The following alphabetical list represents papers published in 2022 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.

2022 Titles :

Abo, K.M., Sainz de Aja, J., Lindstrom-Vautrin, J., Alysandratos, K.D., Richards, A., Garcia-de-Alba, C., Huang, J., Hix, O.T., Werder, R.B., Bullitt, E., Rudolph Jaenisch, et al. (2022). Air-liquid interface culture promotes maturation and allows environmental exposure of pluripotent stem cell-derived alveolar epithelium. JCI Insight 7(6):e155589. Type 2 alveolar epithelial cells (AT2s), facultative progenitor cells of the lung alveolus, play a vital role in the biology of the distal lung. In vitro model systems that incorporate human cells, recapitulate the biology of primary AT2s, and interface with the outside environment could serve as useful tools to elucidate functional characteristics of AT2s in homeostasis and disease. We and others recently adapted human induced pluripotent stem cell-derived AT2s (iAT2s) for air-liquid interface (ALI) culture. Here, we comprehensively characterize the effects of ALI culture on iAT2s and benchmark their transcriptional profile relative to both freshly sorted and cultured primary human fetal and adult AT2s. We find that iAT2s cultured at ALI maintain an AT2 phenotype while upregulating expression of transcripts associated with AT2 maturation. We then leverage this platform to assay the effects of exposure to clinically significant, inhaled toxicants including cigarette smoke and electronic cigarette vapor. Full Text

Al-Radhawi, M.A., Tripathi, S., Zhang, Y., Sontag, E.D., and Levine, H. (2022). Epigenetic factor competition reshapes the EMT landscape. PNAS 119 (42) : e2210844119. The emergence of and transitions between distinct phenotypes in isogenic cells can be attributed to the intricate interplay of epigenetic marks, external signals, and gene-regulatory elements. These elements include chromatin remodelers, histone modifiers, transcription factors, and regulatory RNAs. Mathematical models known as gene-regulatory networks (GRNs) are an increasingly important tool to unravel the workings of such complex networks. In such models, epigenetic factors are usually proposed to act on the chromatin regions directly involved in the expression of relevant genes. However, it has been well-established that these factors operate globally and compete with each other for targets genome-wide. Therefore, a perturbation of the activity of a regulator can redistribute epigenetic marks across the genome and modulate the levels of competing regulators. In this paper, we propose a conceptual and mathematical modeling framework that incorporates both local and global competition effects between antagonistic epigenetic regulators, in addition to local transcription factors, and show the counterintuitive consequences of such interactions. We apply our approach to recent experimental findings on the epithelial-mesenchymal transition (EMT). We show that it can explain the puzzling experimental data, as well as provide verifiable predictions. Full Text

Alvarez-Dominguez, J.R., Winther, S., Hansen, J.B., Lodish, H.F., and Knoll, M. (2022). An adipose lncRAP2-Igf2bp2 complex enhances adipogenesis and energy expenditure by stabilizing target mRNAs. iScience 25(1): 103680. lncRAP2 is a conserved cytoplasmic lncRNA enriched in adipose tissue and required for adipogenesis. Using purification and in vivo interactome analyses, we show that lncRAP2 forms complexes with proteins that stabilize mRNAs and modulate translation, among them Igf2bp2. Surveying transcriptome-wide Igf2bp2 client mRNAs in white adipocytes reveals selective binding to mRNAs encoding adipogenic regulators and energy expenditure effectors, including adiponectin. These same target proteins are downregulated when either Igf2bp2 or lncRAP2 is downregulated, hindering adipocyte lipolysis. Proteomics and ribosome profiling show this occurs predominantly through mRNA accumulation, as lncRAP2-Igf2bp2 complex binding does not impact translation efficiency. Phenome-wide association studies reveal specific associations of genetic variants within both lncRAP2 and Igf2bp2 with body mass and type 2 diabetes, and both lncRAP2 and Igf2bp2 are suppressed in adipose depots of obese and diabetic individuals. Thus, the lncRAP2-Igf2bp2 complex potentiates adipose development and energy expenditure and is associated with susceptibility to obesity-linked diabetes. Full Text

Banani, S.F., Afeyan, L.K., Hawken, S.W., Henninger, J.E., Dall'Agnese, A., Clark, V.E., Platt, J.M., Oksuz, O., Hannett, N.M., Sagi, I., Lee TI, and Young RA. (2022). Genetic variation associated with condensate dysregulation in disease. Developmental Cell. Online Ahead of Print. A multitude of cellular processes involve biomolecular condensates, which has led to the suggestion that diverse pathogenic mutations may dysregulate condensates. Although proof-of-concept studies have identified specific mutations that cause condensate dysregulation, the full scope of the pathological genetic variation that affects condensates is not yet known. Here, we comprehensively map pathogenic mutations to condensate-promoting protein features in putative condensate-forming proteins and find over 36,000 pathogenic mutations that plausibly contribute to condensate dysregulation in over 1,200 Mendelian diseases and 550 cancers. This resource captures mutations presently known to dysregulate condensates, and experimental tests confirm that additional pathological mutations do indeed affect condensate properties in cells. These findings suggest that condensate dysregulation may be a pervasive pathogenic mechanism underlying a broad spectrum of human diseases, provide a strategy to identify proteins and mutations involved in pathologically altered condensates, and serve as a foundation for mechanistic insights into disease and therapeutic hypotheses. Full Text

Barberio, A.E., Smith, S.G., Pires, I.S., Iyer, S., Reinhardt, F., Melo, M.B., Suh, H., Weinberg, R.A., Irvine, D.J., and Hammond, P.T. Layer-by-layer interleukin-12 nanoparticles drive a safe and effective response in ovarian tumors. Bioengineering & Translational Medicine. Early View Online. Ovarian cancer is especially deadly, challenging to treat, and has proven refractory to known immunotherapies. Cytokine therapy is an attractive strategy to drive a proinflammatory immune response in immunologically cold tumors such as many high grade ovarian cancers; however, this strategy has been limited in the past due to severe toxicity. We previously demonstrated the use of a layer-by-layer (LbL) nanoparticle (NP) delivery vehicle in subcutaneous flank tumors to reduce the toxicity of interleukin-12 (IL-12) therapy upon intratumoral injection. However, ovarian cancer cannot be treated by local injection as it presents as dispersed metastases. Herein, we demonstrate the use of systemically delivered LbL NPs using a cancer cell membrane-binding outer layer to effectively target and engage the adaptive immune system as a treatment in multiple orthotopic ovarian tumor models, including immunologically cold tumors. IL-12 therapy from systemically delivered LbL NPs shows reduced severe toxicity and maintained anti-tumor efficacy compared to carrier-free IL-12 or layer-free liposomal NPs leading to a 30% complete survival rate. Full Text

Bellott, D.W., Cho, T.J., Jackson, E.K., Skaletsky, H., Hughes, J.F., and Page, D.C. (2022). SHIMS 3.0: Highly efficient single-haplotype iterative mapping and sequencing using ultra-long nanopore reads. PloS One 17 (6) : e0269692. The reference sequence of structurally complex regions can only be obtained through a highly accurate clone-based approach that we call Single-Haplotype Iterative Mapping and Sequencing (SHIMS). In recent years, improvements to SHIMS have reduced the cost and time required by two orders of magnitude, but internally repetitive clones still require extensive manual effort to transform draft assemblies into reference-quality finished sequences. Here we describe SHIMS 3.0, using ultra-long nanopore reads to augment the Illumina data from SHIMS 2.0 assemblies and resolve internally repetitive structures. This greatly minimizes the need for manual finishing of Illumina-based draft assemblies, allowing a small team with no prior finishing experience to sequence challenging targets with high accuracy. This protocol proceeds from clone-picking to finished assemblies in 2 weeks for about $80 (USD) per clone. We recently used this protocol to produce reference sequence of structurally complex palindromes on chimpanzee and rhesus macaque X chromosomes. Our protocol provides access to structurally complex regions that would otherwise be inaccessible from whole-genome shotgun data or require an impractical amount of manual effort to generate an accurate assembly. Full Text

Boehnke, N., Straehla, J.P., Safford, H.C., Kocak, M., Rees, M.G., Ronan, M., Rosenberg, D., Adelmann, C.H., Chivukula, R.R., Nabar, N., et al. (2022). Massively parallel pooled screening reveals genomic determinants of nanoparticle delivery. Science 377(6604) : eabm5551. To accelerate the translation of cancer nanomedicine, we used an integrated genomic approach to improve our understanding of the cellular processes that govern nanoparticle trafficking. We developed a massively parallel screen that leverages barcoded, pooled cancer cell lines annotated with multiomic data to investigate cell association patterns across a nanoparticle library spanning a range of formulations with clinical potential. We identified both materials properties and cell-intrinsic features that mediate nanoparticle-cell association. Using machine learning algorithms, we constructed genomic nanoparticle trafficking networks and identified nanoparticle-specific biomarkers. We validated one such biomarker: gene expression of SLC46A3, which inversely predicts lipid-based nanoparticle uptake in vitro and in vivo. Our work establishes the power of integrated screens for nanoparticle delivery and enables the identification and utilization of biomarkers to rationally design nanoformulations. Full Text

Cangelosi, A.L., Puszynska, A.M., Roberts, J.M., Armani, A., Nguyen, T.P., Spinelli, J.B., Kunchok, T., Wang, B., Chan, S.H., Lewis, C.A., William C Comb, George W Bell , Aharon Helman, David M Sabatini (2022). Zonated leucine sensing by Sestrin-mTORC1 in the liver controls the response to dietary leucine. Science 377(6601) : 47-56. The mechanistic target of rapamycin complex 1 (mTORC1) kinase controls growth in response to nutrients, including the amino acid leucine. In cultured cells, mTORC1 senses leucine through the leucine-binding Sestrin proteins, but the physiological functions and distribution of Sestrin-mediated leucine sensing in mammals are unknown. We find that mice lacking Sestrin1 and Sestrin2 cannot inhibit mTORC1 upon dietary leucine deprivation and suffer a rapid loss of white adipose tissue (WAT) and muscle. The WAT loss is driven by aberrant mTORC1 activity and fibroblast growth factor 21 (FGF21) production in the liver. Sestrin expression in the liver lobule is zonated, accounting for zone-specific regulation of mTORC1 activity and FGF21 induction by leucine. These results establish the mammalian Sestrins as physiological leucine sensors and reveal a spatial organization to nutrient sensing by the mTORC1 pathway. Full Text

Chaves-Moreira, D., Mitchell, M.A., Arruza, C., Rawat, P., Sidoli, S., Nameki, R., Reddy, J., Corona, R.I., Afeyan, L.K., Klein, I.A., et al. (2022). The transcription factor PAX8 promotes angiogenesis in ovarian cancer through interaction with SOX17. Science Signaling 15(728):eabm2496. PAX8 is a master transcription factor that is essential during embryogenesis and promotes neoplastic growth. It is expressed by the secretory cells lining the female reproductive tract, and its deletion during development results in atresia of reproductive tract organs. Nearly all ovarian carcinomas express PAX8, and its knockdown results in apoptosis of ovarian cancer cells. To explore the role of PAX8 in these tissues, we purified the PAX8 protein complex from nonmalignant fallopian tube cells and high-grade serous ovarian carcinoma cell lines. We found that PAX8 was a member of a large chromatin remodeling complex and preferentially interacted with SOX17, another developmental transcription factor. Depleting either PAX8 or SOX17 from cancer cells altered the expression of factors involved in angiogenesis and functionally disrupted tubule and capillary formation in cell culture and mouse models. PAX8 and SOX17 in ovarian cancer cells promoted the secretion of angiogenic factors by suppressing the expression of SERPINE1, which encodes a proteinase inhibitor with antiangiogenic effects. The findings reveal a non-cell-autonomous function of these transcription factors in regulating angiogenesis in ovarian cancer. Full Text

Chen, A., Liao, S., Cheng, M., Ma, K., Wu, L., Lai, Y., Qiu, X. , Yang, J., Xu, J., Hao, S., et al. (2022). Spatiotemporal transcriptomic atlas of mouse organogenesis using DNA nanoball-patterned arrays. Cell 185, 1777-1792. Spatially resolved transcriptomic technologies are promising tools to study complex biological processes such as mammalian embryogenesis. However, the imbalance between resolution, gene capture, and field of view of current methodologies precludes their systematic application to analyze relatively large and three-dimensional mid- and late-gestation embryos. Here, we combined DNA nanoball (DNB)-patterned arrays and in situ RNA capture to create spatial enhanced resolution omics-sequencing (Stereo-seq). We applied Stereo-seq to generate the mouse organogenesis spatiotemporal transcriptomic atlas (MOSTA), which maps with single-cell resolution and high sensitivity the kinetics and directionality of transcriptional variation during mouse organogenesis. We used this information to gain insight into the molecular basis of spatial cell heterogeneity and cell fate specification in developing tissues such as the dorsal midbrain. Our panoramic atlas will facilitate in-depth investigation of longstanding questions concerning normal and abnormal mammalian development. 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 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

Cho, N.H., Cheveralls, K.C., Brunner, A.D., Kim, K., Michaelis, A.C., Raghavan, P., Kobayashi, H., Savy, L., Li, J.Y., Canaj, H., Weissman, J.S., et al. (2022). OpenCell: Endogenous tagging for the cartography of human cellular organization. Science 375(6585):eabi6983. Elucidating the wiring diagram of the human cell is a central goal of the postgenomic era. We combined genome engineering, confocal live-cell imaging, mass spectrometry, and data science to systematically map the localization and interactions of human proteins. Our approach provides a data-driven description of the molecular and spatial networks that organize the proteome. Unsupervised clustering of these networks delineates functional communities that facilitate biological discovery. We found that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions, and that RNA binding proteins form a specific subgroup defined by unique interaction and localization properties. Paired with a fully interactive website (opencell.czbiohub.org), our work constitutes a resource for the quantitative cartography of human cellular organization. Full Text

Choi, J., and Richards, E.J. (2022). The edge of the nucleus: Variations on a theme. Developmental Cell 57(1):3-4. The plant nuclear lamina utilizes distinct and highly divergent proteins to mediate chromatin interactions at the nuclear edge. In this issue of Developmental Cell, Tang et al. show that members of PNET2, a family of inner nuclear membrane proteins in Arabidopsis, are capable of binding histones and are involved in large-scale genome organization. Full Text

Corradin O., Sallari, R., Hoang, A.T., Kassim, B.S., Ben Hutta, G., Cuoto, L., Quach, B.C., Lovrenert, K., Hays, C., Gryder, B.E., Song, Y., et al. (2022). Convergence of case-specific epigenetic alterations identify a confluence of genetic vulnerabilities tied to opioid overdose. Molecular Psychiatry. Online Ahead of Print. Opioid use disorder is a highly heterogeneous disease driven by a variety of genetic and environmental risk factors which have yet to be fully elucidated. Opioid overdose, the most severe outcome of opioid use disorder, remains the leading cause of accidental death in the United States. We interrogated the effects of opioid overdose on the brain using ChIP-seq to quantify patterns of H3K27 acetylation in dorsolateral prefrontal cortical neurons isolated from 51 opioid-overdose cases and 51 accidental death controls. Among opioid cases, we observed global hypoacetylation and identified 388 putative enhancers consistently depleted for H3K27ac. Machine learning on H3K27ac patterns predicted case-control status with high accuracy. We focused on case-specific regulatory alterations, revealing 81,399 hypoacetylation events, uncovering vast inter-patient heterogeneity. We developed a strategy to decode this heterogeneity based on convergence analysis, which leveraged promoter-capture Hi-C to identify five genes over-burdened by alterations in their regulatory network or "plexus": ASTN2, KCNMA1, DUSP4, GABBR2, ENOX1. These convergent loci are enriched for opioid use disorder risk genes and heritability for generalized anxiety, number of sexual partners, and years of education. Overall, our multi-pronged approach uncovers neurobiological aspects of opioid use disorder and captures genetic and environmental factors perpetuating the opioid epidemic. Full Text

Cutano, V., Ferreira Mendes, J.M., Escudeiro-Lopes, S., Machado, S., Vinaixa Forner, J., Gonzales-Morena, J.M., Prevorovsky, M., Zemlianski, V., Feng, Y., Kralova Viziova, P., Sonia Iyer, et al. (2023). LACTB exerts tumor suppressor properties in epithelial ovarian cancer through regulation of Slug. Life Science Alliance 6(1):e202201510. Epithelial-mesenchymal transition (EMT) is a cellular mechanism used by cancer cells to acquire migratory and stemness properties. In this study, we show, through in vitro, in vivo, and 3D culture experiments, that the mitochondrial protein LACTB manifests tumor suppressor properties in ovarian cancer. We show that LACTB is significantly down-regulated in epithelial ovarian cancer cells and clinical tissues. Re-expression of LACTB negatively effects the growth of cancer cells but not of non-tumorigenic cells. Mechanistically, we show that LACTB leads to differentiation of ovarian cancer cells and loss of their stemness properties, which is achieved through the inhibition of the EMT program and the LACTB-dependent down-regulation of Snail2/Slug transcription factor. This study uncovers a novel role of LACTB in ovarian cancer and proposes new ways of counteracting the oncogenic EMT program in this model system. Full Text

