instructions in the form of positional information. Fate specification can lead to differentiating cells in single steps rather than requiring a long lineage hierarchy. Planarians display constitutive expression of positional information from muscle cells, which is required for patterned maintenance of tissues in tissue turnover. Amputation leads to the rapid resetting of positional information in a process triggered by wound signaling and the resetting of positional information is required for regeneration. These findings suggest a model for planarian regeneration in which adult positional information resets after injury to regulate stem cells to bring about the replacement of missing parts. Full Text

Reddien, P.W. (2021). Principles of regeneration revealed by the planarian eye. Current Opinion in Cell Biology 73 : 19-25. One approach to elucidating the principles of regeneration is to investigate mechanisms that regenerate a target organ. Planarian eyes are discrete, visible structures that are dispensable for viability, making them powerful for studying the logic of regeneration. Fate specification in eye regeneration occurs in stem cells (neoblasts), generating eye progenitors. Eye progenitor production is not responsive to the presence or absence of the eye, with regeneration explained by constant progenitor production in the appropriate positional environment. Eye progenitors display coarse spatial specification. A combination of eye-extrinsic cues and self-organization with differentiated eye cells dictate where migratory eye progenitors target. Finally, guidepost-like cells influence regenerating axons to facilitate the restoration of eye circuitry. These findings from the eye as a case study present a model that explains how regeneration can occur. Full Text

Reddy, J., Fonseca, M.A.S., Corona, R.I., Nameki, R., Segato Dezem, F., Klein, I.A., Chang, H., Chaves-Moreira, D., Afeyan, L.K., Malta, T.M., Richard A Young, et al. (2021). Predicting master transcription factors from pan-cancer expression data. Science Advances 7(48):eabf6123. Critical developmental “master transcription factors” (MTFs) can be subverted during tumorigenesis to control oncogenic transcriptional programs. Current approaches to identifying MTFs rely on ChIP-seq data, which is unavailable for many cancers. We developed the CaCTS (Cancer Core Transcription factor Specificity) algorithm to prioritize candidate MTFs using pan-cancer RNA sequencing data. CaCTS identified candidate MTFs across 34 tumor types and 140 subtypes including predictions for cancer types/subtypes for which MTFs are unknown, including e.g. PAX8, SOX17, and MECOM as candidates in ovarian cancer (OvCa). In OvCa cells, consistent with known MTF properties, these factors are required for viability, lie proximal to superenhancers, co-occupy regulatory elements globally, co-bind loci encoding OvCa biomarkers, and are sensitive to pharmacologic inhibition of transcription. Our predictions of MTFs, especially for tumor types with limited understanding of transcriptional drivers, pave the way to therapeutic targeting of MTFs in a broad spectrum of cancers. Full Text

Reinfeld, B.I., Madden, M.Z., Wolf, M.M., Chytil, A., Bader, J.E., Patterson, A.R., Sugiura, A., Cohen, A.S., Ali, A., Do, B.T., Lewis, C.A., et al. (2021). Cell-programmed nutrient partitioning in the tumour microenvironment. Nature. Online Ahead of Print. Cancer cells characteristically consume glucose through Warburg metabolism(1), a process that forms the basis of tumour imaging by positron emission tomography (PET). Tumour-infiltrating immune cells also rely on glucose, and impaired immune cell metabolism in the tumour microenvironment (TME) contributes to immune evasion by tumour cells(2-4). However, whether the metabolism of immune cells is dysregulated in the TME by cell-intrinsic programs or by competition with cancer cells for limited nutrients remains unclear. Here we used PET tracers to measure the access to and uptake of glucose and glutamine by specific cell subsets in the TME. Notably, myeloid cells had the greatest capacity to take up intratumoral glucose, followed by T cells and cancer cells, across a range of cancer models. By contrast, cancer cells showed the highest uptake of glutamine. This distinct nutrient partitioning was programmed in a cell-intrinsic manner through mTORC1 signalling and the expression of genes related to the metabolism of glucose and glutamine. Inhibiting glutamine uptake enhanced glucose uptake across tumour-resident cell types, showing that glutamine metabolism suppresses glucose uptake without glucose being a limiting factor in the TME. Thus, cell-intrinsic programs drive the preferential acquisition of glucose and glutamine by immune and cancer cells, respectively. Cell-selective partitioning of these nutrients could be exploited to develop therapies and imaging strategies to enhance or monitor the metabolic programs and activities of specific cell populations in the TME. Full Text

Richards, A., Berth, S.H., Brady, S., and Morfini, G. (2021). Engagement of Neurotropic Viruses in Fast Axonal Transport: Mechanisms, Potential Role of Host Kinases and Implications for Neuronal Dysfunction. Frontiers in Cellular Neuroscience 15 : 684762. Much remains unknown about mechanisms sustaining the various stages in the life cycle of neurotropic viruses. An understanding of those mechanisms operating before their replication and propagation could advance the development of effective anti-viral strategies. Here, we review our current knowledge of strategies used by neurotropic viruses to undergo bidirectional movement along axons. We discuss how the invasion strategies used by specific viruses might influence their mode of interaction with selected components of the host's fast axonal transport (FAT) machinery, including specialized membrane-bounded organelles and microtubule-based motor proteins. As part of this discussion, we provide a critical evaluation of various reported interactions among viral and motor proteins and highlight limitations of some in vitro approaches that led to their identification. Based on a large body of evidence documenting activation of host kinases by neurotropic viruses, and on recent work revealing regulation of FAT through phosphorylation-based mechanisms, we posit a potential role of host kinases on the engagement of viruses in retrograde FAT. Finally, we briefly describe recent evidence linking aberrant activation of kinase pathways to deficits in FAT and neuronal degeneration in the context of human neurodegenerative diseases. Based on these findings, we speculate that neurotoxicity elicited by viral infection may involve deregulation of host kinases involved in the regulation of FAT and other cellular processes sustaining neuronal function and survival. Full Text

Rivera-Cuevas, Y., Mayoral, J., Di Cristina, M., Lawrence, A.E., Olafsson, E.B., Patel, R.K., Thornhill, D., Waldman, B.S., Ono, A., Sexton, J.Z., Sebastian Lourido, et al. (2021). Toxoplasma gondii exploits the host ESCRT machinery for parasite uptake of host cytosolic proteins. PLoS Pathogens 17 : e1010138. Toxoplasma gondii is a master manipulator capable of effectively siphoning the resources from the host cell for its intracellular subsistence. However, the molecular underpinnings of how the parasite gains resources from its host remain largely unknown. Residing within a non-fusogenic parasitophorous vacuole (PV), the parasite must acquire resources across the limiting membrane of its replicative niche, which is decorated with parasite proteins including those secreted from dense granules. We discovered a role for the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in host cytosolic protein uptake by T. gondii by disrupting host ESCRT function. We identified the transmembrane dense granule protein TgGRA14, which contains motifs homologous to the late domain motifs of HIV-1 Gag, as a candidate for the recruitment of the host ESCRT machinery to the PV membrane. Using an HIV-1 virus-like particle (VLP) release assay, we found that the motif-containing portion of TgGRA14 is sufficient to substitute for HIV-1 Gag late domain to mediate ESCRT-dependent VLP budding. We also show that TgGRA14 is proximal to and interacts with host ESCRT components and other dense granule proteins during infection. Furthermore, analysis of TgGRA14-deficient parasites revealed a marked reduction in ingestion of a host cytosolic protein compared to WT parasites. Thus, we propose a model in which T. gondii recruits the host ESCRT machinery to the PV where it can interact with TgGRA14 for the internalization of host cytosolic proteins across the PV membrane (PVM). These findings provide new insight into how T. gondii accesses contents of the host cytosol by exploiting a key pathway for vesicular budding and membrane scission. Full Text

Rossiter, N.J., Huggler, K.S., Adelmann, C.H., Keys, H.R., Soens, R.W., Sabatini, D.M., and Cantor, J.R. (2021). CRISPR screens in physiologic medium reveal conditionally essential genes in human cells. Cell Metabolism. Online Ahead of Print. Forward genetic screens across hundreds of cancer cell lines have started to define the genetic dependencies of proliferating human cells and how these vary by genotype and lineage. Most screens, however, have been carried out in culture media that poorly reflect metabolite availability in human blood. Here, we performed CRISPR-based screens in traditional versus human plasma-like medium (HPLM). Sets of conditionally essential genes in human cancer cell lines span several cellular processes and vary with both natural cell-intrinsic diversity and the combination of basal and serum components that comprise typical media. Notably, we traced the causes for each of three conditional CRISPR phenotypes to the availability of metabolites uniquely defined in HPLM versus conventional media. Our findings reveal the profound impact of medium composition on gene essentiality in human cells, and also suggest general strategies for using genetic screens in HPLM to uncover new cancer vulnerabilities and gene-nutrient interactions. Full Text

