The following alphabetical list represents papers published in 2023 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.

2023 Titles :

-Alternative CDC20 translational isoforms tune mitotic arrest duration. Tsang
-Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma. Chan
-Analysis of Gene Expression Patterns and RNA Localization by Fluorescence in Situ Hybridization in Whole Mount Drosophila Testes. Fingerhut
-Approaches to pandemic prevention - the chromatin vaccine. Zhang
-Asymmetric Stem Cell Division and Germline Immortality. Yamashita
-Author Correction: A positive feedback loop controls Toxoplasma chronic differentiation. Licon
-Author Correction: Distinct chemical environments in biomolecular condensates. Kilgore
-Author Correction: Efficient C•G-to-G•C base editors developed using CRISPRi screens, target-library analysis, and machine learning. Koblan
-Bat pluripotent stem cells reveal unusual entanglement between host and viruses. Déjosez
-Blue light receptor CRY1 regulates HSFA1d nuclear localization to promote plant thermotolerance. Gao
-Cancer-associated mesothelial cells are regulated by the anti-Müllerian hormone axis. Chauvin
-CDK7 controls E2F- and MYC-driven proliferative and metabolic vulnerabilities in multiple myeloma. Yao
-CDPK2A and CDPK1 form a signaling module upstream of Toxoplasma motility. Shortt
-A cell-type-specific error-correction signal in the posterior parietal cortex. Green
-Cell type-specific role of CBX2 and its disordered region in spermatogenesis. Kim
-Comparative landscape of genetic dependencies in human and chimpanzee stem cells. She
-Complex haploinsufficiency in pluripotent cells yields somatic cells with DNA methylation abnormalities and pluripotency induction defects.Lasry
-A conserved complex of microneme proteins mediates rhoptry discharge in Toxoplasma. Valleau
-Correction: Differential effects of JNK1 and JNK2 on signal specific induction of apoptosis. Hochedlinger
-Coupled protein quality control during nonsense-mediated mRNA decay. Inglis
-Derepression of Y-linked multicopy protamine-like genes interferes with sperm nuclear compaction in D. melanogaster. Park
-Development of a Patient-Derived 3D Immuno-Oncology Platform to Potentiate Immunotherapy Responses in Ascites-Derived Circulating Tumor Cells Gerton
-Discovery of antibiotics that selectively kill metabolically dormant bacteria. Zheng
-Distinct chemical environments in biomolecular condensates. Kilgore
-Drosophila embryos spatially sort their nutrient stores to facilitate their utilization. Kilwein
-A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens. Guna
-Efficient in vivo genome editing prevents hypertrophic cardiomyopathy in mice. Reichart
-Emergence of a proton exchange-based isomerization and lactonization mechanism in the plant coumarin synthase COSY. Kim
-Emergent dynamics of adult stem cell lineages from single nucleus and single cell RNA-Seq of Drosophila testes. Raz
-Epigenetic alterations identify a confluence of genetic vulnerabilities tied to opioid overdose. Hoang
-Epigenetic Regulation During Plant Development and the Capacity for Epigenetic Memory. Hemenway
-Eukaryotic translation initiation factor eIF4E-5 is required for spermiogenesis in Drosophila melanogaster. Shao
-Fate specification is spatially intermingled across planarian stem cells. Park
-FoxK1 Associated Gene Regulatory Network in Hepatic Insulin Action and It's Relationship to FoxO1 and Insulin Receptor Mediated Transcriptional Regulation. Allu
-Fusome topology and inheritance during insect gametogenesis. Diegmiller
-Genetic predisposition to neuroblastoma results from a regulatory polymorphism that promotes the adrenergic cell state. Weichert-Leahey
-A genome-wide optical pooled screen reveals regulators of cellular antiviral responses. Carlson
-Genomic and biological variation in bat IFNs: An antiviral treatment approach. Al-Eitan
-Genomics and biochemical analyses reveal a metabolon key to β-L-ODAP biosynthesis in Lathyrus sativus. Edwards
-Guidelines for establishing a cytometry laboratory. Belkina
-HP6/Umbrea is dispensable for viability and fertility, suggesting essentiality of newly evolved genes is rare. Grill
-The human inactive X chromosome modulates expression of the active X chromosome.San Roman
-The human Y and inactive X chromosomes similarly modulate autosomal gene expression. San Roman
-Human iPS cell-derived sensory neurons can be infected by SARS-CoV-2. Flamier
-Human microglia maturation is underpinned by specific gene regulatory networks. Han
-The implications of satellite DNA instability on cellular function and evolution. Flynn
-In Vitro Evaluation of the Potential for Drug Interactions by Salidroside. Kasprzyk