Dagan, Y., Yesharim, Y., Bonneau, A.R., Frankovits, T., Schwartz, S., Reddien, P.W., and Wurtzel, O. (2022). m6A is required for resolving progenitor identity during planarian stem cell differentiation. EMBO Journal e109895. Online Ahead of Print. Regeneration and tissue homeostasis require accurate production of missing cell lineages. Cell production is driven by changes to gene expression, which is shaped by multiple layers of regulation. Here, we find that the ubiquitous mRNA base-modification, m6A, is required for proper cell fate choice and cellular maturation in planarian stem cells (neoblasts). We mapped m6A-enriched regions in 7,600 planarian genes and found that perturbation of the m6A pathway resulted in progressive deterioration of tissues and death. Using single-cell RNA sequencing of >20,000 cells following perturbation of the m6A pathway, we identified an increase in expression of noncanonical histone variants, and that inhibition of the pathway resulted in accumulation of undifferentiated cells throughout the animal in an abnormal transcriptional state. Analysis of >1,000 planarian gene expression datasets revealed that the inhibition of the chromatin modifying complex NuRD had almost indistinguishable consequences, unraveling an unappreciated link between m6A and chromatin modifications. Our findings reveal that m6A is critical for planarian stem cell homeostasis and gene regulation in tissue maintenance and regeneration. Full Text

Dall'Agnese, A., Platt, J.M., Zheng, M.M., Friesen, M., Dall'Agnese, G., Blaise, A.M., Spinelli, J.B., Henninger, J.E., Tevonian, E.N., Hannett, N.M., Charalampos Lazaris, Henry R Kilgor, Rudolf Jaenisch, Jacob F Jeppesen, Tong I Lee, Richard A Young, et al. (2022). The dynamic clustering of insulin receptor underlies its signaling and is disrupted in insulin resistance. Nature Communications 13(1):7522. Insulin receptor (IR) signaling is central to normal metabolic control and is dysregulated in metabolic diseases such as type 2 diabetes. We report here that IR is incorporated into dynamic clusters at the plasma membrane, in the cytoplasm and in the nucleus of human hepatocytes and adipocytes. Insulin stimulation promotes further incorporation of IR into these dynamic clusters in insulin-sensitive cells but not in insulin-resistant cells, where both IR accumulation and dynamic behavior are reduced. Treatment of insulin-resistant cells with metformin, a first-line drug used to treat type 2 diabetes, can rescue IR accumulation and the dynamic behavior of these clusters. This rescue is associated with metformin's role in reducing reactive oxygen species that interfere with normal dynamics. These results indicate that changes in the physico-mechanical features of IR clusters contribute to insulin resistance and have implications for improved therapeutic approaches. Full Text

Davidson, S.M., Schmidt, D.R., Heyman, J.E., O'Brien, J.P., Liu, A.C., Israelsen, W.J., Dayton, T.L., Sehgal, R., Bronson, R.T., Freinkman, E., et al. (2022). Pyruvate Kinase M1 Suppresses Development and Progression of Prostate Adenocarcinoma Cancer Research 82(13) : 2403-2416. Altered metabolism helps sustain cancer cell proliferation and survival. Most cancers, including prostate cancers, express the M2 splice isoform of pyruvate kinase (PKM2), which can support anabolic metabolism to support cell proliferation. However, Pkm2 expression is dispensable for the formation and growth of many cancers in vivo. Expression of pyruvate kinase isoform M1 (Pkm1) is restricted to relatively few tissues and has been reported to promote growth of select tumors, but the role of PKM1 in cancer has been less studied than PKM2. To test how differential expression of pyruvate kinase isoforms affects cancer initiation and progression, we generated mice harboring a conditional allele of Pkm1 and crossed these mice, or those with a Pkm2 conditional allele, with a Pten loss-driven prostate cancer model. Pkm1 loss led to increased PKM2 expression and accelerated prostate cancer development, whereas Pkm2 deletion led to increased PKM1 expression and suppressed tumor progression. Metabolic profiling revealed altered nucleotide levels in tumors with high PKM1 expression, and failure of these tumors to progress was associated with DNA replication stress and senescence. Consistent with these data, a small molecule pyruvate kinase activator that mimics a high activity PKM1-like state suppressed progression of established prostate tumors. Analysis of human specimens showed PKM2 expression is retained in most human prostate cancers. Overall, this study uncovers a role for pyruvate kinase isoforms in prostate cancer initiation and progression, and argues that pharmacologic pyruvate kinase activation may be beneficial for treating prostate cancer. Full Text

Diaz-Ortiz, M.E., Seo, Y., Posavi, M., Carceles Cordon, M., Clark, E., Jain, N., Charan, R., Gallagher, M.D., Unger, T.L., Amari, N., et al. (2022). GPNMB confers risk for Parkinson's disease through interaction with α-synuclein. Science 377 (6608):eabk0637. Many risk loci for Parkinson's disease (PD) have been identified by genome-wide association studies (GWASs), but target genes and mechanisms remain largely unknown. We linked the GWAS-derived chromosome 7 locus (sentinel single-nucleotide polymorphism rs199347) to GPNMB through colocalization analyses of expression quantitative trait locus and PD risk signals, confirmed by allele-specific expression studies in the human brain. In cells, glycoprotein nonmetastatic melanoma protein B (GPNMB) coimmunoprecipitated and colocalized with α-synuclein (aSyn). In induced pluripotent stem cell-derived neurons, loss of GPNMB resulted in loss of ability to internalize aSyn fibrils and develop aSyn pathology. In 731 PD and 59 control biosamples, GPNMB was elevated in PD plasma, associating with disease severity. Thus, GPNMB represents a PD risk gene with potential for biomarker development and therapeutic targeting. Full Text

Diehl, F.F., Miettinen, T.P., Elbashir, R., Nabel, C.S., Darnell, A.M., Do, B.T., Manalis, S.R., Lewis, C.A., and Vander Heiden, M.G. (2022). Nucleotide imbalance decouples cell growth from cell proliferation. Nature Cell Biology. Online Ahead of Print. Nucleotide metabolism supports RNA synthesis and DNA replication to enable cell growth and division. Nucleotide depletion can inhibit cell growth and proliferation, but how cells sense and respond to changes in the relative levels of individual nucleotides is unclear. Moreover, the nucleotide requirement for biomass production changes over the course of the cell cycle, and how cells coordinate differential nucleotide demands with cell cycle progression is not well understood. Here we find that excess levels of individual nucleotides can inhibit proliferation by disrupting the relative levels of nucleotide bases needed for DNA replication and impeding DNA replication. The resulting purine and pyrimidine imbalances are not sensed by canonical growth regulatory pathways like mTORC1, Akt and AMPK signalling cascades, causing excessive cell growth despite inhibited proliferation. Instead, cells rely on replication stress signalling to survive during, and recover from, nucleotide imbalance during S phase. We find that ATR-dependent replication stress signalling is activated during unperturbed S phases and promotes nucleotide availability to support DNA replication. Together, these data reveal that imbalanced nucleotide levels are not detected until S phase, rendering cells reliant on replication stress signalling to cope with this metabolic problem and disrupting the coordination of cell growth and division. Full Text

Drouin-Ouellet, J., Li, D., Lu, Y.R., and Echegaray, C.V. (2022). The 2022 International Society for Stem Cell Research (ISSCR) Annual Meeting: Celebrating 20 Years of Achievements. Cell Reprogramming 24 : 212-222. Last June, the stem cell community came together to celebrate the 20th anniversary of the International Society for Stem Cell Research (ISSCR), one of the leading organizations in the field. The hybrid event mixed a varied program filled with plenary talks, concurrent track sessions, poster presentations, exhibit booths, and plenty of opportunities to enhance stem cell research through bonding between academia and industry. This report highlights the Plenary sessions, with the main topics discussed by each speaker. All the impressive research showcased during the meeting is genuine proof of the great advancements the field has witnessed during these last 20 years, and the more to come. Full Text

Eisen, T.J., Li, J., and Bartel, D.P. (2022). The Interplay Between Translational Efficiency, Poly(A) Tails, MicroRNAs, and Neuronal Activation. RNA. Neurons provide a rich setting for studying post-transcriptional control. Here, we investigate the landscape of translational control in neurons and search for mRNA features that explain differences in translational efficiency (TE), considering the interplay between TE, mRNA poly(A)-tail lengths, microRNAs, and neuronal activation. In neurons and brain tissues, TE correlates with tail length, and a few dozen mRNAs appear to undergo cytoplasmic polyadenylation upon light or chemical stimulation. However, the correlation between TE and tail length is modest, explaining <5% of TE variance, and even this modest relationship diminishes when accounting for other mRNA features. Thus, tail length appears to affect TE only minimally. Accordingly, miRNAs, which accelerate deadenylation of their mRNA targets, primarily influence target mRNA levels, with no detectable effect on either steady-state tail lengths or TE. Larger correlates with TE include codon composition and predicted mRNA folding energy. When combined in a model, the identified correlates explain 38-45% of TE variance. These results provide a framework for considering the relative impact of factors that contribute to translational control in neurons. They indicate that when examined in bulk, translational control in neurons largely resembles that of other types of post-embryonic cells. Thus, detection of more specialized control might require analyses that can distinguish translation occurring in neuronal processes from that occurring in cell bodies. Full Text

Fingerhut, J.M., and Yamashita, Y.M. (2022). The regulation and potential functions of intronic satellite DNA. Seminars in Cell & Developmental Biology. Online Ahead of Print. Satellite DNAs are arrays of tandem repeats found in the eukaryotic genome. They are mainly found in pericentromeric heterochromatin and have been believed to be mostly inert, leading satellite DNAs to be erroneously regarded as junk. Recent studies have started to elucidate the function of satellite DNA, yet little is known about the peculiar case where satellite DNA is found within the introns of protein coding genes, resulting in incredibly large introns, a phenomenon termed intron gigantism. Studies in Drosophila demonstrated that satellite DNA-containing introns are transcribed with the gene and require specialized mechanisms to overcome the burdens imposed by the extremely long stretches of repetitive DNA. Whether intron gigantism confers any benefit or serves any functional purpose for cells and/or organisms remains elusive. Here we review our current understanding of intron gigantism: where it is found, the challenges it imposes, how it is regulated and what purpose it may serve. Full Text

Friesen, M., Khalil, A.S., Barrasa, M.I., Jeppesen, J.F., Mooney, D.J., and Jaenisch, R. (2022). Development of a physiological insulin resistance model in human stem cell-derived adipocytes. Science Advances 8(24) : eabn7298. Adipocytes are key regulators of human metabolism, and their dysfunction in insulin signaling is central to metabolic diseases including type II diabetes mellitus (T2D). However, the progression of insulin resistance into T2D is still poorly understood. This limited understanding is due, in part, to the dearth of suitable models of insulin signaling in human adipocytes. Traditionally, adipocyte models fail to recapitulate in vivo insulin signaling, possibly due to exposure to supraphysiological nutrient and hormone conditions. We developed a protocol for human pluripotent stem cell-derived adipocytes that uses physiological nutrient conditions to produce a potent insulin response comparable to in vivo adipocytes. After systematic optimization, this protocol allows robust insulin-stimulated glucose uptake and transcriptional insulin response. Furthermore, exposure of sensitized adipocytes to physiological hyperinsulinemia dampens insulin-stimulated glucose uptake and dysregulates insulin-responsive transcription. Overall, our methodology provides a novel platform for the mechanistic study of insulin signaling and resistance using human pluripotent stem cell-derived adipocytes. Full Text

Funk, L., Su, K.C., Ly, J., Feldman, D., Singh, A., Moodie, B., Blainey, P.C., and Cheeseman, I.M. (2022). The phenotypic landscape of essential human genes. Cell. Online Ahead of Print. Understanding the basis for cellular growth, proliferation, and function requires determining the roles of essential genes in diverse cellular processes, including visualizing their contributions to cellular organization and morphology. Here, we combined pooled CRISPR-Cas9-based functional screening of 5,072 fitness-conferring genes in human HeLa cells with microscopy-based imaging of DNA, the DNA damage response, actin, and microtubules. Analysis of >31 million individual cells identified measurable phenotypes for >90% of gene knockouts, implicating gene targets in specific cellular processes. Clustering of phenotypic similarities based on hundreds of quantitative parameters further revealed co-functional genes across diverse cellular activities, providing predictions for gene functions and associations. By conducting pooled live-cell screening of ∼450,000 cell division events for 239 genes, we additionally identified diverse genes with functional contributions to chromosome segregation. Our work establishes a resource detailing the consequences of disrupting core cellular processes that represents the functional landscape of essential human genes. Full Text

Glinkerman, C.M., Lin, S.T., Ni, J.H., Li, F.S., Zhao, X.H., and Weng, J.K. (2022). Sporopollenin-inspired design and synthesis of robust polymeric materials. Communications Chemistry 5 no. 110. Pine sporopollenin harbors unique monomeric units and inter-unit linkages forming mechanically robust and chemically inert biopolymers. Here, the authors design and synthesize several sporopollenin analogues, and characterize their chemical, thermal and mechanical properties as robust polymers. Sporopollenin is a mechanically robust and chemically inert biopolymer that constitutes the outer protective exine layer of plant spores and pollen grains. Recent investigation of the molecular structure of pine sporopollenin revealed unique monomeric units and inter-unit linkages distinct from other previously known biopolymers, which could be harnessed for new material design. Herein, we report the bioinspired synthesis of a series of sporopollenin analogues. This exercise confirms large portions of our previously proposed pine sporopollenin structural model, while the measured chemical, thermal, and mechanical properties of the synthetic sporopollenins constitute favorable attributes of a new kind of robust material. This study explores a new design framework of robust materials inspired by natural sporopollenins, and provides insights and reagents for future elucidation and engineering of sporopollenin biosynthesis in plants. Full Text

Gu, X., Jouandin, P., Lalgudi, P.V., Binari, R., Valenstein, M.L., Reid, M.A., Allen, A.E., Kamitaki, N., Locasale, J.W., Perrimon, N.,and David M. Sabatini (2022). Sestrin mediates detection of and adaptation to low-leucine diets in Drosophila. Nature. Online Ahead of Print. Mechanistic target of rapamycin complex 1 (mTORC1) regulates cell growth and metabolism in response to multiple nutrients, including the essential amino acid leucine(1). Recent work in cultured mammalian cells established the Sestrins as leucine-binding proteins that inhibit mTORC1 signalling during leucine deprivation(2,3), but their role in the organismal response to dietary leucine remains elusive. Here we find that Sestrin-null flies (Sesn(-/-)) fail to inhibit mTORC1 or activate autophagy after acute leucine starvation and have impaired development and a shortened lifespan on a low-leucine diet. Knock-in flies expressing a leucine-binding-deficient Sestrin mutant (Sesn(L431E)) have reduced, leucine-insensitive mTORC1 activity. Notably, we find that flies can discriminate between food with or without leucine, and preferentially feed and lay progeny on leucine-containing food. This preference depends on Sestrin and its capacity to bind leucine. Leucine regulates mTORC1 activity in glial cells, and knockdown of Sesn in these cells reduces the ability of flies to detect leucine-free food. Thus, nutrient sensing by mTORC1 is necessary for flies not only to adapt to, but also to detect, a diet deficient in an essential nutrient. Full Text

Guna, A., Stevens, T.A., Inglis, A.J., Replogle, J.M., Esantsi, T.K., Muthukumar, G., Shaffer, K.C.L., Wang, M.L., Pogson, A.N., Weissman, J.S., et al. (2022). MTCH2 is a mitochondrial outer membrane protein insertase. Science 378(6617) : 317-322. In the mitochondrial outer membrane, α-helical transmembrane proteins play critical roles in cytoplasmic-mitochondrial communication. Using genome-wide CRISPR screens, we identified mitochondrial carrier homolog 2 (MTCH2), and its paralog MTCH1, and showed that it is required for insertion of biophysically diverse tail-anchored (TA), signal-anchored, and multipass proteins, but not outer membrane β-barrel proteins. Purified MTCH2 was sufficient to mediate insertion into reconstituted proteoliposomes. Functional and mutational studies suggested that MTCH2 has evolved from a solute carrier transporter. MTCH2 uses membrane-embedded hydrophilic residues to function as a gatekeeper for the outer membrane, controlling mislocalization of TAs into the endoplasmic reticulum and modulating the sensitivity of leukemia cells to apoptosis. Our identification of MTCH2 as an insertase provides a mechanistic explanation for the diverse phenotypes and disease states associated with MTCH2 dysfunction. Full Text

Guna, A., Hazu, M., Pinton Tomaleri, G., and Voorhees, R.M. (2022). A TAle of Two Pathways: Tail-Anchored Protein Insertion at the Endoplasmic Reticulum.Cold Spring Harbor Perspectives in Biology.Online Ahead of Print. Tail-anchored (TA) proteins are an essential class of integral membrane proteins required for many aspects of cellular physiology. TA proteins contain a single carboxy-terminal transmembrane domain that must be post-translationally recognized, guided to, and ultimately inserted into the correct cellular compartment. The majority of TA proteins begin their biogenesis in the endoplasmic reticulum (ER) and utilize two parallel strategies for targeting and insertion: the guided-entry of tail-anchored proteins (GET) and ER-membrane protein complex (EMC) pathways. Here we focus on how these two sets of machinery target, transfer, and insert TAs into the lipid bilayer in close collaboration with quality control machinery. Additionally, we highlight the unifying features of the insertion process as revealed by recent structures of the GET and EMC membrane protein complexes. Full Text