Rossman, M.P., Hoi, K., Chan, V., Abraham, B.J., Yang, S., Mullahoo, J., Papanastasiou, M., Wang, Y., Elia, I., Perlin, J.R., Richard A. Young, et al.(2021). Cell-specific transcriptional control of mitochondrial metabolism by TIF1γ drives erythropoiesis. Science 372(6543):716-721. Transcription and metabolism both influence cell function, but dedicated transcriptional control of metabolic pathways that regulate cell fate has rarely been defined. We discovered, using a chemical suppressor screen, that inhibition of the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) rescues erythroid differentiation in bloodless zebrafish moonshine (mon) mutant embryos defective for transcriptional intermediary factor 1 gamma (tif1γ). This rescue depends on the functional link of DHODH to mitochondrial respiration. The transcription elongation factor TIF1γ directly controls coenzyme Q (CoQ) synthesis gene expression. Upon tif1γ loss, CoQ levels are reduced, and a high succinate/α-ketoglutarate ratio leads to increased histone methylation. A CoQ analog rescues mon's bloodless phenotype. These results demonstrate that mitochondrial metabolism is a key output of a lineage transcription factor that drives cell fate decisions in the early blood lineage. Full Text

Schlingmann, K.P., Jouret, F., Shen, K., Nigam, A., Arjona, F., Dafinger, C.,Houillier, P., Jones, D., Kleinerüschkamp, F., Oh, J., David Sabatini, et al. (2021). mTOR-Activating Mutations in RRAGD are Causative for Kidney Tubulopathy and Cardiomyopathy. Journal of the American Society of Nephrology : JASN. Online Ahead of Print. Background Over the last decade, advances in genetic techniques have resulted in the identification of rare hereditary disorders of renal magnesium and salt handling. Nevertheless, approximately 20% of all tubulopathy patients lack a genetic diagnosis. Methods We performed whole-exome and genome sequencings of a patient cohort with a novel inherited salt-losing tubulopathy, hypomagnesemia, and dilated cardiomyopathy. We also conducted subsequent functional analyses in vitro of identified variants of RRAGD, a gene that encodes a small Rag guanosine triphosphatase (GTPase). Results In eight children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo Six of these patients also had dilated cardiomyopathy and three underwent heart transplantation. We identified a heterozygous variant in RRAGD that segregated with the phenotype in eight members of a large family with similar kidney manifestations. The GTPase RagD encoded by RRAGD plays a role in mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron included the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro Conclusions Our findings establish a novel disease, which we call autosomal dominant kidney hypomagnesemia (ADKH-RRAGD), that combines an electrolyte-losing tubulopathy and dilated cardiomyopathy. The condition is caused by variants in the RRAGD gene, which encodes Rag GTPase D; these variants lead to an activation of mTOR signaling, suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function. Full Text

Schmidt, D.R., Patel, R., Kirsch, D.G., Lewis, C.A., Vander Heiden, M.G., and Locasale, J.W. (2021). Metabolomics in cancer research and emerging applications in clinical oncology. CA: a Cancer Journal for Clinicians. Online Ahead of Print. Cancer has myriad effects on metabolism that include both rewiring of intracellular metabolism to enable cancer cells to proliferate inappropriately and adapt to the tumor microenvironment, and changes in normal tissue metabolism. With the recognition that fluorodeoxyglucose-positron emission tomography imaging is an important tool for the management of many cancers, other metabolites in biological samples have been in the spotlight for cancer diagnosis, monitoring, and therapy. Metabolomics is the global analysis of small molecule metabolites that like other -omics technologies can provide critical information about the cancer state that are otherwise not apparent. Here, the authors review how cancer and cancer therapies interact with metabolism at the cellular and systemic levels. An overview of metabolomics is provided with a focus on currently available technologies and how they have been applied in the clinical and translational research setting. The authors also discuss how metabolomics could be further leveraged in the future to improve the management of patients with cancer. Full Text

Shah, V.S., Chivukula, R.R., Lin, B., Waghray, A., and Rajagopal, J. (2021). Cystic Fibrosis and the Cells of the Airway Epithelium: What Are Ionocytes and What Do They Do? Annual Review of Pathology. Online Ahead of Print. Cystic fibrosis (CF) is caused by defects in an anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Recently, a new airway epithelial cell type has been discovered and dubbed the pulmonary ionocyte. Unexpectedly, these ionocytes express higher levels of CFTR than any other airway epithelial cell type. However, ionocytes are not the sole CFTR-expressing airway epithelial cells, and CF-associated disease genes are in fact expressed in multiple airway epithelial cell types. The experimental depletion of ionocytes perturbs epithelial physiology in the mouse trachea, but the role of these rare cells in the pathogenesis of human CF remains mysterious. Ionocytes have been described in diverse tissues (kidney and inner ear) and species (frog and fish). We draw on these prior studies to suggest potential roles of airway ionocytes in health and disease. A complete understanding of ionocytes in the mammalian airway will ultimately depend on cell type-specific genetic manipulation Expected final online publication date for the Annual Review of Pathology: Mechanisms of Disease, Volume 17 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates. Full Text

Sharp, P.A., Chakraborty, A.K., Henninger, J.E., and Young, R.A. (2021). RNA in formation and regulation of transcriptional condensates. RNA 28(1) : 52-57. Macrosopic membraneless organelles containing RNA such as the nucleoli, germ granules and the Cajal body have been known for decades. These biomolecular condensates are liquid-like bodies that can form by a phase transition. Recent evidence has revealed the presence of similar microscopic condensates associated with transcription of genes. This brief article summarizes thoughts about the importance of condensates in regulation of transcription and how RNA molecules, as components of such condensates, control the synthesis of RNA. Models and experimental data suggest that RNAs from enhancers facilitate the formation of a condensate that stabilizes the binding of transcription factors and accounts for a burst of transcription at the promoter. Termination of this burst is pictured as a non-equilibrium feedback loop where additional RNA destabilizes the condensate. Full Text

Sheppard, H.E., Dall'Agnese, A., Park, W.D., Shamim, M.H., Dubrulle, J., Johnson, H.L., Stossi, F., Cogswell, P., Sommer, J., Levy, J., Richard A Young, et al. (2021). Targeted brachyury degradation disrupts a highly specific autoregulatory program controlling chordoma cell identity. Cell Reports Medicine 2(1):100188. Chordomas are rare spinal tumors addicted to expression of the developmental transcription factor brachyury. In chordomas, brachyury is super-enhancer associated and preferentially downregulated by pharmacologic transcriptional CDK inhibition, leading to cell death. To understand the underlying basis of this sensitivity, we dissect the brachyury transcription regulatory network and compare the consequences of brachyury degradation with transcriptional CDK inhibition. Brachyury defines the chordoma super-enhancer landscape and autoregulates through binding its super-enhancer, and its locus forms a transcriptional condensate. Transcriptional CDK inhibition and brachyury degradation disrupt brachyury autoregulation, leading to loss of its transcriptional condensate and transcriptional program. Compared with transcriptional CDK inhibition, which globally downregulates transcription, leading to cell death, brachyury degradation is much more selective, inducing senescence and sensitizing cells to anti-apoptotic inhibition. These data suggest that brachyury downregulation is a core tenet of transcriptional CDK inhibition and motivates developing strategies to target brachyury and its autoregulatory feedback loop. Full Text

Shukla, A., Perales, R., and Kennedy, S. (2021). piRNAs coordinate poly(UG) tailing to prevent aberrant and perpetual gene silencing. Current Biology : CB. Online Ahead of Print. piRNAs coordinate poly(UG) tailing to prevent aberrant and perpetual gene silencing. Noncoding RNAs have emerged as mediators of transgenerational epigenetic inheritance (TEI) in a number of organisms. A robust example of such RNA-directed TEI is the inheritance of gene-silencing states following RNA interference (RNAi) in the metazoan C. elegans. During RNAi inheritance, gene silencing is transmitted by a self-perpetuating cascade of siRNA-directed poly(UG) tailing of mRNA fragments (pUGylation), followed by siRNA synthesis from poly(UG)-tailed mRNA templates (termed pUG RNA/siRNA cycling). Despite the self-perpetuating nature of pUG RNA/siRNA cycling, RNAi inheritance is finite, suggesting that systems likely exist to prevent indefinite RNAi-triggered gene silencing. Here we show that, in the absence of Piwi-interacting RNAs (piRNAs), an animal-specific class of small noncoding RNA, RNAi-based gene silencing can become essentially permanent, lasting at near 100% penetrance for more than 5 years and hundreds of generations. This perpetual gene silencing is mediated by continuous pUG RNA/siRNA cycling. Further, we find that piRNAs coordinate endogenous RNAi pathways to prevent germline-expressed genes, which are not normally subjected to TEI, from entering a state of continual and irreversible epigenetic silencing also mediated by continuous maintenance of pUG RNA/siRNA cycling. Together, our results show that one function of C. elegans piRNAs is to insulate germline-expressed genes from aberrant and runaway inactivation by the pUG RNA/siRNA epigenetic inheritance system. Full Text