-Inaugurating High-Throughput Profiling of Extracellular Vesicles for Earlier Ovarian Cancer Detection. Jo
-The Information Theory of Aging. Lu
-Inhibition of the proline metabolism rate-limiting enzyme P5CS allows proliferation of glutamine-restricted cancer cells. Linder
-Integrative analysis of multimodal mass spectrometry data in MZmine 3. Schmid
-Layer-by-layer interleukin-12 nanoparticles drive a safe and effective response in ovarian tumors. Barberio
-LINE1-Mediated Reverse Transcription and Genomic Integration of SARS-CoV-2 mRNA Detected in Virus-Infected but Not in Viral mRNA-Transfected Cells. Zhang
-Lipoproteins act as vehicles for lipid antigen delivery and activation of invariant natural killer T-cells. Engelen
-Manipulating mitochondrial electron flow enhances tumor immunogenicity. Mangalhara
-Massively parallel base editing to map variant effects in human hematopoiesis. Martin-Rufino
-A maternally programmed intergenerational mechanism enables male offspring to make piRNAs from Y-linked precursor RNAs in Drosophila. Venkei
-Mechanical activation of mitochondria in germ cell differentiation. Gäbelein
-Microfluidics-free single-cell genomics with templated emulsification. Clark
-MMD collaborates with ACSL4 and MBOAT7 to promote polyunsaturated phosphatidylinositol remodeling and susceptibility to ferroptosis. Phadnis
-Molecular mechanisms of tubulogenesis revealed in the sea star hydro-vascular organ. Perillo
-The multifaceted roles of Myb domain-containing proteins in apicomplexan parasites. Schwarz
-Multiplex epigenome editing of MECP2 to rescue Rett syndrome neurons. Qian
-NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency. Sun
-New Tools for Lineage Tracing in Cancer In Vivo. Jones
-Oncogenic CDK13 mutations impede nuclear RNA surveillance. Insco
-Organism-wide, cell-type-specific secretome mapping of exercise training in mice. Wei
-OSR1 disruption contributes to uterine factor infertility via impaired Müllerian duct development and endometrial receptivity.Lofrano-Porto
-Phagocytic cooperativity by tumour macrophages. Maoz
-A phylogeny of the evening primrose family (Onagraceae) using a target enrichment approach with 303 nuclear loci. Overson
-A positive feedback loop controls Toxoplasma chronic differentiation. Licon
-Practical strategies for robust and inexpensive imaging of aqueous-cleared tissues. Williams
-rDNA magnification is a unique feature of germline stem cells. Nelson
-Regulatory architecture of cell identity genes and housekeeping genes. Dall'Agnese
-Regulatory architecture of housekeeping genes is driven by promoter assemblies. Dejosez
-Regulatory T cells play a crucial role in maintaining sperm tolerance and male fertility. Barrachina
-Repeat-associated non-AUG translation induces cytoplasmic aggregation of CAG repeat-containing RNAs. Das
-Replication stress and defective checkpoints make fallopian tube epithelial cells putative drivers of high-grade serous ovarian cancer. Galhenage
-The retrotransposon R2 maintains Drosophila ribosomal DNA repeats. Nelson
-Role of condensates in modulating DNA repair pathways and its implication for chemoresistance. Dall'Agnese
-Screening in serum-derived medium reveals differential response to compounds targeting metabolism. Abbott
-Seeing is believing: the impact of RB on nuclear organization. Krishnan
-Spatiotemporally resolved transcriptomics reveals the subcellular RNA kinetic landscape. Ren
-Specific host metabolite and gut microbiome alterations are associated with bone loss during spaceflight. Bedree
-A statistical approach for identifying primary substrates of ZSWIM8-mediated microRNA degradation in small-RNA sequencing data. Wang
-Structures, functions, and adaptations of the human LINE-1 ORF2 protein. Baldwin
-Sustained Vision Recovery by OSK Gene Therapy in a Mouse Model of Glaucoma. Karg
-Systematic functional interrogation of SARS-CoV-2 host factors using Perturb-seq. Sunshine
-A TAle of Two Pathways: Tail-Anchored Protein Insertion at the Endoplasmic Reticulum. Guna
-Testing for Allele-specific Expression from Human Brain Samples. Diaz-Ortiz
-Three recent sex chromosome-to-autosome fusions in a Drosophila virilis strain with high satellite DNA content. Flynn
-Translational fidelity screens in mammalian cells reveal eIF3 and eIF4G2 as regulators of start codon selectivity. She
-Transcription factors interact with RNA to regulate genes. Oksuz
-transXpress: a Snakemake pipeline for streamlined de novo transcriptome assembly and annotation. Fallon
-Tunable Conductive Hydrogel Scaffolds for Neural Cell Differentiation. Tringides
-Why Ys are not necessarily toxic.Yamashita
-The yeast RNA methylation complex consists of conserved yet reconfigured components with m6A-dependent and independent roles. Ensinck