Gupta, A., Martin-Rufino, J.D., Jones, T.R., Subramanian, V., Qiu, X., Grody, E.I., Bloemendal, A., Weng, C., Niu, S.Y., Min, K.H., et al. (2022). Inferring gene regulation from stochastic transcriptional variation across single cells at steady state. PNAS 119 (34): e2207392119. Regulatory relationships between transcription factors (TFs) and their target genes lie at the heart of cellular identity and function; however, uncovering these relationships is often labor-intensive and requires perturbations. Here, we propose a principled framework to systematically infer gene regulation for all TFs simultaneously in cells at steady state by leveraging the intrinsic variation in the transcriptional abundance across single cells. Through modeling and simulations, we characterize how transcriptional bursts of a TF gene are propagated to its target genes, including the expected ranges of time delay and magnitude of maximum covariation. We distinguish these temporal trends from the time-invariant covariation arising from cell states, and we delineate the experimental and technical requirements for leveraging these small but meaningful cofluctuations in the presence of measurement noise. While current technology does not yet allow adequate power for definitively detecting regulatory relationships for all TFs simultaneously in cells at steady state, we investigate a small-scale dataset to inform future experimental design. This study supports the potential value of mapping regulatory connections through stochastic variation, and it motivates further technological development to achieve its full potential. Full Text

Hallacli, E., Kayatekin, C., Nazeen, S., Wang, X.H., Sheinkopf, Z., Sathyakumar, S., Sarkar, S., Jiang, X., Dong, X., Di Maio, R., Jackson Sandoe, Susan Lindquist, David P Bartel, et al. (2022). The Parkinson's disease protein alpha-synuclein is a modulator of processing bodies and mRNA stability. Cell 185, 2035-2056.e2033.Alpha-synuclein (αS) is a conformationally plastic protein that reversibly binds to cellular membranes. It aggregates and is genetically linked to Parkinson's disease (PD). Here, we show that αS directly modulates processing bodies (P-bodies), membraneless organelles that function in mRNA turnover and storage. The N terminus of αS, but not other synucleins, dictates mutually exclusive binding either to cellular membranes or to P-bodies in the cytosol. αS associates with multiple decapping proteins in close proximity on the Edc4 scaffold. As αS pathologically accumulates, aberrant interaction with Edc4 occurs at the expense of physiologic decapping-module interactions. mRNA decay kinetics within PD-relevant pathways are correspondingly disrupted in PD patient neurons and brain. Genetic modulation of P-body components alters αS toxicity, and human genetic analysis lends support to the disease-relevance of these interactions. Beyond revealing an unexpected aspect of αS function and pathology, our data highlight the versatility of conformationally plastic proteins with high intrinsic disorder. Full Text

Harada, T., Heshmati, Y., Kalfon, J., Perez, M.W., Xavier Ferrucio, J., Ewers, J., Hubbell Engler, B., Kossenkov, A., Ellegast, J.M., Yi, J.S., Li, C.H., et al. (2022). A distinct core regulatory module enforces oncogene expression in KMT2A-rearranged leukemia. Genes & Development. Online Ahead of Print. Acute myeloid leukemia with KMT2A (MLL) rearrangements is characterized by specific patterns of gene expression and enhancer architecture, implying unique core transcriptional regulatory circuitry. Here, we identified the transcription factors MEF2D and IRF8 as selective transcriptional dependencies of KMT2A-rearranged AML, where MEF2D displays partially redundant functions with its paralog, MEF2C. Rapid transcription factor degradation followed by measurements of genome-wide transcription rates and super resolution microscopy revealed that MEF2D and IRF8 form a distinct core regulatory module with a narrow direct transcriptional program that includes activation of the key oncogenes MYC, HOXA9, and BCL2. Our study illustrates a mechanism of context-specific transcriptional addiction whereby a specific AML subclass depends on a highly specialized core regulatory module to directly enforce expression of common leukemia oncogenes. Full Text

Hartooni, N., Sung, J., Jain, A. , and Morgan, D.O. (2022). Single-molecule analysis of specificity and multivalency in binding of short linear substrate motifs to the APC/C. Nature Communications 13(1):341. Robust regulatory signals in the cell often depend on interactions between short linear motifs (SLiMs) and globular proteins. Many of these interactions are poorly characterized because the binding proteins cannot be produced in the amounts needed for traditional methods. To address this problem, we developed a single-molecule off-rate (SMOR) assay based on microscopy of fluorescent ligand binding to immobilized protein partners. We used it to characterize substrate binding to the Anaphase-Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that triggers chromosome segregation. We find that SLiMs in APC/C substrates (the D box and KEN box) display distinct affinities and specificities for the substrate-binding subunits of the APC/C, and we show that multiple SLiMs in a substrate generate a high-affinity multivalent interaction. The remarkably adaptable substrate-binding mechanisms of the APC/C have the potential to govern the order of substrate destruction in mitosis. Full Text

Hemenway, E.A., and Gehring, M. (2022). Epigenetic Regulation During Plant Development and the Capacity for Epigenetic Memory. Annual Review of Plant Biology 74 : The establishment, maintenance, and removal of epigenetic modifications provide an additional layer of regulation, beyond genetically encoded factors, by which plants can control developmental processes and adapt to the environment. Epigenetic inheritance, while historically referring to information not encoded in the DNA sequence that is inherited between generations, can also refer to epigenetic modifications that are maintained within an individual but are reset between generations. Both types of epigenetic inheritance occur in plants, and the functions and mechanisms distinguishing the two are of great interest to the field. Here, we discuss examples of epigenetic dynamics and maintenance during selected stages of growth and development and their functional consequences. Epigenetic states are also dynamic in response to stress, with consequences for transposable element regulation. How epigenetic resetting between generations occurs during normal development and in response to stress is an emerging area of research. Full Text

Hendrickson, E.L., Bor, B., Kerns, K.A., Lamont, E.I., Chang, Y., Liu, J., Cen, L., Schulte, Fabian, Hardt, M., Shi, W., et al. (2022). Transcriptome of Epibiont Saccharibacteria Nanosynbacter lyticus Strain TM7x During the Establishment of Symbiosis. Journal of Bacteriology : e0011222. Online Ahead of Print. Saccharibacteria Nanosynbacter lyticus strain TM7x is a member of the broadly distributed candidate phylum radiation. These bacteria have ultrasmall cell sizes, have reduced genomes, and live as epibionts on the surfaces of other bacteria. The mechanisms by which they establish and maintain this relationship are not yet fully understood. The transcriptomes of the epibiont TM7x and its host bacteria Schaalia odontolytica strain XH001 were captured across the establishment of symbiosis during both the initial interaction and stable symbiosis. The results showed a dynamic interaction with large shifts in gene expression for both species between the initial encounter and stable symbiosis, notably in transporter genes. During stable symbiosis, the host XH001 showed higher gene expression for peptidoglycan biosynthesis, mannosylation, cell cycle and stress-related genes, whereas it showed lower expression of chromosomal partitioning genes. This was consistent with the elongated cell shape seen in XH001 infected with TM7x and our discovery that infection resulted in thickened cell walls. Within TM7x, increased pili, type IV effector genes, and arginine catabolism/biosynthesis gene expression during stable symbiosis implied a key role for these functions in the interaction. Consistent with its survival and persistence in the human microbiome as an obligate epibiont with reduced de novo biosynthetic capacities, TM7x also showed higher levels of energy production and peptidoglycan biosynthesis, but lower expression of stress-related genes, during stable symbiosis. These results imply that TM7x and its host bacteria keep a delicate balance in order to sustain an episymbiotic lifestyle. IMPORTANCE Nanosynbacter lyticus type strain TM7x is the first cultivated member of the Saccharibacteria and the candidate phyla radiation (CPR). It was discovered to be ultrasmall in cell size with a highly reduced genome that establishes an obligate epibiotic relationship with its host bacterium. The CPR is a large, monophyletic radiation of bacteria with reduced genomes that includes Saccharibacteria. The vast majority of the CPR have yet to be cultivated, and our insights into these unique organisms to date have been derived from only a few Saccharibacteria species. Being obligate parasites, it is unknown how these ultrasmall Saccharibacteria, which are missing many de novo biosynthetic pathways, are maintained at a high prevalence within the human microbiome as well as in the environment. Full Text

Herneisen, A.L, Li, Z.H., Chan, A.W., Moreno, S.N.J., and Lourido, S. (2022). Temporal and thermal profiling of the Toxoplasma proteome implicates parasite Protein Phosphatase 1 in the regulation of Ca(2+)-responsive pathways. eLife 11 : e80336. Online Ahead of Print. Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca(2+) signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the replicative and lytic phases of the infectious cycle, as well as the transition between them. Despite the presence of conserved Ca(2+)-responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca(2+)-responsive proteome of the model apicomplexan T. gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca(2+) release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca(2+)-responsive proteins, such as parasite Ca(2+)-dependent protein kinases. Our approach also identified numerous Ca(2+)-responsive proteins that are not predicted to bind Ca(2+), yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP1) as a Ca(2+)-responsive enzyme that relocalized to the parasite apex upon Ca(2+) store release. Conditional depletion of PP1 revealed that the phosphatase regulates Ca(2+) uptake to promote parasite motility. PP1 may thus be partly responsible for Ca(2+)-regulated serine/threonine phosphatase activity in apicomplexan parasites. Full Text

Hsu, A., Duan, Q., Day, D.S., Luo, X., McMahon, S., Huang, Y., Feldman, Z.B., Jiang, Z., Zhang, T., Liang, Y.,Young, R.A., et al. (2022). Targeting transcription in heart failure via CDK7/12/13 inhibition. Nature Communications 13(1) : 4345. Heart failure with reduced ejection fraction (HFrEF) is associated with high mortality, highlighting an urgent need for new therapeutic strategies. As stress-activated cardiac signaling cascades converge on the nucleus to drive maladaptive gene programs, interdicting pathological transcription is a conceptually attractive approach for HFrEF therapy. Here, we demonstrate that CDK7/12/13 are critical regulators of transcription activation in the heart that can be pharmacologically inhibited to improve HFrEF. CDK7/12/13 inhibition using the first-in-class inhibitor THZ1 or RNAi blocks stress-induced transcription and pathologic hypertrophy in cultured rodent cardiomyocytes. THZ1 potently attenuates adverse cardiac remodeling and HFrEF pathogenesis in mice and blocks cardinal features of disease in human iPSC-derived cardiomyocytes. THZ1 suppresses Pol II enrichment at stress-transactivated cardiac genes and inhibits a specific pathologic gene program in the failing mouse heart. These data identify CDK7/12/13 as druggable regulators of cardiac gene transactivation during disease-related stress, suggesting that HFrEF features a critical dependency on transcription that can be therapeutically exploited. Full Text

Hughes, J.F., Skaletsky, H., Nicholls, P.K., Drake, A., Pyntikova, T., Cho, T.J., Bellott, D.W., and Page, D.C. (2022). A gene deriving from the ancestral sex chromosomes was lost from the X and retained on the Y chromosome in eutherian mammals. BMC Biology 20 (1): 133. The mammalian X and Y chromosomes originated from a pair of ordinary autosomes. Over the past ~180 million years, the X and Y have become highly differentiated and now only recombine with each other within a short pseudoautosomal region. While the X chromosome broadly preserved its gene content, the Y chromosome lost ~92% of the genes it once shared with the X chromosome. PRSSLY is a Y-linked gene identified in only a few mammalian species that was thought to be acquired, not ancestral. However, PRSSLY's presence in widely divergent species-bull and mouse-led us to further investigate its evolutionary history. RESULTS: We discovered that PRSSLY is broadly conserved across eutherians and has ancient origins. PRSSLY homologs are found in syntenic regions on the X chromosome in marsupials and on autosomes in more distant animals, including lizards, indicating that PRSSLY was present on the ancestral autosomes but was lost from the X and retained on the Y in eutherian mammals. We found that across eutheria, PRSSLY's expression is testis-specific, and, in mouse, it is most robustly expressed in post-meiotic germ cells. The closest paralog to PRSSLY is the autosomal gene PRSS55, which is expressed exclusively in testes, involved in sperm differentiation and migration, and essential for male fertility in mice. Outside of eutheria, in species where PRSSLY orthologs are not Y-linked, we find expression in a broader range of somatic tissues, suggesting that PRSSLY has adopted a germ-cell-specific function in eutherians. Finally, we generated Prssly mutant mice and found that they are fully fertile but produce offspring with a modest female-biased sex ratio compared to controls. CONCLUSIONS: PRSSLY appears to be the first example of a gene that derives from the mammalian ancestral sex chromosomes that was lost from the X and retained on the Y. Although the function of PRSSLY remains to be determined, it may influence the sex ratio by promoting the survival or propagation of Y-bearing sperm. Full Text

Hunt, G., Boija, A., and Mannervik, M. (2022). p300/CBP sustains Polycomb silencing by non-enzymatic functions. Molecular Cell 82 (19) : 3580-3597. Maintenance of appropriate cell states involves epigenetic mechanisms, including Polycomb-group (PcG)-mediated transcriptional repression. While PcG proteins are known to induce chromatin compaction, how PcG proteins gain access to DNA in compact chromatin to achieve long-term silencing is poorly understood. Here, we show that the p300/CREB-binding protein (CBP) co-activator is associated with two-thirds of PcG regions and required for PcG occupancy at many of these in Drosophila and mouse cells. CBP stabilizes RNA polymerase II (Pol II) at PcG-bound repressive sites and promotes Pol II pausing independently of its histone acetyltransferase activity. CBP and Pol II pausing are necessary for RNA-DNA hybrid (R-loop) formation and nucleosome depletion at Polycomb Response Elements (PREs), whereas transcription beyond the pause region is not. These results suggest that non-enzymatic activities of the CBP co-activator have been repurposed to support PcG-mediated silencing, revealing how chromatin regulator interplay maintains transcriptional states. Full Text

Issigonis, M., Redkar, A.B., Rozario, T., Khan, U.W., Mejia-Sanchez, R., Lapan, S.W., Reddien, P.W., and Newmark, P.A. (2022). A Krüppel-like factor is required for development and regeneration of germline and yolk cells from somatic stem cells in planarians. PLoS Biology 20(7) : e3001472. Sexually reproducing animals segregate their germline from their soma. In addition to gamete-producing gonads, planarian and parasitic flatworm reproduction relies on yolk cell-generating accessory reproductive organs (vitellaria) supporting development of yolkless oocytes. Despite the importance of vitellaria for flatworm reproduction (and parasite transmission), little is known about this unique evolutionary innovation. Here, we examine reproductive system development in the planarian Schmidtea mediterranea, in which pluripotent stem cells generate both somatic and germ cell lineages. We show that a homolog of the pluripotency factor Klf4 is expressed in primordial germ cells (PGCs), presumptive germline stem cells (GSCs), and yolk cell progenitors. Knockdown of this klf4-like (klf4l) gene results in animals that fail to specify or maintain germ cells; surprisingly, they also fail to maintain yolk cells. We find that yolk cells display germ cell-like attributes and that vitellaria are structurally analogous to gonads. In addition to identifying a new proliferative cell population in planarians (yolk cell progenitors) and defining its niche, our work provides evidence supporting the hypothesis that flatworm germ cells and yolk cells share a common evolutionary origin. Full Text

Iyer, J., Gentry, L.K., Bergwell, M., Smith, A., Guagliardo, S., Kropp, P.A., Sankaralingam, P., Liu, Y., Spooner, E., Bowerman, B., et al. (2022). The chromatin remodeling protein CHD-1 and the EFL-1/DPL-1 transcription factor cooperatively down regulate CDK-2 to control SAS-6 levels and centriole number. PLoS Genetics 18(4):e1009799. Centrioles are submicron-scale, barrel-shaped organelles typically found in pairs, and play important roles in ciliogenesis and bipolar spindle assembly. In general, successful execution of centriole-dependent processes is highly reliant on the ability of the cell to stringently control centriole number. This in turn is mainly achieved through the precise duplication of centrioles during each S phase. Aberrations in centriole duplication disrupt spindle assembly and cilia-based signaling and have been linked to cancer, primary microcephaly and a variety of growth disorders. Studies aimed at understanding how centriole duplication is controlled have mainly focused on the post-translational regulation of two key components of this pathway: the master regulatory kinase ZYG-1/Plk4 and the scaffold component SAS-6. In contrast, how transcriptional control mechanisms might contribute to this process have not been well explored. Here we show that the chromatin remodeling protein CHD-1 contributes to the regulation of centriole duplication in the C. elegans embryo. Specifically, we find that loss of CHD-1 or inactivation of its ATPase activity can restore embryonic viability and centriole duplication to a strain expressing insufficient ZYG-1 activity. Interestingly, loss of CHD-1 is associated with increases in the levels of two ZYG-1-binding partners: SPD-2, the centriole receptor for ZYG-1 and SAS-6. Finally, we explore transcriptional regulatory networks governing centriole duplication and find that CHD-1 and a second transcription factor, EFL-1/DPL-1 cooperate to down regulate expression of CDK-2, which in turn promotes SAS-6 protein levels. Disruption of this regulatory network results in the overexpression of SAS-6 and the production of extra centrioles. Full Text

Jaenisch R. (2022). Beatrice Mintz, a giant in mammalian development. PNAS 119 : e2201631119. Beatrice Mintz, known as “Bea” to her friends, was a developmental geneticist. Her approach to science has been deemed as “innovative, unprecedented, extraordinary, unequaled”. Her pioneering work had a major impact on many different areas of science. She began her career addressing one of the most complex and fascinating questions of development: how the many different and diverse tissues in an organism are initiated and develop from a single fertilized egg. Full Text