Sienski, G., Narayan, P., Bonner, J.M., Kory, N., Boland, S., Arczewska, A.A., Ralvenius, W.T., Akay, L., Lockshin, E., He, L., David M. Sabatini , Susan Lindquist , et al. (2021). APOE4 disrupts intracellular lipid homeostasis in human iPSC-derived glia. Science Translational Medicine 13(583):eaaz4564. The E4 allele of the apolipoprotein E gene (APOE) has been established as a genetic risk factor for many diseases including cardiovascular diseases and Alzheimer's disease (AD), yet its mechanism of action remains poorly understood. APOE is a lipid transport protein, and the dysregulation of lipids has recently emerged as a key feature of several neurodegenerative diseases including AD. However, it is unclear how APOE4 perturbs the intracellular lipid state. Here, we report that APOE4, but not APOE3, disrupted the cellular lipidomes of human induced pluripotent stem cell (iPSC)-derived astrocytes generated from fibroblasts of APOE4 or APOE3 carriers, and of yeast expressing human APOE isoforms. We combined lipidomics and unbiased genome-wide screens in yeast with functional and genetic characterization to demonstrate that human APOE4 induced altered lipid homeostasis. These changes resulted in increased unsaturation of fatty acids and accumulation of intracellular lipid droplets both in yeast and in APOE4-expressing human iPSC-derived astrocytes. We then identified genetic and chemical modulators of this lipid disruption. We showed that supplementation of the culture medium with choline (a soluble phospholipid precursor) restored the cellular lipidome to its basal state in APOE4-expressing human iPSC-derived astrocytes and in yeast expressing human APOE4 Our study illuminates key molecular disruptions in lipid metabolism that may contribute to the disease risk linked to the APOE4 genotype. Our study suggests that manipulating lipid metabolism could be a therapeutic approach to help alleviate the consequences of carrying the APOE4 allele. Full Text

Slaidina, M., Gupta, S., Banisch, T.U., and Lehmann, R. (2021). A single-cell atlas reveals unanticipated cell type complexity in Drosophila ovaries. Genome Research 31:1938-1951. Organ function relies on the spatial organization and functional coordination of numerous cell types. The Drosophila ovary is a widely used model system to study the cellular activities underlying organ function, including stem cell regulation, cell signaling and epithelial morphogenesis. However, the relative paucity of cell type-specific reagents hinders investigation of molecular functions at the appropriate cellular resolution. Here, we used single-cell RNA sequencing to characterize all cell types of the stem cell compartment and early follicles of the Drosophila ovary. We computed transcriptional signatures and identified specific markers for nine states of germ cell differentiation and 23 somatic cell types and subtypes. We uncovered an unanticipated diversity of escort cells, the somatic cells that directly interact with differentiating germline cysts. Three escort cell subtypes reside in discrete anatomical positions and express distinct sets of secreted and transmembrane proteins, suggesting that diverse micro-environments support the progressive differentiation of germ cells. Finally, we identified 17 follicle cell subtypes and characterized their transcriptional profiles. Altogether, we provide a comprehensive resource of gene expression, cell type-specific markers, spatial coordinates, and functional predictions for 34 ovarian cell types and subtypes. Full Text

Snyder, E.A., San Roman, A.K., Piña-Aguilar, R.E., Steeves, M.A., McNamara, E.A., Souter, I., Hayes, F.J., Levitsky, L.L., and Lin, A.E. (2021). Genetic counseling for women with 45,X/46,XX mosaicism: Towards more personalized management. European Journal of Medical Genetics, 64(3): March 2021, 104140. Despite numerous clinical series, consistent karyotype-phenotype correlations for Turner syndrome have not been established, although a lower level of 45,X is generally thought to be associated with a milder phenotype. This limits personalized counseling for women with 45,X/46,XX mosaicism. To better understand the phenotypic spectrum associated with various levels of 45,X/46,XX mosaicism, we compared patients evaluated in the Massachusetts General Hospital Turner Syndrome Clinic to determine if cardiac, renal, and thyroid abnormalities correlated with the percentage of 45,X cells present in a peripheral blood karyotype. Of the 118 patients included in the study, 78 (66%) patients had non-mosaic 45,X and 40 (34%) patients had varying levels of 45,X/46,XX mosaicism. Patients with ≤70% 45,X compared with those with >70% 45,X had a significantly lower frequency of cardiac and renal anomalies. The presence of hypothyroidism was somewhat lower for the ≤70% 45,X group, but was not statistically significant. Supplemental tissue testing on another tissue type, typically buccal mucosa, was often useful in counseling patients with 45,X mosaicism. Given the modest sample size of patients with varying levels of mosaicism and the variability of Turner syndrome abnormalities, we hope this preliminary study will inspire a multicenter collaboration, which may lead to modification of clinical guidelines. Because several patients with ≤70% 45,X were ascertained from perinatal care referrals, we still advise women with 45,X mosaicism pursuing pregnancy to receive standard Turner syndrome cardiac surveillance. There is an opportunity to personalize counseling and surveillance for patients based on percentage of 45,X cells on chromosome analysis. Full Text

Sphabmixay, P., Raredon, M.S.B., Wang, A.J., Lee, H., Hammond, P.T., Fang, N.X., and Griffith, L.G. (2021). High resolution stereolithography fabrication of perfusable scaffolds to enable long-term meso-scale hepatic culture for disease modeling. Biofabrication 13:4. Microphysiological systems (MPS), comprising human cell cultured in formats that capture features of the three-dimensional (3D) microenvironments of native human organs under microperfusion, are promising tools for biomedical research. Here we report the development of a mesoscale physiological system (MePS) enabling the long-term 3D perfused culture of primary human hepatocytes at scales of over 10(6)cells per MPS. A central feature of the MePS, which employs a commercially-available multiwell bioreactor for perfusion, is a novel scaffold comprising a dense network of nano- and micro-porous polymer channels, designed to provide appropriate convective and diffusive mass transfer of oxygen and other nutrients while maintaining physiological values of shear stress. The scaffold design is realized by a high resolution stereolithography fabrication process employing a novel resin. This new culture system sustains mesoscopic hepatic tissue-like cultures with greater hepatic functionality (assessed by albumin and urea synthesis, and CYP3A4 activity) and lower inflammation markers compared to comparable cultures on the commercial polystyrene scaffold. To illustrate applications to disease modeling, we established an insulin-resistant phenotype by exposing liver cells to hyperglycemic and hyperinsulinemic media. Future applications of the MePS include the co-culture of hepatocytes with resident immune cells and the integration with multiple organs to model complex liver-associated diseases. Full Text

Spinelli, J.B., Rosen, P.C., Sprenger, H.G., Puszynska, A.M., Mann, J.L., Roessler, J.M., Cangelosi, A.L., Henne, A., Condon, K.J., Zhang, T.,Kunchok, T.,Lewis, C.A., and Sabatini, D.M., et al. (2021). Fumarate is a terminal electron acceptor in the mammalian electron transport chain. Science 374 : 1227-1237. For electrons to continuously enter and flow through the mitochondrial electron transport chain (ETC), they must ultimately land on a terminal electron acceptor (TEA), which is known to be oxygen in mammals. Paradoxically, we find that complex I and dihydroorotate dehydrogenase (DHODH) can still deposit electrons into the ETC when oxygen reduction is impeded. Cells lacking oxygen reduction accumulate ubiquinol, driving the succinate dehydrogenase (SDH) complex in reverse to enable electron deposition onto fumarate. Upon inhibition of oxygen reduction, fumarate reduction sustains DHODH and complex I activities. Mouse tissues display varying capacities to use fumarate as a TEA, most of which net reverse the SDH complex under hypoxia. Thus, we delineate a circuit of electron flow in the mammalian ETC that maintains mitochondrial functions under oxygen limitation. Full Text