Jones, M.G., Rosen, Y., and Yosef, N. (2022). Interactive, integrated analysis of single-cell transcriptomic and phylogenetic data with PhyloVision. Cell Reports Methods 2(4):100200. Recent advances in CRISPR-Cas9 engineering and single-cell assays have enabled the simultaneous measurement of single-cell transcriptomic and phylogenetic profiles. However, there are few computational tools enabling users to integrate and derive insight from a joint analysis of these two modalities. Here, we describe "PhyloVision": an open-source software for interactively exploring data from both modalities and for identifying and interpreting heritable gene modules whose concerted expression are associated with phylogenetic relationships. PhyloVision provides a feature-rich, interactive, and shareable web-based report for investigating these modules while also supporting several other data and meta-data exploration capabilities. We demonstrate the utility of PhyloVision using a published dataset of metastatic lung adenocarcinoma cells, whose phylogeny was resolved using a CRISPR-Cas9-based lineage-tracing system. Together, we anticipate that PhyloVision and the methods it implements will be a useful resource for scalable and intuitive data exploration for any assay that simultaneously measures cell state and lineage. Full Text

Joung, J., Kirchgatterer, P.C., Singh, A., Cho, J.H., Nety, S.P., Larson, R.C., Macrae, R.K., Deasy, R., Tseng, Y.Y., Maus, M.V., et al. (2022). CRISPR activation screen identifies BCL-2 proteins and B3GNT2 as drivers of cancer resistance to T cell-mediated cytotoxicity. Nature Communications 13(1):1606. The cellular processes that govern tumor resistance to immunotherapy remain poorly understood. To gain insight into these processes, here we perform a genome-scale CRISPR activation screen for genes that enable human melanoma cells to evade cytotoxic T cell killing. Overexpression of four top candidate genes (CD274 (PD-L1), MCL1, JUNB, and B3GNT2) conferred resistance in diverse cancer cell types and mouse xenografts. By investigating the resistance mechanisms, we find that MCL1 and JUNB modulate the mitochondrial apoptosis pathway. JUNB encodes a transcription factor that downregulates FasL and TRAIL receptors, upregulates the MCL1 relative BCL2A1, and activates the NF-κB pathway. B3GNT2 encodes a poly-N-acetyllactosamine synthase that targets >10 ligands and receptors to disrupt interactions between tumor and T cells and reduce T cell activation. Inhibition of candidate genes sensitized tumor models to T cell cytotoxicity. Our results demonstrate that systematic gain-of-function screening can elucidate resistance pathways and identify potential targets for cancer immunotherapy. Full Text

Kasprzyk, P.G., Vickery, C., Ye, M., Sewastianik, M., Gong, W., Ding, S., Dziwenka, M., Mozingo, A., Valm, K., Schachner, H., and Jing-Ke Weng (2022). Safety of a Sustainably Produced, Bioengineered, Nature-Identical Salidroside Compound. Nutrients 14(11) : 2330. Bioactive phytochemicals such as salidroside have been studied to understand the beneficial effects of Rhodiola rosea, an herbaceous plant used in traditional medicine to increase energy and treat a variety of health issues. However, Rhodiola plants are often slow-growing, and many are endangered in their native habitats. Thus, there is a need for safe, alternative supplies of key phytochemicals from Rhodiola. The salidroside subject of this safety study is a synthetic biology product from fermentation of a bioengineered E. coli that produces salidroside. Here, we present comprehensive test results that support the safety of salidroside manufactured via a patented sustainable bioengineering manufacturing process. In vitro bacterial reverse mutation assays with the bioengineered salidroside show no mutagenicity in any of the concentrations tested. In vivo toxicity studies in rats show no adverse effects from the salidroside product. Based on the results of these studies, we conclude that the bioengineered salidroside discussed here is not genotoxic and demonstrates a no-observed-adverse-effect level (NOAEL) at least 2000 mg/kg bw/day in male and female Sprague-Dawley rats. This study supports that the salidroside compound produced using bioengineered E. coli is a viable alternative to salidroside produced from harvested Rhodiola plants for use as a dietary supplement, food ingredient, or potentially as a pharmaceutical product. Full Text

Kataura, T., Sedlackova, L., Otten, E.G., Kumari, R., Shapira, D., Scialo, F., Stefanatos, R., Ishikawa, K.I., Kelly, G., Seranova, E., Dorothea Maetzel , Rudolf Jaenisch, et al. (2022). Autophagy promotes cell survival by maintaining NAD levels. Developmental Cell 57 (22): 2584-2598. Autophagy is an essential catabolic process that promotes the clearance of surplus or damaged intracellular components. Loss of autophagy in age-related human pathologies contributes to tissue degeneration through a poorly understood mechanism. Here, we identify an evolutionarily conserved role of autophagy from yeast to humans in the preservation of nicotinamide adenine dinucleotide (NAD) levels, which are critical for cell survival. In respiring mouse fibroblasts with autophagy deficiency, loss of mitochondrial quality control was found to trigger hyperactivation of stress responses mediated by NADases of PARP and Sirtuin families. Uncontrolled depletion of the NAD(H) pool by these enzymes ultimately contributed to mitochondrial membrane depolarization and cell death. Pharmacological and genetic interventions targeting several key elements of this cascade improved the survival of autophagy-deficient yeast, mouse fibroblasts, and human neurons. Our study provides a mechanistic link between autophagy and NAD metabolism and identifies targets for interventions in human diseases associated with autophagic, lysosomal, and mitochondrial dysfunction. Full Text

Kersten, R.D., Mydy, L.S., Fallon, T.R., de Waal, F., Shafiq, K., Wotring, J.W., Sexton, J.Z., and Weng, J.K. (2022). Gene-Guided Discovery and Ribosomal Biosynthesis of Moroidin Peptides. Journal of the American Chemical Society. Online Ahead of Print. Moroidin is a bicyclic plant octapeptide with tryptophan side-chain cross-links, originally isolated as a pain-causing agent from the Australian stinging tree Dendrocnide moroides. Moroidin and its analog celogentin C, derived from Celosia argentea, are inhibitors of tubulin polymerization and, thus, lead structures for cancer therapy. However, low isolation yields from source plants and challenging organic synthesis hinder moroidin-based drug development. Here, we present biosynthesis as an alternative route to moroidin-type bicyclic peptides and report that they are ribosomally synthesized and posttranslationally modified peptides (RiPPs) derived from BURP-domain peptide cyclases in plants. By mining 793 plant transcriptomes for moroidin core peptide motifs within BURP-domain precursor peptides, we identified a moroidin cyclase in Japanese kerria, which catalyzes the installation of the tryptophan-indole-centered macrocyclic bonds of the moroidin bicyclic motif in the presence of cupric ions. Based on the kerria moroidin cyclase, we demonstrate the feasibility of producing diverse moroidins including celogentin C in transgenic tobacco plants and report specific cytotoxicity of celogentin C against a lung adenocarcinoma cancer cell line. Our study sets the stage for future biosynthetic development of moroidin-based therapeutics and highlights that mining plant transcriptomes can reveal bioactive cyclic peptides and their underlying cyclases from new source plants. Full Text

Keys, H.R., and Knouse, K.A. (2022). Genome-scale CRISPR screening in a single mouse liver. Cell Genomics 2(12):100217. A complete understanding of the genetic determinants underlying mammalian physiology and disease is limited by the capacity for high-throughput genetic dissection in the living organism. Genome-wide CRISPR screening is a powerful method for uncovering the genetic regulation of cellular processes, but the need to stably deliver single guide RNAs to millions of cells has largely restricted its implementation to ex vivo systems. There thus remains a need for accessible high-throughput functional genomics in vivo. Here, we establish genome-wide screening in the liver of a single mouse and use this approach to uncover regulation of hepatocyte fitness. We uncover pathways not identified in cell culture screens, underscoring the power of genetic dissection in the organism. The approach we developed is accessible, scalable, and adaptable to diverse phenotypes and applications. We have hereby established a foundation for high-throughput functional genomics in a living mammal, enabling comprehensive investigation of physiology and disease. Full Text

Kilgore, H.R., and Young, R.A. (2022). Learning the chemical grammar of biomolecular condensates. Nature Chemical Biology. Online Ahead of Print. Biomolecular condensates compartmentalize and regulate assemblies of biomolecules engaged in vital physiological processes in cells. Specific proteins and nucleic acids engaged in shared functions occur in any one kind of condensate, suggesting that these compartments have distinct chemical specificities. Indeed, some small-molecule drugs concentrate in specific condensates due to chemical properties engendered by particular amino acids in the proteins in those condensates. Here we argue that the chemical properties that govern molecular interactions between a small molecule and biomolecules within a condensate can be ascertained for both the small molecule and the biomolecules. We propose that learning this 'chemical grammar', the rules describing the chemical features of small molecules that engender attraction or repulsion by the physicochemical environment of a specific condensate, should enable design of drugs with improved efficacy and reduced toxicity. Full Text

Kingston, E.R., Blodgett, L.W., and Bartel, D.P. (2022). Endogenous transcripts direct microRNA degradation in Drosophila, and this targeted degradation is required for proper embryonic development. Molecular Cell. Online Ahead of Print. MicroRNAs (miRNAs) typically direct degradation of their mRNA targets. However, some targets have unusual miRNA-binding sites that direct degradation of cognate miRNAs. Although this target-directed miRNA degradation (TDMD) is thought to shape the levels of numerous miRNAs, relatively few sites that endogenously direct degradation have been identified. Here, we identify six sites, five in mRNAs and one in a noncoding RNA named Marge, which serve this purpose in Drosophila cells or embryos. These six sites direct miRNA degradation without collateral target degradation, helping explain the effectiveness of this miRNA-degradation pathway. Mutations that disrupt this pathway are lethal, with many flies dying as embryos. Concomitant derepression of miR-3 and its paralog miR-309 appears responsible for some of this lethality, whereas the loss of Marge-directed degradation of miR-310 miRNAs causes defects in embryonic cuticle development. Thus, TDMD is implicated in the viability of an animal and is required for its proper development. Full Text

Kong, J.H., Vasquez, C.G., Agrawal, S., Malaney, P., Mikedis, M.M., Moffitt, A.B., von Diezmann, L., and Termini, C.M. (2022). Creating accessibility in academic negotiations. Trends in Biochemical Sciences. Online Ahead of Print. The process of evaluating and negotiating a tenure-track job offer is unstructured and highly variable, making it susceptible to bias and inequitable outcomes. We outline common aspects of and recommendations for negotiating an academic job offer in the life sciences to support equitable recruitment of diverse faculty. Full Text

Krzisch, M.A., Yuan, B., Whitfield, T.W., Fu, D., Garrett-Engele, C.M., Khalil, A.S., Lungjangwa, T., Kristin R Andrykovich , Rosalie G J Rietjens , Bhav Jain, Rudolf Jaenisch , et al. (2022). Fragile X Syndrome Patient-Derived Neurons Developing in the Mouse Brain Show FMR1-Dependent Phenotypes. Biological Psychiatry. Online Ahead of Print. Fragile X syndrome (FXS) is characterized by physical abnormalities, anxiety, intellectual disability, hyperactivity, autistic behaviors, and seizures. Abnormal neuronal development in FXS is poorly understood. Data on patients with FXS remain scarce, and FXS animal models have failed to yield successful therapies. In vitro models do not fully recapitulate the morphology and function of human neurons. METHODS: To mimic human neuron development in vivo, we coinjected neural precursor cells derived from FXS patient-derived induced pluripotent stem cells and neural precursor cells derived from corrected isogenic control induced pluripotent stem cells into the brain of neonatal immune-deprived mice. RESULTS: The transplanted cells populated the brain and a proportion differentiated into neurons and glial cells. Immunofluorescence and single and bulk RNA sequencing analyses showed accelerated maturation of FXS neurons after an initial delay. Additionally, we found increased percentages of Arc- and Egr-1-positive FXS neurons and wider dendritic protrusions of mature FXS striatal medium spiny neurons. CONCLUSIONS: This transplantation approach provides new insights into the alterations of neuronal development in FXS by facilitating physiological development of cells in a 3-dimensional context. Full Text

Kumon, T., and Lampson, M.A. (2022). Evolution of eukaryotic centromeres by drive and suppression of selfish genetic elements. Seminars in Cell & Developmental Biology. Online Ahead of Print. Despite the universal requirement for faithful chromosome segregation, eukaryotic centromeres are rapidly evolving. It is hypothesized that rapid centromere evolution represents an evolutionary arms race between selfish genetic elements that drive, or propagate at the expense of organismal fitness, and mechanisms that suppress fitness costs. Selfish centromere DNA achieves preferential inheritance in female meiosis by recruiting more effector proteins that alter spindle microtubule interaction dynamics. Parallel pathways for effector recruitment are adaptively evolved to suppress functional differences between centromeres. Opportunities to drive are not limited to female meiosis, and selfish transposons, plasmids and B chromosomes also benefit by maximizing their inheritance. Rapid evolution of selfish genetic elements can diversify suppressor mechanisms in different species that may cause hybrid incompatibility. Full Text

Lambert, A.W., Fiore, C., Chutake, Y., Verhaar, E.R., Strasser, P.C., Chen, M.W., Farouq, D., Das, S., Li, X., Eaton, E.N., Robert A. Weinberg, et al, (2022). ΔNp63/p73 drive metastatic colonization by controlling a regenerative epithelial stem cell program in quasi-mesenchymal cancer stem cells. Developmental Cell 57(24): 2714-2730. Cancer stem cells (CSCs) may serve as the cellular seeds of tumor recurrence and metastasis, and they can be generated via epithelial-mesenchymal transitions (EMTs). Isolating pure populations of CSCs is difficult because EMT programs generate multiple alternative cell states, and phenotypic plasticity permits frequent interconversions between these states. Here, we used cell-surface expression of integrin β4 (ITGB4) to isolate highly enriched populations of human breast CSCs, and we identified the gene regulatory network operating in ITGB4(+) CSCs. Specifically, we identified ΔNp63 and p73, the latter of which transactivates ΔNp63, as centrally important transcriptional regulators of quasi-mesenchymal CSCs that reside in an intermediate EMT state. We found that the transcriptional program controlled by ΔNp63 in CSCs is largely distinct from the one that it orchestrates in normal basal mammary stem cells and, instead, it more closely resembles a regenerative epithelial stem cell response to wounding. Moreover, quasi-mesenchymal CSCs repurpose this program to drive metastatic colonization via autocrine EGFR signaling. Full Text

Lan, T.C.T., Allan, M.F., Malsick, L.E., Woo, J.Z., Zhu, C., Zhang, F., Khandwala, S., Nyeo, S.S.Y., Sun, Y., Guo, J.U., Rouskin, S., et al. (2022). Secondary structural ensembles of the SARS-CoV-2 RNA genome in infected cells. Nature Communications 13(1):1128. SARS-CoV-2 is a betacoronavirus with a single-stranded, positive-sense, 30-kilobase RNA genome responsible for the ongoing COVID-19 pandemic. Although population average structure models of the genome were recently reported, there is little experimental data on native structural ensembles, and most structures lack functional characterization. Here we report secondary structure heterogeneity of the entire SARS-CoV-2 genome in two lines of infected cells at single nucleotide resolution. Our results reveal alternative RNA conformations across the genome and at the critical frameshifting stimulation element (FSE) that are drastically different from prevailing population average models. Importantly, we find that this structural ensemble promotes frameshifting rates much higher than the canonical minimal FSE and similar to ribosome profiling studies. Our results highlight the value of studying RNA in its full length and cellular context. The genomic structures detailed here lay groundwork for coronavirus RNA biology and will guide the design of SARS-CoV-2 RNA-based therapeutics. Full Text

Laqtom, N.N., Dong, W., Medoh, U.N., Cangelosi, A.L., Dharamdasani, V., Chan, S.H., Kunchok, T., Lewis, C.A., Heinze, I., Tang, R., et al. (2022). CLN3 is required for the clearance of glycerophosphodiesters from lysosomes. Nature 609(7929):1005-1011. Lysosomes have many roles, including degrading macromolecules and signalling to the nucleus(1). Lysosomal dysfunction occurs in various human conditions, such as common neurodegenerative diseases and monogenic lysosomal storage disorders (LSDs)(2-4). For most LSDs, the causal genes have been identified but, in some, the function of the implicated gene is unknown, in part because lysosomes occupy a small fraction of the cellular volume so that changes in lysosomal contents are difficult to detect. Here we develop the LysoTag mouse for the tissue-specific isolation of intact lysosomes that are compatible with the multimodal profiling of their contents. We used the LysoTag mouse to study CLN3, a lysosomal transmembrane protein with an unknown function. In children, the loss of CLN3 causes juvenile neuronal ceroid lipofuscinosis (Batten disease), a lethal neurodegenerative LSD. Untargeted metabolite profiling of lysosomes from the brains of mice lacking CLN3 revealed a massive accumulation of glycerophosphodiesters (GPDs)-the end products of glycerophospholipid catabolism. GPDs also accumulate in the lysosomes of CLN3-deficient cultured cells and we show that CLN3 is required for their lysosomal egress. Loss of CLN3 also disrupts glycerophospholipid catabolism in the lysosome. Finally, we found elevated levels of glycerophosphoinositol in the cerebrospinal fluid of patients with Batten disease, suggesting the potential use of glycerophosphoinositol as a disease biomarker. Our results show that CLN3 is required for the lysosomal clearance of GPDs and reveal Batten disease as a neurodegenerative LSD with a defect in glycerophospholipid metabolism. Full Text