Stahl-Rommel, S., Li, D., Sung, M., Li, R., Vijayakumar, A., Atabay, K.D., Bushkin, G.G., Castro, C.L., Foley, K.D., Copeland, D.S., et al. (2021). A CRISPR-based assay for the study of eukaryotic DNA repair onboard the International Space Station. PloS One 16 : e0253403. As we explore beyond Earth, astronauts may be at risk for harmful DNA damage caused by ionizing radiation. Double-strand breaks are a type of DNA damage that can be repaired by two major cellular pathways: non-homologous end joining, during which insertions or deletions may be added at the break site, and homologous recombination, in which the DNA sequence often remains unchanged. Previous work suggests that space conditions may impact the choice of DNA repair pathway, potentially compounding the risks of increased radiation exposure during space travel. However, our understanding of this problem has been limited by technical and safety concerns, which have prevented integral study of the DNA repair process in space. The CRISPR/Cas9 gene editing system offers a model for the safe and targeted generation of double-strand breaks in eukaryotes. Here we describe a CRISPR-based assay for DNA break induction and assessment of double-strand break repair pathway choice entirely in space. As necessary steps in this process, we describe the first successful genetic transformation and CRISPR/Cas9 genome editing in space. These milestones represent a significant expansion of the molecular biology toolkit onboard the International Space Station. Full Text

Sullivan, M.R., Darnell, A.M., Reilly, M.F., Kunchok, T. , Joesch-Cohen, L., Rosenberg, D., Ali, A., Rees, M.G., Roth, J.A., Lewis, C.A., et al. (2021). Methionine synthase is essential for cancer cell proliferation in physiological folate environments. Nature Metabolism 3(11):1500-1511. Folate metabolism can be an effective target for cancer treatment. However, standard cell culture conditions utilize folic acid, a non-physiological folate source for most tissues. We find that the enzyme that couples folate and methionine metabolic cycles, methionine synthase, is required for cancer cell proliferation and tumour growth when 5-methyl tetrahydrofolate (THF), the major folate found in circulation, is the extracellular folate source. In such physiological conditions, methionine synthase incorporates 5-methyl THF into the folate cycle to maintain intracellular levels of the folates needed for nucleotide production. 5-methyl THF can sustain intracellular folate metabolism in the absence of folic acid. Therefore, cells exposed to 5-methyl THF are more resistant to methotrexate, an antifolate drug that specifically blocks folic acid incorporation into the folate cycle. Together, these data argue that the environmental folate source has a profound effect on folate metabolism, determining how both folate cycle enzymes and antifolate drugs impact proliferation. Full Text

Sun C., Rosenstock, T.R., Cohen, M.A., and Sarkar, S. (2021). Autophagy Dysfunction as a Phenotypic Readout in hiPSC-Derived Neuronal Cell Models of Neurodegenerative Diseases. Methods in Molecular Biology. Online Ahead of Print. Autophagy is an evolutionarily conserved catabolic pathway for the degradation of cytoplasmic constituents in eukaryotic cells. It is the primary disposal route for selective removal of undesirable cellular materials like aggregation-prone proteins and damaged organelles for maintaining cellular homeostasis, and for bulk degradation of intracellular macromolecules and recycling the breakdown products for providing energy homeostasis during starvation. These functions of autophagy are attributed to cellular survival and thus pertinent for human health; however, malfunction of this process is detrimental to the cells, particularly for post-mitotic neurons. Thus, basal autophagy is vital for maintaining neuronal homeostasis, whereas autophagy dysfunction contributes to neurodegeneration. Defective autophagy has been demonstrated in several neurodegenerative diseases wherein pharmacological induction of autophagy is beneficial in many of these disease models. Elucidating the mechanisms underlying defective autophagy is imperative for the development of therapies targeting this process. Disease-affected human neuronal cells can be established from patient-derived human induced pluripotent stem cells (hiPSCs) that provide a clinically relevant platform for studying disease mechanisms and drug discovery. Thus, modeling autophagy dysfunction as a phenotypic readout in patient-derived neurons provides a more direct platform for investigating the mechanisms underlying defective autophagy and evaluating the therapeutic efficacy of autophagy inducers. Toward this, several hiPSC-derived neuronal cell models of neurodegenerative diseases have been employed. In this review, we highlight the key methodologies pertaining to hiPSC maintenance and neuronal differentiation, and studying autophagy at an endogenous level in hiPSC-derived neuronal cells. Full Text Full Text

Swartz S.Z., Tan, T.H., Perillo, M., Fakhri, N., Wessel, G.M., Wikramanayake, A.H., and Cheeseman, I.M. (2021). Polarized Dishevelled dissolution and reassembly drives embryonic axis specification in sea star oocytes. Current Biology. Online Ahead of Print. The organismal body axes that are formed during embryogenesis are intimately linked to intrinsic asymmetries established at the cellular scale in oocytes.(1) However, the mechanisms that generate cellular asymmetries within the oocyte and then transduce that polarity to organismal scale body axes are poorly understood outside of select model organisms. Here, we report an axis-defining event in meiotic oocytes of the sea star Patiria miniata. Dishevelled (Dvl) is a cytoplasmic Wnt pathway effector required for axis development in diverse species,(2-4) but the mechanisms governing its function and distribution remain poorly defined. Using time-lapse imaging, we find that Dvl localizes uniformly to puncta throughout the cell cortex in Prophase I-arrested oocytes but becomes enriched at the vegetal pole following meiotic resumption through a dissolution-reassembly mechanism. This process is driven by an initial disassembly phase of Dvl puncta, followed by selective reformation of Dvl assemblies at the vegetal pole. Rather than being driven by Wnt signaling, this localization behavior is coupled to meiotic cell cycle progression and influenced by Lamp1+ endosome association and Frizzled receptors pre-localized within the oocyte cortex. Our results reveal a cell cycle-linked mechanism by which maternal cellular polarity is transduced to the embryo through spatially regulated Dvl dynamics. Full Text

Swartz S.Z., Nguyen, H.T., McEwan, B.C., Adamo, M.E., Cheeseman, I.M., and Kettenbach, A.N. (2021). Selective dephosphorylation by PP2A-B55 directs the meiosis I - meiosis II transition in oocytes. eLife 10:e70588. Meiosis is a specialized cell cycle that requires sequential changes to the cell division machinery to facilitate changing functions. To define the mechanisms that enable the oocyte-to-embryo transition, we performed time-course proteomics in synchronized sea star oocytes from prophase I through the first embryonic cleavage. Although we find that protein levels are broadly stable, our analysis reveals that dynamic waves of phosphorylation underlie each meiotic stage. We find that the phosphatase PP2A-B55 is reactivated at the meiosis I/II transition resulting in the preferential dephosphorylation of threonine residues. Selective dephosphorylation is critical for directing the MI / MII transition as altering PP2A-B55 substrate preferences disrupts key cell cycle events after meiosis I. In addition, threonine to serine substitution of a conserved phosphorylation site in the substrate INCENP prevents its relocalization at anaphase I. Thus, through its inherent phospho-threonine preference, PP2A-B55 imposes specific phosphoregulated behaviors that distinguish the two meiotic divisions. Full Text

Tang X., Jaenisch, R., and Sur, M. (2021). The role of GABAergic signalling in neurodevelopmental disorders. Nature Reviews Neuroscience. Online ahead of print. GABAergic inhibition shapes the connectivity, activity and plasticity of the brain. A series of exciting new discoveries provides compelling evidence that disruptions in a number of key facets of GABAergic inhibition have critical roles in the aetiology of neurodevelopmental disorders (NDDs). These facets include the generation, migration and survival of GABAergic neurons, the formation of GABAergic synapses and circuit connectivity, and the dynamic regulation of the efficacy of GABAergic signalling through neuronal chloride transporters. In this Review, we discuss recent work that elucidates the functions and dysfunctions of GABAergic signalling in health and disease, that uncovers the contribution of GABAergic neural circuit dysfunction to NDD aetiology and that leverages such mechanistic insights to advance precision medicine for the treatment of NDDs. Full Text

Tarasovetc, E.V., Allu, P.K., Wimbish, R.T., DeLuca, J.G., Cheeseman, I.M., Black, B.E., and Grishchuk, E.L. (2021). Permitted and restricted steps of human kinetochore assembly in mitotic cell extracts. Molecular Biology of the Cell. Online Ahead of Print. Mitotic kinetochores assemble via the hierarchical recruitment of numerous cytosolic components to the centromere region of each chromosome. However, how these orderly and localized interactions are achieved without spurious macromolecular assemblies forming from soluble kinetochore components in the cell cytosol remains poorly understood. We developed assembly assays to monitor the recruitment of GFP-tagged recombinant proteins and native proteins from human cell extracts to inner kinetochore components immobilized on microbeads. In contrast to prior work in yeast and Xenopus egg extracts, we find that human mitotic cell extracts fail to support de novo assembly of microtubule-binding sub-complexes. A subset of interactions, such as those between CENP-A-containing nucleosomes and CENP-C, are permissive under these conditions. However, the subsequent phospho-dependent binding of the Mis12 complex is less efficient, whereas recruitment of the Ndc80 complex is blocked, leading to weak microtubule-binding activity of assembled particles. Using molecular variants of the Ndc80 complex, we show that auto-inhibition of native Ndc80 complex restricts its ability to bind to the CENP-T/W complex, whereas inhibition of the Ndc80 microtubule-binding is driven by a different mechanism. Together, our work reveals regulatory mechanisms that guard against the spurious formation of cytosolic microtubule-binding kinetochore particles. Full Text

Tomasello, D.L., Kim, J.L., Khodour, Y., McCammon, J.M., Mitalipova, M., Jaenisch, R., Futerman, A.H., and Sive, H. (2022). 16pdel lipid changes in iPSC-derived neurons and function of FAM57B in lipid metabolism and synaptogenesis. iScience 25 (1): 103551. The complex 16p11.2 deletion syndrome (16pdel) is accompanied by neurological disorders, including epilepsy, autism spectrum disorder, and intellectual disability. We demonstrated that 16pdel iPSC differentiated neurons from affected people show augmented local field potential activity and altered ceramide-related lipid species relative to unaffected. FAM57B, a poorly characterized gene in the 16p11.2 interval, has emerged as a candidate tied to symptomatology. We found that FAM57B modulates ceramide synthase (CerS) activity, but is not a CerS per se. In FAM57B mutant human neuronal cells and zebrafish brain, composition and levels of sphingolipids and glycerolipids associated with cellular membranes are disrupted. Consistently, we observed aberrant plasma membrane architecture and synaptic protein mislocalization, which were accompanied by depressed brain and behavioral activity. Together, these results suggest that haploinsufficiency of FAM57B contributes to changes in neuronal activity and function in 16pdel syndrome through a crucial role for the gene in lipid metabolism. Full Text

Tomezsko, P., Swaminathan, H., and Rouskin, S. (2021). DMS-MaPseq for Genome-Wide or Targeted RNA Structure Probing In Vitro and In Vivo. Methods in Molecular Biology 2254, 219-238. DMS-MaPseq is a chemical probing method combined with high throughput sequencing used to study RNA structure. Here we present a flexible protocol for adherent and suspension mammalian cells to analyze RNA structure in vitro or in vivo. The protocol provides instruction on either a targeted sequencing of a lncRNA of interest or a transcriptome-wide approach that provides structural data on all expressed RNAs, including lncRNAs. This technique is particularly useful for comparing in vitro and in vivo structure of RNAs, determining how mutations and polymorphisms with phenotypic effects influence RNA structure and analyzing RNA structure across the entire transcriptome. Full Text

Torrens-Spence, M.P., Glinkerman, C.M., Günther, J., and Weng, J.K. (2021). Imine chemistry in plant metabolism. Current Opinion in Plant Biology 60 : 101999. Imine chemistry represents an important category of chemical reactions involved in the biosynthesis of plant natural products, ranging from the newly discovered mobile defense hormone N-hydroxy-pipecolic acid to the red-to-yellow tyrosine-derived betalain pigments. Spontaneous imine formation reactions have also served as the basis for the evolution of numerous plant metabolic enzymes, such as specialized Pictet-Spenglerases that produce the backbone structures of benzylisoquinoline and monoterpene indole alkaloids and pyridoxal 5'-phosphate-dependent enzymes of diverse functions. Here, we review occurrences of imine chemistry in plant metabolism and their chemical and biochemical mechanisms. A better understanding of plant imine chemistry will ultimately facilitate synthetic biology approaches to further expand the scope of imine natural product biosynthesis for broad biotechnological applications. Full Text

Trapecar, M., Wogram, E., Svoboda, D., Communal, C., Omer, A., Lungjangwa, T., Sphabmixay, P., Velazquez, J., Schneider, K., Wright, C.W., Rudolph Jaenisch, et al. (2021). Human physiomimetic model integrating microphysiological systems of the gut, liver, and brain for studies of neurodegenerative diseases. Science Advances 7(5):eabd1707. Slow progress in the fight against neurodegenerative diseases (NDs) motivates an urgent need for highly controlled in vitro systems to investigate organ-organ- and organ-immune-specific interactions relevant for disease pathophysiology. Of particular interest is the gut/microbiome-liver-brain axis for parsing out how genetic and environmental factors contribute to NDs. We have developed a mesofluidic platform technology to study gut-liver-cerebral interactions in the context of Parkinson's disease (PD). It connects microphysiological systems (MPSs) of the primary human gut and liver with a human induced pluripotent stem cell-derived cerebral MPS in a systemically circulated common culture medium containing CD4(+) regulatory T and T helper 17 cells. We demonstrate this approach using a patient-derived cerebral MPS carrying the PD-causing A53T mutation, gaining two important findings: (i) that systemic interaction enhances features of in vivo-like behavior of cerebral MPSs, and (ii) that microbiome-associated short-chain fatty acids increase expression of pathology-associated pathways in PD. Full Text

Tsai, P.Y., Lee, M.S., Jadhav, U., Naqvi, I., Madha, S., Adler, A., Mistry, M., Naumenko, S., Lewis, C.A., Hitchcock, D.S., et al. (2021). Adaptation of pancreatic cancer cells to nutrient deprivation is reversible and requires glutamine synthetase stabilization by mTORC1. PNAS 118(10):e2003014118. Pancreatic ductal adenocarcinoma (PDA) is a lethal, therapy-resistant cancer that thrives in a highly desmoplastic, nutrient-deprived microenvironment. Several studies investigated the effects of depriving PDA of either glucose or glutamine alone. However, the consequences on PDA growth and metabolism of limiting both preferred nutrients have remained largely unknown. Here, we report the selection for clonal human PDA cells that survive and adapt to limiting levels of both glucose and glutamine. We find that adapted clones exhibit increased growth in vitro and enhanced tumor-forming capacity in vivo. Mechanistically, adapted clones share common transcriptional and metabolic programs, including amino acid use for de novo glutamine and nucleotide synthesis. They also display enhanced mTORC1 activity that prevents the proteasomal degradation of glutamine synthetase (GS), the rate-limiting enzyme for glutamine synthesis. This phenotype is notably reversible, with PDA cells acquiring alterations in open chromatin upon adaptation. Silencing of GS suppresses the enhanced growth of adapted cells and mitigates tumor growth. These findings identify nongenetic adaptations to nutrient deprivation in PDA and highlight GS as a dependency that could be targeted therapeutically in pancreatic cancer patients.Full Text

Ukadike, K.C., Ni, K., Wang, X., Taylor, M.S.., LaCava, J., Pachman, L.M., Eckert, M., Stevens, A., Lood, C., and Mustelin, T. (2021). IgG and IgA autoantibodies against L1 ORF1p expressed in granulocytes correlate with granulocyte consumption and disease activity in pediatric systemic lupus erythematosus. Arthritis Research & Therapy 23 : no.153. Most patients with systemic lupus erythematosus (SLE) have IgG autoantibodies against the RNA-binding p40 (ORF1p) protein encoded by the L1 retroelement. This study tested if these autoantibodies are also present in children with pediatric SLE (pSLE) and if the p40 protein itself could be detected in immune cells. METHODS: Autoantibodies in the plasma of pSLE patients (n = 30), healthy children (n = 37), and disease controls juvenile idiopathic arthritis (JIA) (n = 32) and juvenile dermatomyositis (JDM) (n = 60), were measured by ELISA. Expression of p40 in immune cells was assessed by flow cytometry. Markers of neutrophil activation and death were quantitated by ELISA. RESULTS: IgG and IgA autoantibodies reactive with p40 were detected in the pSLE patients, but were low in healthy controls and in JIA or JDM. pSLE patients with active disease (13 of them newly diagnosed) had higher titers than the same patients after effective therapy (p = 0.0003). IgG titers correlated with SLEDAI (r = 0.65, p = 0.0001), ESR (r = 0.43, p = 0.02), and anti-dsDNA antibodies (r = 0.49, p < 0.03), and inversely with complement C3 (r = -0.55, p = 0.002) and C4 (r = -0.51, p = 0.006). p40 protein was detected in a subpopulation of CD66b(+) granulocytes in pSLE, as well as in adult SLE patients. Myeloperoxidase and neutrophil elastase complexed with DNA and the neutrophil-derived S100A8/A9 were elevated in plasma from pSLE patients with active disease and correlated with anti-p40 autoantibodies and disease activity. CONCLUSIONS: Children with active SLE have elevated IgG and IgA autoantibodies against L1 p40, and this protein can be detected in circulating granulocytes in both pediatric and adult SLE patients. P40 expression and autoantibody levels correlate with disease activity. Markers of neutrophil activation and death also correlate with these autoantibodies and with disease activity, suggesting that neutrophils express L1 and are a source of p40. Full Text