Li, D., Gandhi, D., Kumon, T., and Yamashita, Y.M. (2022). Ribosomal DNA Instability as a Potential Cause of Karyotype Evolution. Molecular Biology & Evolution 39(11):msac221. Karyotype refers to the configuration of the genome into a set of chromosomes. The karyotype difference between species is expected to impede various biological processes, such as chromosome segregation and meiotic chromosome pairing, potentially contributing to incompatibility. Karyotypes can rapidly change between closely related species and even among populations of the same species. However, the forces driving karyotype evolution are poorly understood. Here we describe a unique karyotype of a Drosophila melanogaster strain isolated from the Seychelles archipelago. This strain has lost the ribosomal DNA (rDNA) locus on the X chromosome. Because the Y chromosome is the only other rDNA-bearing chromosome, all females carry at least one Y chromosome as the source of rDNA. Interestingly, we found that the strain also carries a truncated Y chromosome (YS) that is stably maintained in the population despite its inability to support male fertility. Our modeling and cytological analysis suggest that the Y chromosome has a larger negative impact on female fitness than the YS chromosome. Moreover, we generated an independent strain that lacks X rDNA and has a karyotype of XXY females and XY males. This strain quickly evolved multiple karyotypes: two new truncated Y chromosomes (similar to YS), as well as two independent X chromosome fusions that contain the Y-derived rDNA fragment, eliminating females' dependence on the Y chromosome. Considering that Robertsonian fusions frequently occur at rDNA loci in humans, we propose that rDNA loci instability may be one of driving forces of karyotype evolution. Full Text

Li, H., Namburi, P., Olson, J.M., Borio, M., Lemieux, M.E., Beyeler, A., Calhoon, G.G., Hitora-Imamura, N., Coley, A.A., Libster, A., King, H.O., et al. (2022). Neurotensin orchestrates valence assignment in the amygdala. Nature 608(7923):586-592. The ability to associate temporally segregated information and assign positive or negative valence to environmental cues is paramount for survival. Studies have shown that different projections from the basolateral amygdala (BLA) are potentiated following reward or punishment learning(1-7). However, we do not yet understand how valence-specific information is routed to the BLA neurons with the appropriate downstream projections, nor do we understand how to reconcile the sub-second timescales of synaptic plasticity(8-11) with the longer timescales separating the predictive cues from their outcomes. Here we demonstrate that neurotensin (NT)-expressing neurons in the paraventricular nucleus of the thalamus (PVT) projecting to the BLA (PVT-BLA:NT) mediate valence assignment by exerting NT concentration-dependent modulation in BLA during associative learning. We found that optogenetic activation of the PVT-BLA:NT projection promotes reward learning, whereas PVT-BLA projection-specific knockout of the NT gene (Nts) augments punishment learning. Using genetically encoded calcium and NT sensors, we further revealed that both calcium dynamics within the PVT-BLA:NT projection and NT concentrations in the BLA are enhanced after reward learning and reduced after punishment learning. Finally, we showed that CRISPR-mediated knockout of the Nts gene in the PVT-BLA pathway blunts BLA neural dynamics and attenuates the preference for active behavioural strategies to reward and punishment predictive cues. In sum, we have identified NT as a neuropeptide that signals valence in the BLA, and showed that NT is a critical neuromodulator that orchestrates positive and negative valence assignment in amygdala neurons by extending valence-specific plasticity to behaviourally relevant timescales. Full Text

Li X., Gordon, P.J., Gaynes, J.A., Fuller, A.W., Ringuette, R., Santiago, C.P., Wallace, V., Blackshaw, S., Li, P., and Levine, E.M. (2022). Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis. eLife 11:e78342. An important question in organogenesis is how tissue-specific transcription factors interact with signaling pathways. In some cases, transcription factors define the context for how signaling pathways elicit tissue- or cell-specific responses, and in others, they influence signaling through transcriptional regulation of signaling components or accessory factors. We previously showed that during optic vesicle patterning, the Lim-homeodomain transcription factor Lhx2 has a contextual role by linking the Sonic Hedgehog (Shh) pathway to downstream targets without regulating the pathway itself. Here, we show that during early retinal neurogenesis in mice, Lhx2 is a multilevel regulator of Shh signaling. Specifically, Lhx2 acts cell autonomously to control the expression of pathway genes required for efficient activation and maintenance of signaling in retinal progenitor cells. The Shh co-receptors Cdon and Gas1 are candidate direct targets of Lhx2 that mediate pathway activation, whereas Lhx2 directly or indirectly promotes the expression of other pathway components important for activation and sustained signaling. We also provide genetic evidence suggesting that Lhx2 has a contextual role by linking the Shh pathway to downstream targets. Through these interactions, Lhx2 establishes the competence for Shh signaling in retinal progenitors and the context for the pathway to promote early retinal neurogenesis. The temporally distinct interactions between Lhx2 and the Shh pathway in retinal development illustrate how transcription factors and signaling pathways adapt to meet stage-dependent requirements of tissue formation. Full Text

Lin, B., Luo, J., and Lehmann, R. (2022). An AMPK phosphoregulated RhoGEF feedback loop tunes cortical flow-driven amoeboid migration in vivo. Science Advances 8(37) : eabo0323. Development, morphogenesis, immune system function, and cancer metastasis rely on the ability of cells to move through diverse tissues. To dissect migratory cell behavior in vivo, we developed cell type-specific imaging and perturbation techniques for Drosophila primordial germ cells (PGCs). We find that PGCs use global, retrograde cortical actin flows for orientation and propulsion during guided developmental homing. PGCs use RhoGEF2, a RhoA-specific RGS-RhoGEF, as a dose-dependent regulator of cortical flow through a feedback loop requiring its conserved PDZ and PH domains for membrane anchoring and local RhoA activation. This feedback loop is regulated for directional migration by RhoGEF2 availability and requires AMPK rather than canonical Gα(12/13) signaling. AMPK multisite phosphorylation of RhoGEF2 near a conserved EB1 microtubule-binding SxIP motif releases RhoGEF2 from microtubule-dependent inhibition. Thus, we establish the mechanism by which global cortical flow and polarized RhoA activation can be dynamically adapted during natural cell navigation in a changing environment. Full Text

Ling, L., Li, F., Yang, P., Oates, R.D., Silber, S., Kurischko, C., Luca, F.C., Leu, N.A., Zhang, J., Yue, Q., Helen Skaletsky, Laura G Brown, David C Page, et al. (2022). Genetic characterization of a missense mutation in the X-linked TAF7L gene identified in an oligozoospermic man. Biology of Reproduction.Online Ahead of Print. While hundreds of knockout mice show infertility as a major phenotype, causative genic mutations of male infertility in humans remain rather limited. Here we report the identification of a missense mutation (D136G) in the X-linked TAF7L gene as a potential cause of oligozoospermia in men. The human aspartate (D136) is evolutionally conserved across species, and its change to glycine (G) is predicted to be detrimental. Genetic complementation experiments in budding yeast demonstrate that the conserved aspartate or its analogous asparagine (N) residue in yeast TAF7 is essential for cell viability and thus its mutation to glycine is lethal. Although the corresponding D144G substitution in the mouse Taf7l gene does not affect male fertility, RNA-seq analyses reveal alterations in transcriptome profiles in the Taf7l (D144G) mutant testes. These results support this TAF7L mutation as a risk factor for oligozoospermia in humans. Full Text

Ma, H., de Zwaan, E., Guo, Y.E., Cejas, P., Thiru, P., van de Bunt, M., Jeppesen, J.F.Alessandra Dall'Agnese  , Brian J Abraham  , Dongdong Fu  , Carrie Garrett-Engele  , Tong Ihn Lee , Syamala, S., Dall'Agnese, A., Abraham, B.J., Linda G Griffith, and Richard A Young  , Rudolf Jaenisch   (2022). The nuclear receptor THRB facilitates differentiation of human PSCs into more mature hepatocytes. Cell Stem Cell. Online Ahead of Print. To understand the mechanisms regulating the in vitro maturation of hPSC-derived hepatocytes, we developed a 3D differentiation system and compared gene regulatory elements in human primary hepatocytes with those in hPSC-hepatocytes that were differentiated in 2D or 3D conditions by RNA-seq, ATAC-seq, and H3K27Ac ChIP-seq. Regulome comparisons showed a reduced enrichment of thyroid receptor THRB motifs in accessible chromatin and active enhancers without a reduced transcription of THRB. The addition of thyroid hormone T3 increased the binding of THRB to the CYP3A4 proximal enhancer, restored the super-enhancer status and gene expression of NFIC, and reduced the expression of AFP. The resultant hPSC-hepatocytes showed gene expression, epigenetic status, and super-enhancer landscape closer to primary hepatocytes and activated regulatory regions including non-coding SNPs associated with liver-related diseases. Transplanting the hPSC-hepatocytes resulted in the engraftment of human hepatocytes into the mouse liver without disrupting normal liver histology. This work implicates the environmental factor-nuclear receptor axis in regulating the maturation of hPSC-hepatocytes. Full Text

McGeary S.E., Bisaria, N., Pham, T.M., Wang, P.Y., and Bartel, D.P. (2022). MicroRNA 3'-compensatory pairing occurs through two binding modes, with affinity shaped by nucleotide identity and position. eLife 11 : e69803. MicroRNAs (miRNAs), in association with Argonaute (AGO) proteins, direct repression by pairing to sites within mRNAs. Compared to pairing preferences of the miRNA seed region (nucleotides 2-8), preferences of the miRNA 3' region are poorly understood, due to the sparsity of measured affinities for the many pairing possibilities. We used RNA bind-n-seq with purified AGO2-miRNA complexes to measure relative affinities of >1000 3'-pairing architectures for each miRNA. In some cases, optimal 3' pairing increased affinity by >500 fold. Some miRNAs had two high-affinity 3'-pairing modes-one of which included additional nucleotides bridging seed and 3' pairing to enable high-affinity pairing to miRNA nucleotide 11. The affinity of binding and the position of optimal pairing both tracked with the occurrence of G or oligo(G/C) nucleotides within the miRNA. These and other results advance understanding of miRNA targeting, providing insight into how optimal 3' pairing is determined for each miRNA. Full Text

Morales, J., Allegakoen, D.V., Garcia, J.A., Kwong, K., Sahu, P.K., Fajardo, D.A., Pan, Y., Horlbeck, M.A., Guna, A., Jonathan S Weissman, Gustafson, W.C., et al. (2022). GATOR2-dependent mTORC1 activity is a therapeutic vulnerability in FOXO1 fusion-positive rhabdomyosarcoma. JCI Insight 7(23):e162207. Oncogenic FOXO1 gene fusions drive a subset of rhabdomyosarcoma (RMS) with poor survival; to date, these cancer drivers are therapeutically intractable. To identify new therapies for this disease, we undertook an isogenic CRISPR-interference screen to define PAX3-FOXO1-specific genetic dependencies and identified genes in the GATOR2 complex. GATOR2 loss in RMS abrogated aa-induced lysosomal localization of mTORC1 and consequent downstream signaling, slowing G1-S cell cycle transition. In vivo suppression of GATOR2 impaired the growth of tumor xenografts and favored the outgrowth of cells lacking PAX3-FOXO1. Loss of a subset of GATOR2 members can be compensated by direct genetic activation of mTORC1. RAS mutations are also sufficient to decouple mTORC1 activation from GATOR2, and indeed, fusion-negative RMS harboring such mutations exhibit aa-independent mTORC1 activity. A bisteric, mTORC1-selective small molecule induced tumor regressions in fusion-positive patient-derived tumor xenografts. These findings highlight a vulnerability in FOXO1 fusion-positive RMS and provide rationale for the clinical evaluation of bisteric mTORC1 inhibitors, currently in phase I testing, to treat this disease. Isogenic genetic screens can, thus, identify potentially exploitable vulnerabilities in fusion-driven pediatric cancers that otherwise remain mostly undruggable. Full Text

Morelli, E., Fulciniti, M., Samur, M.K., Ribeiro, C., Wert-Lamas, L., Henninger, J.E., Gulla, A., Aktas Samur, A., Todoerti, K., Talluri, S., Richard Young, et al. (2022). A MIR17HG-derived Long Noncoding RNA Provides an Essential Chromatin Scaffold for Protein Interaction and Myeloma Growth. Blood. Online Ahead of Print. Long noncoding RNAs (lncRNA) can drive tumorigenesis and are susceptible to therapeutic intervention. Here, we used a large-scale CRISPR interference viability screen to interrogate cell growth dependency to lncRNA genes in multiple myeloma (MM), and identified a prominent role for the miR-17-92 cluster host gene (MIR17HG). We show that a MIR17HG-derived lncRNA, named lnc-17-92, is the main mediator of cell growth dependency acting in a microRNA- and DROSHA- independent manner. Lnc-17-92 provides a chromatin scaffold for the functional interaction between c-MYC and WDR82, thus promoting the expression of ACACA, which encodes the rate-limiting enzyme of de novo lipogenesis acetyl-coA carboxylase 1 (ACC1). Targeting MIR17HG pre-RNA with clinically applicable antisense molecules disrupts the transcriptional and functional activities of lnc-17-92, causing potent anti-tumor effects both in vitro and in vivo in three pre-clinical animal models, including a clinically relevant PDX-NSG mouse model. This study establishes a novel oncogenic function of MIR17HG and provides potent inhibitors for translation to clinical trials. Full Text

Mudge, J.M., Ruiz-Orera, J., Prensner, J.R., Brunet, M.A., Calvet, F., Jungreis, I., Gonzalez, J.M., Magrane, M., Martinez, T.F., Schulz, J.F., Jonathan S Weissman, et al. (2022). Standardized annotation of translated open reading frames. Nature Biotechnology 40, 994-999. Ribosome profiling (Ribo-seq) has extended our understanding of the translational ‘vocabulary’ of the human genome, uncovering thousands of open reading frames (ORFs) within long noncoding RNAs (lncRNAs) and presumed untranslated regions (UTRs) of protein-coding genes. However, reference gene annotation projects have been circumspect in their incorporation of these ORFs because of uncertainties about their experimental reproducibility and physiological roles. Yet, it is clear that certain ‘Ribo-seq ORFs’ make stable proteins, others mediate gene regulation, and many have medical implications. Ultimately, the absence of standardized ORF annotation has created a circular problem: while Ribo-seq ORFs remain unrecognized by reference annotation databases, this lack of recognition will thwart studies examining their roles. Here, we outline a community-led effort involving Ensembl/GENCODE, the HUGO Gene Nomenclature Committee (HGNC), UniProtKB, HUPO/HPP and PeptideAtlas to produce a standardized catalog of 7,264 human Ribo-seq ORFs; a path to bring protein-level evidence for Ribo-seq ORFs into reference annotation databases; and a roadmap to facilitate research in the global community. Full Text

Naranjo, S., Cabana, C.M., LaFave, L.M., Romero, R., Shanahan, S.L., Bhutkar, A., Westcott, P.M.K., Schenkel, J.M., Ghosh, A., Liao, L.Z., Dian Yang, et al. (2022). Modeling diverse genetic subtypes of lung adenocarcinoma with a next-generation alveolar type 2 organoid platform. Genes & Development. Online Ahead of Print. Lung cancer is the leading cause of cancer-related death worldwide. Lung adenocarcinoma (LUAD), the most common histological subtype, accounts for 40% of all cases. While existing genetically engineered mouse models (GEMMs) recapitulate the histological progression and transcriptional evolution of human LUAD, they are time-consuming and technically demanding. In contrast, cell line transplant models are fast and flexible, but these models fail to capture the full spectrum of disease progression. Organoid technologies provide a means to create next-generation cancer models that integrate the most advantageous features of autochthonous and transplant-based systems. However, robust and faithful LUAD organoid platforms are currently lacking. Here, we describe optimized conditions to continuously expand murine alveolar type 2 (AT2) cells, a prominent cell of origin for LUAD, in organoid culture. These organoids display canonical features of AT2 cells, including marker gene expression, the presence of lamellar bodies, and an ability to differentiate into the AT1 lineage. We used this system to develop flexible and versatile immunocompetent organoid-based models of KRAS, BRAF, and ALK mutant LUAD. Notably, organoid-based tumors display extensive burden and complete penetrance and are histopathologically indistinguishable from their autochthonous counterparts. Altogether, this organoid platform is a powerful, versatile new model system to study LUAD. Full Text

Navarro, A.P., and Cheeseman, I.M. (2022). Identification of a Golgi-localized peptide reveals a minimal Golgi targeting motif. Molecular Biology of the Cell. Online Ahead of Print. Prior work has identified signal sequences and motifs that are necessary and sufficient to target proteins to specific subcellular regions and organelles such as the plasma membrane, nucleus, Endoplasmic Reticulum, and mitochondria. In contrast, minimal sequence motifs that are sufficient for Golgi localization remain largely elusive. In this work, we identified a 37 amino acid alternative open reading frame (altORF) within the mRNA of the centromere protein, CENP-R. This altORF peptide localizes specifically to the cytoplasmic surface of the Golgi apparatus. Through mutational analysis, we identify a minimal 10 amino acid sequence and a critical cysteine residue that are necessary and sufficient for Golgi localization. Pharmacological perturbations suggest that this peptide undergoes lipid modification to promote its localization. Together, our work defines a minimal sequence that is sufficient for Golgi targeting and provide a valuable Golgi marker for live cell imaging. Full Text

Navarro, A.P., and Cheeseman, I.M. (2022). Dynamic cell cycle-dependent phosphorylation modulates CENP-L-CENP-N centromere recruitment. Molecular Biology of the Cell 40(7) : 994-999. The kinetochore is a macromolecular structure that is required to ensure proper chromosome segregation during each cellular division. The kinetochore is assembled upon a platform of the 16-subunit Constitutive Centromere Associated Network (CCAN), which is present at centromeres throughout the cell cycle. The nature and regulation of CCAN assembly, interactions, and dynamics required to facilitate changing centromere properties and requirements remain to be fully elucidated. The CENP-LN complex is a CCAN component that displays a unique cell cycle-dependent localization behavior, peaking in S phase. Here, we demonstrate that phosphorylation of CENP-L and CENP-N controls CENP-LN complex formation and localization in a cell cycle-dependent manner. Mimicking constitutive phosphorylation of either CENP-L or CENP-N or simultaneously preventing phosphorylation of both proteins prevents CENP-LN localization and disrupts chromosome segregation. Together, our work suggests that cycles of phosphorylation and dephosphorylation are critical for CENP-LN complex recruitment and dynamics at kinetochores to enable cell cycle-dependent CCAN reorganization. Full Text