van Gastel, N., Spinelli, J.B., Haigis, M.C., and Scadden, D.T. (2021). Analysis of Leukemia Cell Metabolism through Stable Isotope Tracing in Mice. Bio-Protocol 11(19). Once thought to be a mere consequence of the state of a cell, intermediary metabolism is now recognized as a key regulator of mammalian cell fate and function. In addition, cell metabolism is often disturbed in malignancies such as cancer, and targeting metabolic pathways can provide new therapeutic options. Cell metabolism is mostly studied in cell cultures in vitro, using techniques such as metabolomics, stable isotope tracing, and biochemical assays. Increasing evidence however shows that the metabolic profile of cells is highly dependent on the microenvironment, and metabolic vulnerabilities identified in vitro do not always translate to in vivo settings. Here, we provide a detailed protocol on how to perform in vivo stable isotope tracing in leukemia cells in mice, focusing on glutamine metabolism in acute myeloid leukemia (AML) cells. This method allows studying the metabolic profile of leukemia cells in their native bone marrow niche. Full Text

Wang, F., Graham, E.T., Naowarojna, N., Shi, Z., Wang, Y., Xie, G., Zhou, L., Salmon, W., Jia, J.M., Wang, X., et al. (2021). PALP: A rapid imaging technique for stratifying ferroptosis sensitivity in normal and tumor tissues in situ. Cell Chemical Biology. Online Ahead of Print. Ferroptosis is an emerging cancer suppression strategy. However, how to select cancer patients for treating with ferroptosis inducers remains challenging. Here, we develop photochemical activation of membrane lipid peroxidation (PALP), which uses targeted lasers to induce localized polyunsaturated fatty acyl (PUFA)-lipid peroxidation for reporting ferroptosis sensitivity in cells and tissues. PALP captured by BODIPY-C11 can be suppressed by lipophilic antioxidants and iron chelation, and is dependent on PUFA-lipid levels. Moreover, we develop PALPv2, for studying lipid peroxidation on selected membranes along the z axis in live cells using two-photon microscopes. Using PALPv1, we detect PUFA-lipids in multiple tissues, and validate a PUFA-phospholipid reduction during muscle aging as previously reported. Patterns of PALPv1 signals across multiple cancer cell types in vitro and in vivo are concordant with their ferroptosis susceptibility and PUFA-phospholipid levels. We envision that PALP will enable rapid stratification of ferroptosis sensitivity in cancer patients and facilitate PUFA-lipid research. Full Text

Weinberg, R.A. (2021). David M. Livingston (1941-2021). Cancer Cell 39(12) : 1560-1561. David M. Livingston left us on October 17, 2021. He was best known for his fundamental work on retinoblastoma protein and its effects on cell-cycle progression and, in recent decades, on the biochemistry and function of the homologous repair-associated BRCA1 and BRCA2 proteins. The latter, as we now know, contribute to the pathogenesis of a significant fraction of human breast cancers. Full Text

Weng, J.K., Lynch, J.H., Matos, J.O., and Dudareva, N. (2021). Adaptive mechanisms of plant specialized metabolism connecting chemistry to function. Nature Chemical Biology 17(10):1037-1045. As sessile organisms, plants evolved elaborate metabolic systems that produce a plethora of specialized metabolites as a means to survive challenging terrestrial environments. Decades of research have revealed the genetic and biochemical basis for a multitude of plant specialized metabolic pathways. Nevertheless, knowledge is still limited concerning the selective advantages provided by individual and collective specialized metabolites to the reproductive success of diverse host plants. Here we review the biological functions conferred by various classes of plant specialized metabolites in the context of the interaction of plants with their surrounding environment. To achieve optimal multifunctionality of diverse specialized metabolic processes, plants use various adaptive mechanisms at subcellular, cellular, tissue, organ and interspecies levels. Understanding these mechanisms and the evolutionary trajectories underlying their occurrence in nature will ultimately enable efficient bioengineering of desirable metabolic traits in chassis organisms. Full Text

Wigbers, M.C., Tan, T.H., Brauns, F., Liu, J.H., Swartz, S.Z., Frey, E., and Fakhri, N. A hierarchy of protein patterns robustly decodes cell shape information. Nature Physics 15 Feruary 2021, 1-7. Many cellular processes, such as cell division(1-3), cell motility(4), wound healing(5) and tissue folding(6,7), rely on the precise positioning of proteins on the membrane. Such protein patterns emerge from a combination of protein interactions, transport, conformational state changes and chemical reactions at the molecular level(8). Recent experimental and theoretical work clearly demonstrates the role of geometry, including membrane curvature(9-11) and local cytosolic-to-membrane ratios(12,13), and advective cortical flow in modulating membrane protein patterns. However, it remains unclear how these proteins achieve robust spatiotemporal organization on the membrane during the dynamic cell shape changes involved in physiological processes. Here we use oocytes of the starfish Patiria miniata as a model system to elucidate a shape-adaptation mechanism that robustly controls spatiotemporal protein dynamics on the membrane in spite of cell shape deformations. By combining experiments with biophysical theory, we show how cell shape information contained in a cytosolic gradient can be decoded by a bistable regulator of the enzyme Rho, which is associated with contractility. This bistable front in turn controls a mechanochemical response by locally triggering excitable dynamics of Rho. We posit that such a shape-adaptation mechanism based on a hierarchy of protein patterns may constitute a general physical principle for cell shape sensing and control. Full Text

Wilke, A.C., Doebele, C., Zindel, A., Lee, K.S., Rieke, S.A., Ceribelli, M., Comoglio, F., Phelan, J.D., Wang, J.Q., Pikman, Y., Caroline A. Lewis, et al. (2020). SHMT2 inhibition disrupts the TCF3 transcriptional survival program in Burkitt lymphoma. Blood. Online Ahead of Print. Burkitt lymphoma (BL) is an aggressive lymphoma type that is currently treated by intensive chemoimmunotherapy. Despite the favorable clinical outcome of the majority of BL patients, chemotherapy-related toxicity and disease relapse remain as major clinical challenges, emphasizing the need for innovative therapies. Using genome-scale CRISPR-Cas9 screens, we identified B-cell receptor (BCR) signaling, specific transcriptional regulators and one-carbon metabolism as vulnerabilities in BL. We focused on serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in one-carbon metabolism. Inhibition of SHMT2 by either knockdown or pharmacological compounds induced anti-BL effects in vitro and in vivo. Mechanistically, SHMT2 inhibition led to a significant reduction of intracellular glycine and formate levels, which inhibited the mTOR pathway and thereby triggered autophagic degradation of the oncogenic transcription factor TCF3. As a consequence, this led to a collapse of tonic B-cell receptor signaling, which is controlled by TCF3 and is essential for BL cell survival. In terms of clinical translation, we furthermore identified drugs such as methotrexate that synergized with SHMT inhibitors (SHMT2i). Overall, our study has uncovered the dependency landscape in BL, identified and validated SHMT2 as a drug target and revealed a mechanistic link between SHMT2 and the transcriptional master regulator TCF3, opening up new perspectives for innovative therapies. Full Text