Neumayr, C., Haberle, V., Serebreni, L., Karner, K., Hendy, O., Boija, A., Henninger, J.E., Li, C.H., Stejskal, K., Lin, G., et al. (2022). Differential cofactor dependencies define distinct types of human enhancers. Nature. Online Ahead of Print. All multicellular organisms rely on differential gene transcription regulated by genomic enhancers, which function through cofactors that are recruited by transcription factors(1,2). Emerging evidence suggests that not all cofactors are required at all enhancers(3-5), yet whether these observations reflect more general principles or distinct types of enhancers remained unknown. Here we categorized human enhancers by their cofactor dependencies and show that these categories provide a framework to understand the sequence and chromatin diversity of enhancers and their roles in different gene-regulatory programmes. We quantified enhancer activities along the entire human genome using STARR-seq(6) in HCT116 cells, following the rapid degradation of eight cofactors. This analysis identified different types of enhancers with distinct cofactor requirements, sequences and chromatin properties. Some enhancers were insensitive to the depletion of the core Mediator subunit MED14 or the bromodomain protein BRD4 and regulated distinct transcriptional programmes. In particular, canonical Mediator(7) seemed dispensable for P53-responsive enhancers, and MED14-depleted cells induced endogenous P53 target genes. Similarly, BRD4 was not required for the transcription of genes that bear CCAAT boxes and a TATA box (including histone genes and LTR12 retrotransposons) or for the induction of heat-shock genes. This categorization of enhancers through cofactor dependencies reveals distinct enhancer types that can bypass broadly utilized cofactors, which illustrates how alternative ways to activate transcription separate gene expression programmes and provide a conceptual framework to understand enhancer function and regulatory specificity. Full Text

Ng, T.S.C., Allen, H.H., Rashidian, M., and Miller, M.A. (2022). Probing immune infiltration dynamics in cancer by in vivo imaging. Current Opinion in Chemical Biology 67:102117. Cancer immunotherapies typically aim to stimulate the accumulation and activity of cytotoxic T-cells or pro-inflammatory antigen-presenting cells, reduce immunosuppressive myeloid cells or regulatory T-cells, or elicit some combination of effects thereof. Notwithstanding the encouraging results, immunotherapies such as PD-1/PD-L1-targeted immune checkpoint blockade act heterogeneously across individual patients. It remains challenging to predict and monitor individual responses, especially across multiple sites of metastasis or sites of potential toxicity. To address this need, in vivo imaging of both adaptive and innate immune cell populations has emerged as a tool to quantify spatial leukocyte accumulation in tumors non-invasively. Here we review recent progress in the translational development of probes for in vivo leukocyte imaging, focusing on complementary perspectives provided by imaging of T-cells, phagocytic macrophages, and their responses to therapy. Full Text

Pal, D., Visconti, F., Sepúlveda-Ramírez, S.P., Swartz, S.Z. , and Shuster, C.B. (2022). Use of Echinoderm Gametes and Early Embryos for Studying Meiosis and Mitosis. Methods in Molecular Biology 2415, 1-17. The early embryos of sea urchins and other echinoderms have served as experimental models for the study of cell division since the nineteenth century. Their rapid development, optical clarity, and ease of manipulation continue to offer advantages for studying spindle assembly and cytokinesis. In the absence of transgenic lines, alternative strategies must be employed to visualize microtubules and actin. Here, we describe methods to visualize actin and microtubule using either purified, recombinant proteins, or probes in in vitro-transcribed mRNAs. Full Text

Park, S.Y., Rao, C., Coyte, K.Z., Kuziel, G.A., Zhang, Y., Huang, W., Franzosa, E.A., Weng, J.K., Huttenhower, C., and Rakoff-Nahoum, S. (2022). Strain-level fitness in the gut microbiome is an emergent property of glycans and a single metabolite. Cell 185(3) : 513-529. The human gut microbiota resides within a diverse chemical environment challenging our ability to understand the forces shaping this ecosystem. Here, we reveal that fitness of the Bacteroidales, the dominant order of bacteria in the human gut, is an emergent property of glycans and one specific metabolite, butyrate. Distinct sugars serve as strain-variable fitness switches activating context-dependent inhibitory functions of butyrate. Differential fitness effects of butyrate within the Bacteroides are mediated by species-level variation in Acyl-CoA thioesterase activity and nucleotide polymorphisms regulating an Acyl-CoA transferase. Using in vivo multi-omic profiles, we demonstrate Bacteroides fitness in the human gut is associated together, but not independently, with Acyl-CoA transferase expression and butyrate. Our data reveal that each strain of the Bacteroides exists within a unique fitness landscape based on the interaction of chemical components unpredictable by the effect of each part alone mediated by flexibility in the core genome. Full Text

Pattwell, S.S., Arora, S., Nuechterlein, N., Zager, M., Loeb, K.R., Cimino, P.J., Holland, N.C., Reche-Ley, N., Bolouri, H., Almiron Bonnin, D.A., Vaishnavi V Phadnis, et al. (2022). Oncogenic role of a developmentally regulated NTRK2 splice variant. Science Advances 8(40) : eabo6789. Temporally regulated alternative splicing choices are vital for proper development, yet the wrong splice choice may be detrimental. Here, we highlight a previously unidentified role for the neurotrophin receptor splice variant TrkB.T1 in neurodevelopment, embryogenesis, transformation, and oncogenesis across multiple tumor types in humans and mice. TrkB.T1 is the predominant NTRK2 isoform across embryonic organogenesis, and forced overexpression of this embryonic pattern causes multiple solid and nonsolid tumors in mice in the context of tumor suppressor loss. TrkB.T1 also emerges as the predominant NTRK isoform expressed in a wide range of adult and pediatric tumors, including those harboring tropomyosin receptor kinase fusions. Affinity purification-mass spectrometry proteomic analysis reveals distinct interactors with known developmental and oncogenic signaling pathways such as Wnt, transforming growth factor-β, Sonic Hedgehog, and Ras. From alterations in splicing factors to changes in gene expression, the discovery of isoform specific oncogenes with embryonic ancestry has the potential to shape the way we think about developmental systems and oncology. Full Text

Povilus, R.A., and Friedman, W.E. (2022). Transcriptomes across fertilization and seed development in the water lily Nymphaea thermarum (Nymphaeales): evidence for epigenetic patterning during reproduction. Plant Reproduction. Online Ahead of Print. The first record of gene expression during seed development within the Nymphaeales provides evidence for a variety of biological processes, including dynamic epigenetic patterning during sexual reproduction in the water lily Nymphaea thermarum. Studies of gene expression during seed development have been performed for a growing collection of species from a phylogenetically broad sampling of flowering plants (angiosperms). However, angiosperm lineages whose origins predate the divergence of monocots and eudicots have been largely overlooked. In order to provide a new resource for understanding the early evolution of seed development in flowering plants, we sequenced transcriptomes of whole ovules and seeds from three key stages of reproductive development in the waterlily Nymphaea thermarum, an experimentally tractable member of the Nymphaeales. We first explore patterns of gene expression, beginning with mature ovules and continuing through fertilization into early- and mid-stages of seed development. We find patterns of gene expression that corroborate histological/morphological observations of seed development in this species, such as expression of genes involved in starch synthesis and transcription factors that have been associated with embryo and endosperm development in other species. We also find evidence for processes that were previously not known to be occurring during seed development in this species, such as epigenetic modification. We then examine the expression of genes associated with patterning DNA and histone methylation-processes that are essential for seed development in distantly related and structurally diverse monocots and eudicots. Around 89% of transcripts putatively homologous to DNA and histone methylation modifiers are expressed during seed development in N. thermarum, including homologs of genes known to pattern imprinting-related epigenetic modifications. Our results suggest that dynamic epigenetic patterning is a deeply conserved aspect of angiosperm seed development. Full Text

Qiu, X., Zhang, Y., Martin-Rufino, J.D., Weng, C., Hosseinzadeh, S., Yang, D., Pogson, A.N., Hein, M.Y., Hoi Joseph Min, K., Wang, L., Joseph M. Replogle, Jonathan S.Weissman, et al. (2022). Mapping transcriptomic vector fields of single cells. Cell. Online Ahead of Print. Single-cell (sc)RNA-seq, together with RNA velocity and metabolic labeling, reveals cellular states and transitions at unprecedented resolution. Fully exploiting these data, however, requires kinetic models capable of unveiling governing regulatory functions. Here, we introduce an analytical framework dynamo (https://github.com/aristoteleo/dynamo-release), which infers absolute RNA velocity, reconstructs continuous vector fields that predict cell fates, employs differential geometry to extract underlying regulations, and ultimately predicts optimal reprogramming paths and perturbation outcomes. We highlight dynamo's power to overcome fundamental limitations of conventional splicing-based RNA velocity analyses to enable accurate velocity estimations on a metabolically labeled human hematopoiesis scRNA-seq dataset. Furthermore, differential geometry analyses reveal mechanisms driving early megakaryocyte appearance and elucidate asymmetrical regulation within the PU.1-GATA1 circuit. Leveraging the least-action-path method, dynamo accurately predicts drivers of numerous hematopoietic transitions. Finally, in silico perturbations predict cell-fate diversions induced by gene perturbations. Dynamo, thus, represents an important step in advancing quantitative and predictive theories of cell-state transitions. Full Text

Reddien, P.W. (2022). Positional Information and Stem Cells Combine to Result in Planarian Regeneration. Cold Spring Harbor Perspectives in Biology 14(4):a040717. 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

Replogle, J.M., Bonnar, J.L., Pogson, A.N., Liem, C.R., Maier, N.K., Ding, Y., Russell, B.J., Wang, X., Leng, K., Guna, A., Jonathan S Weissman, et al. (2022). Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors. eLife 11:e81856. CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions in cell biology, genetics, and biotechnology, but wider deployment of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and challenges in generating cell models with consistent CRISPRi-mediated knockdown. Here, we present next-generation CRISPRi sgRNA libraries and effector expression constructs that enable strong and consistent knockdown across mammalian cell models. First, we combine empirical sgRNA selection with a dual-sgRNA library design to generate an ultra-compact (1-3 elements per gene), highly active CRISPRi sgRNA library. Next, we compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides an excellent balance between strong on-target knockdown and minimal nonspecific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines with stable expression of Zim3-dCas9 and robust on-target knockdown. Our results and publicly available reagents establish best practices for CRISPRi genetic screening. Full Text

Replogle, J.M., Saunders, R.A., Pogson, A.N., Hussmann, J.A., Lenail, A., Guna, A., Mascibroda, L., Wagner, E.J., Adelman, K., Lithwick-Yanai, G., Jonathan S. Weissman, et al. (2022). Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq. Cell. Online Ahead of Print. A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (CRISPR-based screens with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. Here, we perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.5 million human cells. We use transcriptional phenotypes to predict the function of poorly characterized genes, uncovering new regulators of ribosome biogenesis (including CCDC86, ZNF236, and SPATA5L1), transcription (C7orf26), and mitochondrial respiration (TMEM242). In addition to assigning gene function, single-cell transcriptional phenotypes allow for in-depth dissection of complex cellular phenomena-from RNA processing to differentiation. We leverage this ability to systematically identify genetic drivers and consequences of aneuploidy and to discover an unanticipated layer of stress-specific regulation of the mitochondrial genome. Our information-rich genotype-phenotype map reveals a multidimensional portrait of gene and cellular function. Full Text

Richards, A., Friesen, M., Khalil, A., Barrasa, M.I., Gehrke, L., and Jaenisch, R. (2022). SARS-CoV-2 infection of human pluripotent stem cell-derived liver organoids reveals potential mechanisms of liver pathology. iScience 25 (10) : 105146. Although respiratory symptoms are the most prevalent disease manifestation of infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), infection can also damage other organs, including the brain, gut, and liver. Symptoms of liver damage are observed in nearly half of patients that succumb to severe SARS-CoV-2 infection. Here we use human-induced pluripotent stem cell-derived liver organoids (HLOs) to recapitulate and characterize liver pathology following virus exposure. Utilizing single-cell sequencing technology, we identified robust transcriptomic changes that occur in SARS-CoV-2 infected liver cells as well as uninfected bystander cells. Our results show a significant induction of many inflammatory pathways, including IFN-α, INF-γ, and IL-6 signaling. Our results further identify IL-6 signaling as a potential mechanism for liver-mediated activation of circulating macrophages. Full Text

Samelson A.J., Tran, Q.D., Robinot, R., Carrau, L., Rezelj, V.V., Kain, A.M., Chen, M., Ramadoss, G.N., Guo, X., Lim, S.A., Jonathan S Weissman , et al. (2022). BRD2 inhibition blocks SARS-CoV-2 infection by reducing transcription of the host cell receptor ACE2. Nature Cell Biology 24(1):24-34. SARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted a targeted CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. Here we show that the protein BRD2 is required for ACE2 transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous ACE2 expression and SARS-CoV-2 infection of human cells, including those of human nasal epithelia. Moreover, pharmacological BRD2 inhibition with the drug ABBV-744 inhibited SARS-CoV-2 replication in Syrian hamsters. We also found that BRD2 controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates the antiviral response. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a therapeutic target for COVID-19. Full Text

Sankaran, V.G., Weissman, J.S., and Zon, L.I. (2022). Cellular barcoding to decipher clonal dynamics in disease. Science 378 : no. 6616. Cellular barcodes are distinct DNA sequences that enable one to track specific cells across time or space. Recent advances in our ability to detect natural or synthetic cellular barcodes, paired with single-cell readouts of cell state, have markedly increased our knowledge of clonal dynamics and genealogies of the cells that compose a variety of tissues and organs. These advances hold promise to redefine our view of human disease. Here, we provide an overview of cellular barcoding approaches, discuss applications to gain new insights into disease mechanisms, and provide an outlook on future applications. We discuss unanticipated insights gained through barcoding in studies of cancer and blood cell production and describe how barcoding can be applied to a growing array of medical fields, particularly with the increasing recognition of clonal contributions in human diseases. Full Text

Sankari, S., Babu, V.M.P., Bian, K., Alhhazmi, A., Andorfer, M.C., Avalos, D.M., Smith, T.A., Yoon, K., Drennan, C.L., Yaffe, M.B., Sebastian Lourido, and Graham C Walker (2022). A haem-sequestering plant peptide promotes iron uptake in symbiotic bacteria. Nature Microbiology. Online Ahead of Print. Symbiotic partnerships with rhizobial bacteria enable legumes to grow without nitrogen fertilizer because rhizobia convert atmospheric nitrogen gas into ammonia via nitrogenase. After Sinorhizobium meliloti penetrate the root nodules that they have elicited in Medicago truncatula, the plant produces a family of about 700 nodule cysteine-rich (NCR) peptides that guide the differentiation of endocytosed bacteria into nitrogen-fixing bacteroids. The sequences of the NCR peptides are related to the defensin class of antimicrobial peptides, but have been adapted to play symbiotic roles. Using a variety of spectroscopic, biophysical and biochemical techniques, we show here that the most extensively characterized NCR peptide, 24 amino acid NCR247, binds haem with nanomolar affinity. Bound haem molecules and their iron are initially made biologically inaccessible through the formation of hexamers (6 haem/6 NCR247) and then higher-order complexes. We present evidence that NCR247 is crucial for effective nitrogen-fixing symbiosis. We propose that by sequestering haem and its bound iron, NCR247 creates a physiological state of haem deprivation. This in turn induces an iron-starvation response in rhizobia that results in iron import, which itself is required for nitrogenase activity. Using the same methods as for L-NCR247, we show that the D-enantiomer of NCR247 can bind and sequester haem in an equivalent manner. The special abilities of NCR247 and its D-enantiomer to sequester haem suggest a broad range of potential applications related to human health. Full Text

Satyaki, P.R.V., and Gehring, M. (2022). RNA Pol IV induces antagonistic parent-of-origin effects on Arabidopsis endosperm. PLoS Biology 20(4):e3001602 Gene expression in endosperm-a seed tissue that mediates transfer of maternal resources to offspring-is under complex epigenetic control. We show here that plant-specific RNA polymerase IV (Pol IV) mediates parental control of endosperm gene expression. Pol IV is required for the production of small interfering RNAs that typically direct DNA methylation. We compared small RNAs (sRNAs), DNA methylation, and mRNAs in Arabidopsis thaliana endosperm from heterozygotes produced by reciprocally crossing wild-type (WT) plants to Pol IV mutants. We find that maternally and paternally acting Pol IV induce distinct effects on endosperm. Loss of maternal or paternal Pol IV impacts sRNAs and DNA methylation at different genomic sites. Strikingly, maternally and paternally acting Pol IV have antagonistic impacts on gene expression at some loci, divergently promoting or repressing endosperm gene expression. Antagonistic parent-of-origin effects have only rarely been described and are consistent with a gene regulatory system evolving under parental conflict. Full Text