Williams, B.P., Bechen, L.L., Pohlmann, D.A., and Gehring, M. (2021). Somatic DNA demethylation generates tissue-specific methylation states and impacts flowering time.Plant Cell. Online Ahead of Print. Cytosine methylation is a reversible epigenetic modification of DNA. In plants, removal of cytosine methylation is accomplished by the four members of the DEMETER (DME) family of 5-methylcytosine DNA glycosylases, named DME, DEMETER-LIKE2 (DML2), DML3, and REPRESSOR OF SILENCING1 (ROS1) in Arabidopsis thaliana. Demethylation by DME is critical for seed development, preventing experiments to determine the function of the entire gene family in somatic tissues by mutant analysis. Here, we bypassed the reproductive defects of dme mutants to create somatic quadruple homozygous mutants of the entire DME family. dme; ros1; dml2; dml3 (drdd) leaves exhibit hypermethylated regions compared to wild-type leaves and rdd triple mutants, indicating functional redundancy among all four demethylases. Targets of demethylation include regions co-targeted by RNA-directed DNA methylation and, surprisingly, CG gene body methylation, indicating dynamic methylation at these less-understood sites. Additionally, many tissue-specific methylation differences are absent in drdd, suggesting a role for active demethylation in generating divergent epigenetic states across wild-type tissues. Furthermore, drdd plants display an early flowering phenotype, which involves 5' hypermethylation and transcriptional down-regulation of FLOWERING LOCUS C. Active DNA demethylation is therefore required for proper methylation across somatic tissues and defines the epigenetic landscape of intergenic and coding regions. Full Text

Wilson, M.M., Callens, C., Le Gallo, M., Mironov, S., Ding, Q., Salamagnon, A., Chavarria, T.E., Viel, R., Peasah, A.D., Bhutkar, A., Robert A. Weinberg, et al. (2021). An EMT-primary cilium-GLIS2 signaling axis regulates mammogenesis and claudin-low breast tumorigenesis. Science Advances 7 (44):eabf6063. The epithelial-mesenchymal transition (EMT) and primary ciliogenesis induce stem cell properties in basal mammary stem cells (MaSCs) to promote mammogenesis, but the underlying mechanisms remain incompletely understood. Here, we show that EMT transcription factors promote ciliogenesis upon entry into intermediate EMT states by activating ciliogenesis inducers, including FGFR1. The resulting primary cilia promote ubiquitination and inactivation of a transcriptional repressor, GLIS2, which localizes to the ciliary base. We show that GLIS2 inactivation promotes MaSC stemness, and GLIS2 is required for normal mammary gland development. Moreover, GLIS2 inactivation is required to induce the proliferative and tumorigenic capacities of the mammary tumor–initiating cells (MaTICs) of claudin-low breast cancers. Claudin-low breast tumors can be segregated from other breast tumor subtypes based on a GLIS2-dependent gene expression signature. Collectively, our findings establish molecular mechanisms by which EMT programs induce ciliogenesis to control MaSC and MaTIC stemness, mammary gland development, and claudin-low breast cancer formation. Full Text

Wright, B.W., Yi, Z., Weissman, J.S., and Chen, J. (2021). The dark proteome: translation from noncanonical open reading frames. Trends in Cell Biology. Online Ahead of Print. Omics-based technologies have revolutionized our understanding of the coding potential of the genome. In particular, these studies revealed widespread unannotated open reading frames (ORFs) throughout genomes and that these regions have the potential to encode novel functional (micro-)proteins and/or hold regulatory roles. However, despite their genomic prevalence, relatively few of these noncanonical ORFs have been functionally characterized, likely in part due to their under-recognition by the broader scientific community. The few that have been investigated in detail have demonstrated their essentiality in critical and divergent biological processes. As such, here we aim to discuss recent advances in understanding the diversity of noncanonical ORFs and their roles, as well as detail biologically important examples within the context of the mammalian genome. Full Text

Xiang, K., and Bartel, D.P. (2021). The molecular basis of coupling between poly(A)-tail length and translational efficiency. eLife 10:e66493. In animal oocytes and early embryos, mRNA poly(A)-tail length strongly influences translational efficiency (TE), but later in development this coupling between tail length and TE disappears. Here, we elucidate how this coupling is first established and why it disappears. Overexpressing cytoplasmic poly(A)-binding protein (PABPC) in Xenopus oocytes specifically improved translation of short-tailed mRNAs, thereby diminishing coupling between tail length and TE. Thus, strong coupling requires limiting PABPC, implying that in coupled systems longer-tail mRNAs better compete for limiting PABPC. In addition to expressing excess PABPC, post-embryonic mammalian cell lines had two other properties that prevented strong coupling: terminal-uridylation-dependent destabilization of mRNAs lacking bound PABPC, and a regulatory regime wherein PABPC contributes minimally to TE. Thus, these results revealed three fundamental mechanistic requirements for coupling and defined the context-dependent functions for PABPC, which promotes TE but not mRNA stability in coupled systems and mRNA stability but not TE in uncoupled systems. Full Text

Yan, X., Stuurman, N., Ribeiro, S.A., Tanenbaum, M.E., Horlbeck, M.A., Liem, C.R., Jost, M., Weissman, J.S. , and Vale, R.D. (2021). High-content imaging-based pooled CRISPR screens in mammalian cells. The Journal of Cell Biology 220(2):e202008158. CRISPR (clustered regularly interspaced short palindromic repeats)-based gene inactivation provides a powerful means for linking genes to particular cellular phenotypes. CRISPR-based screening typically uses large genomic pools of single guide RNAs (sgRNAs). However, this approach is limited to phenotypes that can be enriched by chemical selection or FACS sorting. Here, we developed a microscopy-based approach, which we name optical enrichment, to select cells displaying a particular CRISPR-induced phenotype by automated imaging-based computation, mark them by photoactivation of an expressed photoactivatable fluorescent protein, and then isolate the fluorescent cells using fluorescence-activated cell sorting (FACS). A plugin was developed for the open source software μManager to automate the phenotypic identification and photoactivation of cells, allowing ∼1.5 million individual cells to be screened in 8 h. We used this approach to screen 6,092 sgRNAs targeting 544 genes for their effects on nuclear size regulation and identified 14 bona fide hits. These results present a scalable approach to facilitate imaging-based pooled CRISPR screens. Full Text

Yang, H., Wang, H., and Jaenisch, R. (2021). Response to "Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation". Genome Biology 22(1):98. Gurumurthy et al. [1] recently reported that a method developed by Yang et al. to generate floxed allele (designated as “two donor method” by Gurumurthy et al.) [2] had poor reproducibility. They claimed that three centers could not reproduce our results on generating conditional alleles of the Mecp2 locus and that the “two-donor method” had very low success rate on other loci.Here, we provide our responses to these claims. Full Text

Yang, J.Y., Fang, W., Miranda-Sanchez, F., Brown, J.M., Kauffman, K.M., Acevero, C.M., Bartel, D.P., Polz, M.F., and Kelly, L. (2021). Degradation of host translational machinery drives tRNA acquisition in viruses. Cell Systems. Online Ahead of Print. Viruses are traditionally thought to be under selective pressure to maintain compact genomes and thus depend on host cell translational machinery for reproduction. However, some viruses encode abundant tRNA and other translation-related genes, potentially optimizing for codon usage differences between phage and host. Here, we systematically interrogate selective advantages that carrying 18 tRNAs may convey to a T4-like Vibriophage. Host DNA and RNA degrade upon infection, including host tRNAs, which are replaced by those of the phage. These tRNAs are expressed at levels slightly better adapted to phage codon usage, especially that of late genes. The phage is unlikely to randomly acquire as diverse an array of tRNAs as observed (p = 0.0017). Together, our results support that the main driver behind phage tRNA acquisition is pressure to sustain translation as host machinery degrades, a process resulting in a dynamically adapted codon usage strategy during the course of infection. Full Text

Yeager, R., Bushkin, G.G., Singer, E., Fu, R., Cooperman, B., and McMurray, M. (2021). Post-Transcriptional Control of Mating-Type Gene Expression during Gametogenesis in Saccharomyces cerevisiae. Biomolecules 11(8):1223. Gametogenesis in diploid cells of the budding yeast Saccharomyces cerevisiae produces four haploid meiotic products called spores. Spores are dormant until nutrients trigger germination, when they bud asexually or mate to return to the diploid state. Each sporulating diploid produces a mix of spores of two haploid mating types, a and α. In asexually dividing haploids, the mating types result from distinct, mutually exclusive gene expression programs responsible for production of mating pheromones and the receptors to sense them, all of which are silent in diploids. It was assumed that spores only transcribe haploid- and mating-type-specific genes upon germination. We find that dormant spores of each mating type harbor transcripts representing all these genes, with the exception of Mata1, which we found to be enriched in a spores. Mata1 transcripts, from a rare yeast gene with two introns, were mostly unspliced. If the retained introns reflect tethering to the MATa locus, this could provide a mechanism for biased inheritance. Translation of pheromones and receptors were repressed at least until germination. We find antisense transcripts to many mating genes that may be responsible. These findings add to the growing number of examples of post-transcriptional regulation of gene expression during gametogenesis. Full Text