Scimone, M.L., Cloutier, J.K., Maybrun, C.L., and Reddien, P.W. (2022). The planarian wound epidermis gene equinox is required for blastema formation in regeneration. Nature Communications 13(1):2726. Regeneration often involves the formation of a blastema, an outgrowth or regenerative bud formed at the plane of injury where missing tissues are produced. The mechanisms that trigger blastema formation are therefore fundamental for regeneration. Here, we identify a gene, which we named equinox, that is expressed within hours of injury in the planarian wound epidermis. equinox encodes a predicted secreted protein that is conserved in many animal phyla. Following equinox inhibition, amputated planarians fail to maintain wound-induced gene expression and to subsequently undergo blastema outgrowth. Associated with these defects is an inability to reestablish lost positional information needed for missing tissue specification. Our findings link the planarian wound epidermis, through equinox, to regeneration of positional information and blastema formation, indicating a broad regulatory role of the wound epidermis in diverse regenerative contexts. Full Text

Sengupta S., Das, S., Crespo, A.C., Cornel, A.M., Patel, A.G., Mahadevan, N.R., Campisi, M., Ali, A.K., Sharma, B., Rowe, J.H., Cohen, M.A., Rudolph Jaenisch, et al. (2022). Mesenchymal and adrenergic cell lineage states in neuroblastoma possess distinct immunogenic phenotypes. Nature Cancer. Online Ahead of Print. Apart from the anti-GD2 antibody, immunotherapy for neuroblastoma has had limited success due to immune evasion mechanisms, coupled with an incomplete understanding of predictors of response. Here, from bulk and single-cell transcriptomic analyses, we identify a subset of neuroblastomas enriched for transcripts associated with immune activation and inhibition and show that these are predominantly characterized by gene expression signatures of the mesenchymal lineage state. By contrast, tumors expressing adrenergic lineage signatures are less immunogenic. The inherent presence or induction of the mesenchymal state through transcriptional reprogramming or therapy resistance is accompanied by innate and adaptive immune gene activation through epigenetic remodeling. Mesenchymal lineage cells promote T cell infiltration by secreting inflammatory cytokines, are efficiently targeted by cytotoxic T and natural killer cells and respond to immune checkpoint blockade. Together, we demonstrate that distinct immunogenic phenotypes define the divergent lineage states of neuroblastoma and highlight the immunogenic potential of the mesenchymal lineage. Full Text

Sfeir, A., Fishell, G., Schier, A.F., Dustin, M.L., Gan, W.B., Joyner, A., Lehmann, R., Ron, D., Roth, D., Talbot, W.S., et al. (2022). Basic science under threat: Lessons from the Skirball Institute. Cell 185 (5):755-758. Support for basic science has been eclipsed by initiatives aimed at specific medical problems. The latest example is the dismantling of the Skirball Institute at NYU School of Medicine. Here, we reflect on the achievements and mission underlying the Skirball to gain insight into the dividends of maintaining a basic science vision within the academic enterprises. Full Text

Shah, V.S., Chivukula, R.R., Lin, B., Waghray, A., and Rajagopal, J. (2022). Cystic Fibrosis and the Cells of the Airway Epithelium: What Are Ionocytes and What Do They Do? Annual Review of Pathology 17, 23-46. 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. Full Text

Shpokayte, M., McKissick, O., Guan, X., Yuan, B., Rahsepar, B., Fernandez, F.R., Ruesch, E., Grella, S.L., White, J.A., Liu, X.S., et al. (2022). Hippocampal cells segregate positive and negative engrams. Communications Biology 5(1) : 1009. The hippocampus is involved in processing a variety of mnemonic computations specifically the spatiotemporal components and emotional dimensions of contextual memory. Recent studies have demonstrated cellular heterogeneity along the hippocampal axis. The ventral hippocampus has been shown to be important in the processing of emotion and valence. Here, we combine transgenic and all-virus based activity-dependent tagging strategies to visualize multiple valence-specific engrams in the vHPC and demonstrate two partially segregated cell populations and projections that respond to appetitive and aversive experiences. Next, using RNA sequencing and DNA methylation sequencing approaches, we find that vHPC appetitive and aversive engram cells display different transcriptional programs and DNA methylation landscapes compared to a neutral engram population. Additionally, optogenetic manipulation of tagged cell bodies in vHPC is not sufficient to drive appetitive or aversive behavior in real-time place preference, stimulation of tagged vHPC terminals projecting to the amygdala and nucleus accumbens (NAc), but not the prefrontal cortex (PFC), showed the capacity drive preference and avoidance. These terminals also were able to change their capacity to drive behavior. We conclude that the vHPC contains genetically, cellularly, and behaviorally segregated populations of cells processing appetitive and aversive memory engrams. Full Text

Smith, R.S., Takagishi, S.R., Amici, D.R., Metz, K., Gayatri, S., Alasady, M.J., Wu, Y., Brockway, S., Taiberg, S.L., Khalatyan, N., Susan Lindquist, et al. (2022). HSF2 cooperates with HSF1 to drive a transcriptional program critical for the malignant state. Science Advances 8(11) : eabj6526. Heat shock factor 1 (HSF1) is well known for its role in the heat shock response (HSR), where it drives a transcriptional program comprising heat shock protein (HSP) genes, and in tumorigenesis, where it drives a program comprising HSPs and many noncanonical target genes that support malignancy. Here, we find that HSF2, an HSF1 paralog with no substantial role in the HSR, physically and functionally interacts with HSF1 across diverse types of cancer. HSF1 and HSF2 have notably similar chromatin occupancy and regulate a common set of genes that include both HSPs and noncanonical transcriptional targets with roles critical in supporting malignancy. Loss of either HSF1 or HSF2 results in a dysregulated response to nutrient stresses in vitro and reduced tumor progression in cancer cell line xenografts. Together, these findings establish HSF2 as a critical cofactor of HSF1 in driving a cancer cell transcriptional program to support the anabolic malignant state. Full Text

Smith, T.A., Lopez-Perez, G.S., Herneisen, A.L., Shortt, E., and Lourido, S. (2022). Screening the Toxoplasma kinome with high-throughput tagging identifies a regulator of invasion and egress. Nature Microbiology. Online Ahead of Print. Protein kinases regulate fundamental aspects of eukaryotic cell biology, making them attractive chemotherapeutic targets in parasites like Plasmodium spp. and Toxoplasma gondii. To systematically examine the parasite kinome, we developed a high-throughput tagging (HiT) strategy to endogenously label protein kinases with an auxin-inducible degron and fluorophore. Hundreds of tagging vectors were assembled from synthetic sequences in a single reaction and used to generate pools of mutants to determine localization and function. Examining 1,160 arrayed clones, we assigned 40 protein localizations and associated 15 kinases with distinct defects. The fitness of tagged alleles was also measured by pooled screening, distinguishing delayed from acute phenotypes. A previously unstudied kinase, associated with a delayed phenotype, was shown to be a regulator of invasion and egress. We named the kinase Store Potentiating/Activating Regulatory Kinase (SPARK), based on its impact on intracellular Ca(2+) stores. Despite homology to mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1), SPARK lacks a lipid-binding domain, suggesting a rewiring of the pathway in parasites. HiT screening extends genome-wide approaches into complex cellular phenotypes, providing a scalable and versatile platform to dissect parasite biology. Full Text

Spinelli, J.B., and Zaganjor, E. (2022). Mitochondrial efficiency directs cell fate. Nature Cell Biology 24, 125-126. Mitochondria are asymmetrically distributed to the daughter cells according to their age. A study now identifies metabolic features associated with mitochondrial age that regulate cell fate decisions. Full Text

Stair, M.I., Carrasco, S.E., Annamalai, D., Jordan, E.B., Mannion, A., Feng, Y., Fabian, N., Ge, Z., Muthupalani, S., Dzink-Fox, J., Krzisch, M.A., et al. (2022). The Epidemiology of Invasive, Multiple-antibiotic-resistant Klebsiella pneumoniae Infection in a Breeding Colony of Immunocompromised NSG Mice. Comparative Medicine. Online Ahead of Print. Klebsiella pneumoniae (Kp) is a gram-negative opportunistic pathogen that causes severe pneumonia, pyelonephritis, and sepsis in immunocompromised hosts. During a 4-mo interval, several NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) breeders and pups in our facilities were diagnosed with Kp infections. An initial 6 adult and 1 juvenile NSG mice were submitted for necropsy and histologic examination because of acute onset of diarrhea and death. The evaluation revealed typhlocolitis in 2 of the mice and tritrichomoniasis in all 7. Escherichia coli positive for polyketide synthase (pks+) and Kp were isolated from the intestines. Given a history of sepsis due to pks(+) E. coli in NSG mice in our facilities and determination of its antimicrobial susceptibility, trimethoprim-sulfamethoxazole (TMP-SMX) was administered to the colony in the drinking water for 4 wk. After this intervention, an additional 21 mice became ill or died; 11 of these mice had suppurative pneumonia, meningoencephalitis, hepatitis, metritis, pyelonephritis, or sepsis. Kp was cultured from pulmonary abscesses or blood of 10 of the mice. Whole-genome sequencing (WGS) indicated that the Kp isolates contained genes associated with phenotypes found in pore-forming Kp isolates cultured from humans with ulcerative colitis and primary sclerosing cholangitis. None of the Kp isolates exhibited a hyperviscous phenotype, but 13 of 14 were resistant to TMP-SMX. Antimicrobial susceptibility testing indicated sensitivity of the Kp to enrofloxacin, which was administered in the drinking water. Antibiotic sensitivity profiles were confirmed by WGS of the Kp strains; key virulence and resistance genes to quaternary ammonia compounds were also identified. Enrofloxacin treatment resulted in a marked reduction in mortality, and the study using the NSG mice was completed successfully. Our findings implicate intestinal translocation of Kp as the cause of pneumonia and systemic infections in NSG mice and highlight the importance of identification of enteric microbial pathogens and targeted antibiotic selection when treating bacterial infections in immunocompromised mice. Full Text

Tolani, B., Celli, A., Yao, Y., Tan, Y.Z., Fetter, R., Liem, C.R., de Smith, A.J., Vasanthakumar, T., Bisignano, P., Cotton, A.D., Jonathan S. Weissman et al. (2022). Ras-mutant cancers are sensitive to small molecule inhibition of V-type ATPases in mice. Nature Biotechnology 40(12):1834-1844. Mutations in Ras family proteins are implicated in 33% of human cancers, but direct pharmacological inhibition of Ras mutants remains challenging. As an alternative to direct inhibition, we screened for sensitivities in Ras-mutant cells and discovered 249C as a Ras-mutant selective cytotoxic agent with nanomolar potency against a spectrum of Ras-mutant cancers. 249C binds to vacuolar (V)-ATPase with nanomolar affinity and inhibits its activity, preventing lysosomal acidification and inhibiting autophagy and macropinocytosis pathways that several Ras-driven cancers rely on for survival. Unexpectedly, potency of 249C varies with the identity of the Ras driver mutation, with the highest potency for KRASG13D and G12V both in vitro and in vivo, highlighting a mutant-specific dependence on macropinocytosis and lysosomal pH. Indeed, 249C potently inhibits tumor growth without adverse side effects in mouse xenografts of KRAS-driven lung and colon cancers. A comparison of isogenic SW48 xenografts with different KRAS mutations confirmed that KRASG13D/+ (followed by G12V/+) mutations are especially sensitive to 249C treatment. These data establish proof-of-concept for targeting V-ATPase in cancers driven by specific KRAS mutations such as KRASG13D and G12V. Full Text

Tomasello, D.L., and Wlodkowic, D. (2022). Noninvasive Electrophysiology: Emerging Prospects in Aquatic Neurotoxicity Testing. Environmental Science & Technology. Online Ahead of Print. The significance of neurotoxicological risks associated with anthropogenic pollution is gaining increasing recognition worldwide. In this regard, perturbations in behavioral traits upon exposure to environmentally relevant concentrations of neurotoxic and neuro-modulating contaminants have been linked to diminished ecological fitness of many aquatic species. Despite an increasing interest in behavioral testing in aquatic ecotoxicology there is, however, a notable gap in understanding of the neurophysiological foundations responsible for the altered behavioral phenotypes. One of the canonical approaches to explain the mechanisms of neuro-behavioral changes is functional analysis of neuronal transmission. In aquatic animals it requires, however, invasive, complex, and time-consuming electrophysiology techniques. In this perspective, we highlight emerging prospects of noninvasive, in situ electrophysiology based on multielectrode arrays (MEAs). This technology has only recently been pioneered for the detection and analysis of transient electrical signals in the central nervous system of small model organisms such as zebrafish. The analysis resembles electroencephalography (EEG) applications and provides an appealing strategy for mechanistic explorative studies as well as routine neurotoxicity risk assessment. We outline the prospective future applications and existing challenges of this emerging analytical strategy that is poised to bring new vistas for aquatic ecotoxicology such as greater mechanistic understanding of eco-neurotoxicity and thus more robust risk assessment protocols. Full Text

Tringidess, C.M., Boulingre, M., Khalil, A., Lungjangwa, T., Jaenisch, R., and Mooney, D.J. (2022). Tunable Conductive Hydrogel Scaffolds for Neural Cell Differentiation. Advanced Healthcare Materials: e2202221. Multielectrode arrays would benefit from intimate engagement with neural cells, but typical arrays do not present a physical environment that mimics that of neural tissues. We hypothesized that a porous, conductive hydrogel scaffold with appropriate mechanical and conductive properties could support neural cells in 3D, while tunable electrical and mechanical properties could modulate the growth and differentiation of the cellular networks. By incorporating carbon nanomaterials into an alginate hydrogel matrix, and then freeze-drying the formulations, scaffolds which mimic neural tissue properties were formed. Neural progenitor cells (NPCs) incorporated in the scaffolds formed neurite networks which spanned the material in 3D, and differentiated into astrocytes and myelinating oligodendrocytes. Viscoelastic and more conductive scaffolds produced more dense neurite networks, with an increased percentage of astrocytes and higher myelination. Application of exogenous electrical stimulation to the scaffolds increased the percentage of astrocytes and the supporting cells localized differently with the surrounding neurons. The tunable biomaterial scaffolds can support neural co-cultures for over 12 weeks, and enable a physiologically-mimicking in vitro platform to study the formation of neuronal networks. As these materials have sufficient electrical properties to be used as electrodes in implantable arrays, they may allow for the creation of biohybrid neural interfaces and living electrodes. Full Text

Tsvetkov, P., Coy, S., Petrova, B., Dreishpoon, M., Verma, A., Abdusamad, M., Rossen, J., Joesch-Cohen, L., Humeidi, R., Spangler, R.D., Evgeni Frenkel , et al. (2022). Copper induces cell death by targeting lipoylated TCA cycle proteins. Science 375:1254-1261. Copper is an essential cofactor for all organisms, and yet it becomes toxic if concentrations exceed a threshold maintained by evolutionarily conserved homeostatic mechanisms. How excess copper induces cell death, however, is unknown. Here, we show in human cells that copper-dependent, regulated cell death is distinct from known death mechanisms and is dependent on mitochondrial respiration. We show that copper-dependent death occurs by means of direct binding of copper to lipoylated components of the tricarboxylic acid (TCA) cycle. This results in lipoylated protein aggregation and subsequent iron-sulfur cluster protein loss, which leads to proteotoxic stress and ultimately cell death. These findings may explain the need for ancient copper homeostatic mechanisms. Full Text

Valenstein M.L., Rogala, K.B., Lalgudi, P.V., Brignole, E.J., Gu, X., Saxton, R.A., Chantranupong, L., Kolibius, J., Quast, J.P., and Sabatini, D.M. (2022). Structure of the nutrient-sensing hub GATOR2. Nature. Online Ahead of Print. Mechanistic target of rapamycin complex 1 (mTORC1) controls growth by regulating anabolic and catabolic processes in response to environmental cues, including nutrients(1,2). Amino acids signal to mTORC1 through the Rag GTPases, which are regulated by several protein complexes, including GATOR1 and GATOR2. GATOR2, which has five components (WDR24, MIOS, WDR59, SEH1L and SEC13), is required for amino acids to activate mTORC1 and interacts with the leucine and arginine sensors SESN2 and CASTOR1, respectively(3-5). Despite this central role in nutrient sensing, GATOR2 remains mysterious as its subunit stoichiometry, biochemical function and structure are unknown. Here we used cryo-electron microscopy to determine the three-dimensional structure of the human GATOR2 complex. We found that GATOR2 adopts a large (1.1 MDa), two-fold symmetric, cage-like architecture, supported by an octagonal scaffold and decorated with eight pairs of WD40 β-propellers. The scaffold contains two WDR24, four MIOS and two WDR59 subunits circularized via two distinct types of junction involving non-catalytic RING domains and α-solenoids. Integration of SEH1L and SEC13 into the scaffold through β-propeller blade donation stabilizes the GATOR2 complex and reveals an evolutionary relationship to the nuclear pore and membrane-coating complexes(6). The scaffold orients the WD40 β-propeller dimers, which mediate interactions with SESN2, CASTOR1 and GATOR1. Our work reveals the structure of an essential component of the nutrient-sensing machinery and provides a foundation for understanding the function of GATOR2 within the mTORC1 pathway. Full Text

Vela, D., Sharp, A., Zhang, R., Nguyen, T., Hoang, A., and Pianykh, O.S. (2022). Temporal quality degradation in AI models. Scientific Reports 12(1) : 11654. As AI models continue to advance into many real-life applications, their ability to maintain reliable quality over time becomes increasingly important. The principal challenge in this task stems from the very nature of current machine learning models, dependent on the data as it was at the time of training. In this study, we present the first analysis of AI "aging": the complex, multifaceted phenomenon of AI model quality degradation as more time passes since the last model training cycle. Using datasets from four different industries (healthcare operations, transportation, finance, and weather) and four standard machine learning models, we identify and describe the main temporal degradation patterns. We also demonstrate the principal differences between temporal model degradation and related concepts that have been explored previously, such as data concept drift and continuous learning. Finally, we indicate potential causes of temporal degradation, and suggest approaches to detecting aging and reducing its impact. Full Text