Zhang, J., Yuan, J., Li, Z., Fu, C., Xu, M., Yang, J., Jiang, X., Zhou, B., Ye, X., and Chengchao Xu (2021). Exploring and exploiting plant cyclic peptides for drug discovery and development. Medicinal Research Reviews. Online Ahead of Print. Ever since the discovery of insulin, natural peptides have become an important resource for therapeutic development. Decades of research has led to the discovery of a long list of peptide drugs with broad applications in clinics, from antibiotics to hypertension treatment to pain management. Many of these US FDA-approved peptide drugs are derived from microorganisms and animals. By contrast, the great potential of plant cyclic peptides as therapeutics remains largely unexplored. These macrocyclic peptides typically have rigid structures, good bioavailability and membrane permeability, making them appealing candidates for drug development and engineering. In this review, we introduce the three major classes of plant cyclic peptides and summarize their potential medical applications. We discuss how we can leverage the genome information of many different plants to quickly search for new cyclic peptides and how we can take advantage of the insights gained from their biosynthetic pathways to transform the process of production and drug development. These recent developments have provided a new angle for exploring and exploiting plant cyclic peptides, and we believe that many more peptide drugs derived from plants are about to come.Full Text

Zhang, L., Richards, A., Barrasa, M.I., Hughes, S.H., Young, R.A., and Jaenisch, R. (2021). Response to Parry et al.: Strong evidence for genomic integration of SARS-CoV-2 sequences and expression in patient tissues. PNAS 118(33):e2109497118. Full Text

Zhang, L., Richards, A., Barrasa, M.I., Hughes, S.H., Young, R.A., and Jaenisch, R. (2021). Reply to Briggs et al.: Genomic integration and expression of SARS-CoV-2 sequences can explain prolonged or recurrent viral RNA detection. PNAS 118(44):e2114995118. Full Text

Zhang, L., Richards, A., Barrasa, M.I., Hughes, S.H., Young, R.A., and Jaenisch, R. (2021). Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues. PNAS 118(21):e2105968118. Prolonged detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and recurrence of PCR-positive tests have been widely reported in patients after recovery from COVID-19, but some of these patients do not appear to shed infectious virus. We investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the DNA of human cells in culture and that transcription of the integrated sequences might account for some of the positive PCR tests seen in patients. In support of this hypothesis, we found that DNA copies of SARS-CoV-2 sequences can be integrated into the genome of infected human cells. We found target site duplications flanking the viral sequences and consensus LINE1 endonuclease recognition sequences at the integration sites, consistent with a LINE1 retrotransposon-mediated, target-primed reverse transcription and retroposition mechanism. We also found, in some patient-derived tissues, evidence suggesting that a large fraction of the viral sequences is transcribed from integrated DNA copies of viral sequences, generating viral-host chimeric transcripts. The integration and transcription of viral sequences may thus contribute to the detection of viral RNA by PCR in patients after infection and clinical recovery. Because we have detected only subgenomic sequences derived mainly from the 3' end of the viral genome integrated into the DNA of the host cell, infectious virus cannot be produced from the integrated subgenomic SARS-CoV-2 sequences.Full Text

Zhang, S., Iyer, S. , Ran, H., Dolgalev, I., Gu, S., Wei, W., Foster, C.J.R., Loomis, C.A., Olvera, N., Dao, F., Weinberg, R.A., et al. (2021). Genetically Defined, Syngeneic Organoid Platform for Developing Combination Therapies for Ovarian Cancer. Cancer Discovery 11(2):362-383. The paucity of genetically informed, immunocompetent tumor models impedes evaluation of conventional, targeted, and immune therapies. By engineering mouse fallopian tube epithelial organoids using lentiviral gene transduction and/or CRISPR/Cas9 mutagenesis, we generated multiple high-grade serous tubo-ovarian cancer (HGSC) models exhibiting mutational combinations seen in patients with HGSC. Detailed analysis of homologous recombination (HR)-proficient (Trp53(-/-);Ccne1(OE);Akt2(OE);Kras(OE) ), HR-deficient (Trp53(-/-);Brca1(-/-);Myc(OE) ), and unclassified (Trp53(-/-);Pten(-/-);Nf1(-/-) ) organoids revealed differences in in vitro properties (proliferation, differentiation, and "secretome"), copy-number aberrations, and tumorigenicity. Tumorigenic organoids had variable sensitivity to HGSC chemotherapeutics, and evoked distinct immune microenvironments that could be modulated by neutralizing organoid-produced chemokines/cytokines. These findings enabled development of a chemotherapy/immunotherapy regimen that yielded durable, T cell-dependent responses in Trp53(-/-);Ccne1(OE);Akt2(OE);Kras HGSC; in contrast, Trp53(-/-);Pten(-/-);Nf1(-/-) tumors failed to respond. Mouse and human HGSC models showed genotype-dependent similarities in chemosensitivity, secretome, and immune microenvironment. Genotype-informed, syngeneic organoid models could provide a platform for the rapid evaluation of tumor biology and therapeutics. SIGNIFICANCE: The lack of genetically informed, diverse, immunocompetent models poses a major barrier to therapeutic development for many malignancies. Using engineered fallopian tube organoids to study the cell-autonomous and cell-nonautonomous effects of specific combinations of mutations found in HGSC, we suggest an effective combination treatment for the currently intractable CCNE1-amplified subgroup. Full Text

Zhu, K., Xu, A., Xia, W., Li, P., Zhang, B., Jiang, H., Zhou, S., and Wang, R. (2021). Association Between NAT2 Polymorphism and Lung Cancer Risk: A Systematic Review and Meta-Analysis. Frontiers in Oncology 11 : 567762. Lung cancer is the leading cause of cancer-related death worldwide and has a high incidence rate. N-Acetyltransferase 2 (NAT2) is a polymorphic xenobiotic enzyme, which can catalyze N-acetylation and O-acetylation of various carcinogens such as aromatic, heterocyclic amines and hydrazines. At present, many studies have explored the effects of NAT2 polymorphism on lung cancer, but we found inconsistent results. We researched 18 published studies, involving 4,016 patients and 5,469 controls, to more accurately assess the effects of NAT2 polymorphism on lung cancer risk and to investigate whether smoking is associated. We used STATA software to analyze the extracted data and used STATA for subgroup analysis, sensitivity analysis, and to perform publication bias tests. To determine the correlation, we used the crude odds ratio (ORs) with 95% confidence interval (CIs). Our study was prospectively registered in PROSPERO (CRD42020159737). The odds ratio was 1.53 (95% CI: 1.21-1.95, I² = 45.2%, P=0.104) for the NAT2 slow + intermediate phenotype versus rapid phenotype. The results suggested that people with NAT2 non-rapid (slow + intermediate) phenotype have a significantly increased risk of lung cancer. In addition, NAT2 rapid phenotype was significantly associated with reduced risk of lung cancer, compared with slow phenotype or intermediate phenotype (slow phenotype vs . rapid phenotype: OR: 1.61, 95% CI: 1.07-2.42, I²= 50%, P= 0.075; intermediate phenotype vs . rapid phenotype: OR: 1.47, 95% CI: 1.15-1.88, I²= 40.3%, P= 0.137). Full Text

Zimmerman, M.W., Durbin, A.D., He, S., Oppel, F., Shi, H., Tao, T., Li, Z., Berezovskaya, A., Liu, Y., Zhang, J., Richard A Young, et al. (2021). Retinoic acid rewires the adrenergic core regulatory circuitry of childhood neuroblastoma. Science Advances 7(43):eabe0834. Neuroblastoma cell identity depends on a core regulatory circuit (CRC) of transcription factors that collaborate with MYCN to drive the oncogenic gene expression program. For neuroblastomas dependent on the adrenergic CRC, treatment with retinoids can inhibit cell growth and induce differentiation. Here, we show that when MYCN-amplified neuroblastoma cells are treated with retinoic acid, histone H3K27 acetylation and methylation become redistributed to decommission super-enhancers driving the expression of PHOX2B and GATA3, together with the activation of new super-enhancers that drive high levels of MEIS1 and SOX4 expression. These findings indicate that treatment with retinoids can reprogram the enhancer landscape, resulting in down-regulation of MYCN expression, while establishing a new retino-sympathetic CRC that causes proliferative arrest and sympathetic differentiation. Thus, we provide mechanisms that account for the beneficial effects of retinoids in high-risk neuroblastoma and explain the rapid down-regulation of expression of MYCN despite massive levels of amplification of this gene. Full Text