Wang, A.J., Allen, A., Sofman, M., Sphabmixay, P., Yildiz, E., and Griffith, L.G. (2022). Engineering Modular 3D Liver Culture Microenvironments In Vitro to Parse the Interplay between Biophysical and Biochemical Microenvironment Cues on Hepatic Phenotypes. Advanced Nanobiomed Research 2(1) : 2100049. In vitro models of human liver functions are used across a diverse range of applications in preclinical drug development and disease modeling, with particular increasing interest in models that capture facets of liver inflammatory status. This study investigates how the interplay between biophysical and biochemical microenvironment cues influence phenotypic responses, including inflammation signatures, of primary human hepatocytes (PHH) cultured in a commercially available perfused bioreactor. A 3D printing-based alginate microwell system was designed to form thousands of hepatic spheroids in a scalable manner as a comparator 3D culture modality to the bioreactor. Soft, synthetic extracellular matrix (ECM) hydrogel scaffolds with biophysical properties mimicking features of liver were engineered to replace polystyrene scaffolds, and the biochemical microenvironment was modulated with a defined set of growth factors and signaling modulators. The supplemented media significantly increased tissue density, albumin secretion, and CYP3A4 activity but also upregulated inflammatory markers. Basal inflammatory markers were lower for cells maintained in ECM hydrogel scaffolds or spheroid formats than polystyrene scaffolds, while hydrogel scaffolds exhibited the most sensitive response to inflammation as assessed by multiplexed cytokine and RNA-seq analyses. Together, these engineered 3D liver microenvironments provide insights for probing human liver functions and inflammatory response in vitro. Full Text

Wang, C., Xu, Q., Zhang, X., Day, D.S., Abraham, B.J., Lun, K., Chen, L., Huang, J., and Ji, X. (2022). BRD2 interconnects with BRD3 to facilitate Pol II transcription initiation and elongation to prime promoters for cell differentiation. Cellular and Molecular Life Sciences : CMLS 79 (6) : 338. The bromodomain and extraterminal motif (BET) proteins are critical drug targets for diseases. The precise functions and relationship of BRD2 with other BET proteins remain elusive mechanistically. Here, we used acute protein degradation and quantitative genomic and proteomic approaches to investigate the primary functions of BRD2 in transcription. We report that BRD2 is required for TAF3-mediated Pol II initiation at promoters with low levels of H3K4me3 and for R-loop suppression during Pol II elongation. Single and double depletion revealed that BRD2 and BRD3 function additively, independently, or perhaps antagonistically in Pol II transcription at different promoters. Furthermore, we found that BRD2 regulates the expression of different genes during embryonic body differentiation processes by promoter priming in embryonic stem cells. Therefore, our results suggest complex interconnections between BRD2 and BRD3 at promoters to fine-tune Pol II initiation and elongation for control of cell state. Full Text

Watase, G.J., Nelson, J.O., and Yamashita, Y.M. (2022). Nonrandom sister chromatid segregation mediates rDNA copy number maintenance in Drosophila. Science Advances 8(30) : eabo4443. Although considered to be exact copies of each other, sister chromatids can segregate nonrandomly in some cases. For example, sister chromatids of the X and Y chromosomes segregate nonrandomly during asymmetric division of male germline stem cells (GSCs) in Drosophila melanogaster. Here, we demonstrate that the ribosomal DNA (rDNA) loci, which are located on the X and Y chromosomes, and an rDNA binding protein Indra are required for nonrandom sister chromatid segregation (NRSS). We provide the evidence that NRSS, following unequal sister chromatid exchange, is a mechanism by which GSCs recover rDNA copy number, counteracting the spontaneous copy number loss that occurs during aging. Our study reveals an unexpected role for NRSS in maintaining germline immortality through maintenance of a vulnerable genomic element, rDNA. Full Text

Wei, X., Fu, S., Li, H., Liu, Y., Wang, S., Feng, W., Yang, Y., Liu, X., Zeng, Y.Y., Cheng, M., Qiu, X.,et al. (2022). Single-cell Stereo-seq reveals induced progenitor cells involved in axolotl brain regeneration. Science 377(6610): eabp9444. The molecular mechanism underlying brain regeneration in vertebrates remains elusive. We performed spatial enhanced resolution omics sequencing (Stereo-seq) to capture spatially resolved single-cell transcriptomes of axolotl telencephalon sections during development and regeneration. Annotated cell types exhibited distinct spatial distribution, molecular features, and functions. We identified an injury-induced ependymoglial cell cluster at the wound site as a progenitor cell population for the potential replenishment of lost neurons, through a cell state transition process resembling neurogenesis during development. Transcriptome comparisons indicated that these induced cells may originate from local resident ependymoglial cells. We further uncovered spatially defined neurons at the lesion site that may regress to an immature neuron-like state. Our work establishes spatial transcriptome profiles of an anamniote tetrapod brain and decodes potential neurogenesis from ependymoglial cells for development and regeneration, thus providing mechanistic insights into vertebrate brain regeneration. Full Text

Winter, J.M., Fresenius, H.L., Cunningham, C.N., Wei, P., Keys, H.R., Berg, J.A., Bott, A.J., Yadav, T., Ryan, J.A., Sirohi, D., et al. (2022). Collateral deletion of the mitochondrial AAA+ ATPase ATAD1 sensitizes cancer cells to proteasome dysfunction. eLife 11:e82860. The tumor suppressor gene PTEN is the second most commonly deleted gene in cancer. Such deletions often include portions of the chromosome 10q23 locus beyond the bounds of PTEN itself, which frequently disrupts adjacent genes. Coincidental loss of PTEN-adjacent genes might impose vulnerabilities that could either affect patient outcome basally or be exploited therapeutically. Here we describe how the loss of ATAD1, which is adjacent to and frequently co-deleted with PTEN, predisposes cancer cells to apoptosis triggered by proteasome dysfunction and correlates with improved survival in cancer patients. ATAD1 directly and specifically extracts the pro-apoptotic protein BIM from mitochondria to inactivate it. Cultured cells and mouse xenografts lacking ATAD1 are hypersensitive to clinically used proteasome inhibitors, which activate BIM and trigger apoptosis. This work furthers our understanding of mitochondrial protein homeostasis and could lead to new therapeutic options for the hundreds of thousands of cancer patients who have tumors with chromosome 10q23 deletion. Full Text

Wright, B.W., Yi, Z., Weissman, J.S., and Chen, J. (2022). The dark proteome: translation from noncanonical open reading frames. Trends in Cell Biology 32, 243-25. 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

Wu, D., Poddar, A., Ninou, E., Hwang, E., Cole, M.A., Liu, S.J., Horlbeck, M.A., Chen, J., Replogle, J.M., Carosso, G.A., Jonathan S. Weissman, et al. (2022). Dual genome-wide coding and lncRNA screens in neural induction of induced pluripotent stem cells. Cell Genomics 2(11):100177. Human chromosomes are pervasively transcribed, but systematic understanding of coding and lncRNA genome function in cell differentiation is lacking. Using CRISPR interference (CRISPRi) in human induced pluripotent stem cells, we performed dual genome-wide screens - assessing 18,905 protein-coding and 10,678 lncRNA loci - and identified 419 coding and 201 lncRNA genes that regulate neural induction. Integrative analyses revealed distinct properties of coding and lncRNA genome function, including a 10-fold enrichment of lncRNA genes for roles in differentiation compared to proliferation. Further, we applied Perturb-seq to obtain granular insights into neural induction phenotypes. While most coding hits stalled or aborted differentiation, lncRNA hits were enriched for the genesis of diverse cellular states, including those outside the neural lineage. In addition to providing a rich resource (danlimlab.shinyapps.io/dualgenomewide) for understanding coding and lncRNA gene function in development, these results indicate that the lncRNA genome regulates lineage commitment in a manner fundamentally distinct from coding genes. Full Text

Yamashita, Y.M. (2022). Satellite DNA. Seminars in Cell & Developmental Biology 128 : 1-. Satellite DNA is non-coding, tandemly repeated DNA that can make up more than 50% of the eukaryotic genomes. Despite this abundance, its non-coding nature and poor sequence conservation led to the idea that satellite DNA is mostly junk, with its abundance only reflecting cells’ inability to purge them. Historically, in the ‘pre-genome sequencing era’, satellite DNA and other repetitive DNA were the only type of DNA that offered a sufficient amount of materials for the researchers to work with (due to their repetitiveness), generating considerable volume of studies on satellite DNA and repetitive elements. Those early work had generated foundational knowledge, which was all hinting at its function in a retrospect ([2], [4], [5] just to list a few). Yet, once genome sequence became available, researchers drastically shifted to studying single copy DNA sequences, discarding ‘unmappable’ repetitive sequences. This created a decades-gap in the study of satellite DNA. Meantime, due to technical limitations, the repetitive DNA of clear functions (e.g. centromeric satellite DNA) remained mostly inaccessible.Now we are living in an exciting era when technical breakthroughs of long-read DNA sequencing and the effort of researchers are enabling the study of satellite DNA and other repetitive DNAs. I am delighted to be able to organize the special issue of satellite DNA, where we are able to collectively review the most current knowledge of satellite DNA. Full Text

Yang, D., Jones, M.G., Naranjo, S., Rideout, W.M., 3rd, Min, K.H.J., Ho, R., Wu, W., Replogle, J.M., Page, J.L., Quinn, J.J., Qiu, X., Michael Z Chen, and Jonathan S Weissman , et al. (2022). Lineage tracing reveals the phylodynamics, plasticity, and paths of tumor evolution. Cell. Online Ahead of Print. Tumor evolution is driven by the progressive acquisition of genetic and epigenetic alterations that enable uncontrolled growth and expansion to neighboring and distal tissues. The study of phylogenetic relationships between cancer cells provides key insights into these processes. Here, we introduced an evolving lineage-tracing system with a single-cell RNA-seq readout into a mouse model of Kras;Trp53(KP)-driven lung adenocarcinoma and tracked tumor evolution from single-transformed cells to metastatic tumors at unprecedented resolution. We found that the loss of the initial, stable alveolar-type2-like state was accompanied by a transient increase in plasticity. This was followed by the adoption of distinct transcriptional programs that enable rapid expansion and, ultimately, clonal sweep of stable subclones capable of metastasizing. Finally, tumors develop through stereotypical evolutionary trajectories, and perturbing additional tumor suppressors accelerates progression by creating novel trajectories. Our study elucidates the hierarchical nature of tumor evolution and, more broadly, enables in-depth studies of tumor progression. Full Text

Yu, F., Cato, L.D., Weng, C., Liggett, L.A., Jeon, S., Xu, K., Chiang, C.W.K., Wiemels, J.L., Weissman, J.S., de Smith, A.J., et al. (2022). Variant to function mapping at single-cell resolution through network propagation. Nature Biotechnology. Online Ahead of Print. Genome-wide association studies in combination with single-cell genomic atlases can provide insights into the mechanisms of disease-causal genetic variation. However, identification of disease-relevant or trait-relevant cell types, states and trajectories is often hampered by sparsity and noise, particularly in the analysis of single-cell epigenomic data. To overcome these challenges, we present SCAVENGE, a computational algorithm that uses network propagation to map causal variants to their relevant cellular context at single-cell resolution. We demonstrate how SCAVENGE can help identify key biological mechanisms underlying human genetic variation, applying the method to blood traits at distinct stages of human hematopoiesis, to monocyte subsets that increase the risk for severe Coronavirus Disease 2019 (COVID-19) and to intermediate lymphocyte developmental states that predispose to acute leukemia. Our approach not only provides a framework for enabling variant-to-function insights at single-cell resolution but also suggests a more general strategy for maximizing the inferences that can be made using single-cell genomic data. Full Text

Yunes, S.A., Willoughby, J.L.S., Kwan, J.H., Biagi, J.M., Pokharel, N., Chin, H.G., York, E.A., Su, K.C., George, K., Shah, J.V., et al. (2022). Factor quinolinone inhibitors disrupt spindles and multiple LSF (TFCP2)-protein interactions in mitosis, including with microtubule-associated proteins. PloS One 17(6) : e0268857. Factor quinolinone inhibitors (FQIs), a first-in-class set of small molecule inhibitors targeted to the transcription factor LSF (TFCP2), exhibit promising cancer chemotherapeutic properties. FQI1, the initial lead compound identified, unexpectedly induced a concentration-dependent delay in mitotic progression. Here, we show that FQI1 can rapidly and reversibly lead to mitotic arrest, even when added directly to mitotic cells, implying that FQI1-mediated mitotic defects are not transcriptionally based. Furthermore, treatment with FQIs resulted in a striking, concentration-dependent diminishment of spindle microtubules, accompanied by a concentration-dependent increase in multi-aster formation. Aberrant γ-tubulin localization was also observed. These phenotypes suggest that perturbation of spindle microtubules is the primary event leading to the mitotic delays upon FQI1 treatment. Previously, FQIs were shown to specifically inhibit not only LSF DNA-binding activity, which requires LSF oligomerization to tetramers, but also other specific LSF-protein interactions. Other transcription factors participate in mitosis through non-transcriptional means, and we recently reported that LSF directly binds α-tubulin and is present in purified cellular tubulin preparations. Consistent with a microtubule role for LSF, here we show that LSF enhanced the rate of tubulin polymerization in vitro, and FQI1 inhibited such polymerization. To probe whether the FQI1-mediated spindle abnormalities could result from inhibition of mitotic LSF-protein interactions, mass spectrometry was performed using as bait an inducible, tagged form of LSF that is biotinylated by endogenous enzymes. The global proteomics analysis yielded expected associations for a transcription factor, notably with RNA processing machinery, but also to nontranscriptional components. In particular, and consistent with spindle disruption due to FQI treatment, mitotic, FQI1-sensitive interactions were identified between the biotinylated LSF and microtubule-associated proteins that regulate spindle assembly, positioning, and dynamics, as well as centrosome-associated proteins. Probing the mitotic LSF interactome using small molecule inhibitors therefore supported a non-transcriptional role for LSF in mediating progression through mitosis. Full Text

Zhang, Y., Donaher, J.L., Das, S., Li, X., Reinhardt, F., Krall, J.A., Lambert, A.W., Thiru, P., Keys, H.R., Khan, M., Robert A Weinberg, et al. (2022). Genome-wide CRISPR screen identifies PRC2 and KMT2D-COMPASS as regulators of distinct EMT trajectories that contribute differentially to metastasis. Nature Cell Biology.Online Ahead of Print. Epithelial-mesenchymal transition (EMT) programs operate within carcinoma cells, where they generate phenotypes associated with malignant progression. In their various manifestations, EMT programs enable epithelial cells to enter into a series of intermediate states arrayed along the E-M phenotypic spectrum. At present, we lack a coherent understanding of how carcinoma cells control their entrance into and continued residence in these various states, and which of these states favour the process of metastasis. Here we characterize a layer of EMT-regulating machinery that governs E-M plasticity (EMP). This machinery consists of two chromatin-modifying complexes, PRC2 and KMT2D-COMPASS, which operate as critical regulators to maintain a stable epithelial state. Interestingly, loss of these two complexes unlocks two distinct EMT trajectories. Dysfunction of PRC2, but not KMT2D-COMPASS, yields a quasi-mesenchymal state that is associated with highly metastatic capabilities and poor survival of patients with breast cancer, suggesting that great caution should be applied when PRC2 inhibitors are evaluated clinically in certain patient cohorts. These observations identify epigenetic factors that regulate EMP, determine specific intermediate EMT states and, as a direct consequence, govern the metastatic ability of carcinoma cells. Full Text

Zhu C., Lee, J.Y., Woo, J.Z., Xu, L., Nguyenla, X., Yamashiro, L.H., Ji, F., Biering, S.B., Van Dis, E., Gonzalez, F., Silvi Rouskin, et al. (2022). An intranasal ASO therapeutic targeting SARS-CoV-2. Nature Communications 13(1):4503. The COVID-19 pandemic is exacting an increasing toll worldwide, with new SARS-CoV-2 variants emerging that exhibit higher infectivity rates and that may partially evade vaccine and antibody immunity. Rapid deployment of non-invasive therapeutic avenues capable of preventing infection by all SARS-CoV-2 variants could complement current vaccination efforts and help turn the tide on the COVID-19 pandemic. Here, we describe a novel therapeutic strategy targeting the SARS-CoV-2 RNA using locked nucleic acid antisense oligonucleotides (LNA ASOs). We identify an LNA ASO binding to the 5' leader sequence of SARS-CoV-2 that disrupts a highly conserved stem-loop structure with nanomolar efficacy in preventing viral replication in human cells. Daily intranasal administration of this LNA ASO in the COVID-19 mouse model potently suppresses viral replication (>80-fold) in the lungs of infected mice. We find that the LNA ASO is efficacious in countering all SARS-CoV-2 "variants of concern" tested both in vitro and in vivo. Hence, inhaled LNA ASOs targeting SARS-CoV-2 represents a promising therapeutic approach to reduce or prevent transmission and decrease severity of COVID-19 in infected individuals. LNA ASOs are chemically stable and can be flexibly modified to target different viral RNA sequences and could be stockpiled for future coronavirus pandemics. Full Text