Abbott K.L., Ali, A., Casalena, D., Do, B.T., Ferreira, R., Cheah, J.H., Soule, C.K., Deik, A., Kunchok, T., Caroline A. Lewis, et al. (2023). Screening in serum-derived medium reveals differential response to compounds targeting metabolism. Cell Chemical Biology. Online Now. A challenge for screening new anticancer drugs is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels, which can influence cell metabolism and drug sensitivity. A general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To address this, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We screened several small molecule libraries and found that compounds targeting metabolic enzymes were differentially effective in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients. Full Text

Al-Eitan, L., Mihyar, A., Zhang, L., Bisht, P., and Jaenisch, R. (2023). Genomic and biological variation in bat IFNs: An antiviral treatment approach. Reviews in Medical Virology : e2488. Bat-borne viruses have attracted considerable research, especially in relation to the Covid-19 pandemic. Although bats can carry multiple zoonotic viruses that are lethal to many mammalian species, they appear to be asymptomatic to viral infection despite the high viral loads contained in their bodies. There are several differences between bats and other mammals. One of the major differences between bats and other mammals is the bats' ability to fly, which is believed to have induced evolutionary changes. It may have also favoured them as suitable hosts for viruses. This is related to their tolerance to viral infection. Innate immunity is the first line of defence against viral infection, but bats have metamorphosed the type of responses induced by innate immunity factors such as interferons. The expression patterns of interferons differ, as do those of interferon-related genes such as interferon regulatory factors and interferon-stimulated genes that contribute to the antiviral response of infected cells. In addition, the signalling pathways related to viral infection and immune responses have been subject to evolutionary changes, including mutations compared to their homologues in other mammals and gene selection. This article discusses the differences in the interferon-mediated antiviral response in bats compared to that of other mammals and how these differences are correlated to viral tolerance in bats. The effect of bat interferons related genes on human antiviral response against bat-borne viruses is also discussed. Full Text

Allu, P.K.R., Cardamone, M.D., Gomes, A.S., Dall'agnese, A., Cederquist, C., Pan, H., Dreyfuss, J.M., Enerbäck, S., and Kahn, C.R. (2023). FoxK1 Associated Gene Regulatory Network in Hepatic Insulin Action and It's Relationship to FoxO1 and Insulin Receptor Mediated Transcriptional Regulation. Molecular Metabolism :101825. Online Ahead of Print. OBJECTIVE: Insulin acts on the liver via changes in gene expression to maintain glucose and lipid homeostasis. This study aimed to the Forkhead box protein K1 (FOXK1) associated gene regulatory network as a transcriptional regulator of hepatic insulin action and to determine its role versus FoxO1 and possible actions of the insulin receptor at the DNA level. METHODS: Genome-wide analysis of FoxK1 binding were studied by chromatin immunoprecipitation sequencing and compared to those for IR and FoxO1. These were validated by knockdown experiments and gene expression analysis. RESULTS: Chromatin immunoprecipitation (ChIP) sequencing shows that FoxK1 binds to the proximal promoters and enhancers of over 4000 genes, and insulin enhances this interaction for about 75% of them. These include genes involved in cell cycle, senescence, steroid biosynthesis, autophagy, and metabolic regulation, including glucose metabolism and mitochondrial function and are enriched in a TGTTTAC consensus motif. Some of these genes are also bound by FoxO1. Comparing this FoxK1 ChIP-seq data to that of the insulin receptor (IR) reveals that FoxK1 may act as the transcription factor partner for some of the previously reported roles of IR in gene regulation, including for LARS1 and TIMM22, which are involved in rRNA processing and cell cycle. CONCLUSION: These data demonstrate that FoxK1 is an important regulator of gene expression in response to insulin in liver and may act in concert with FoxO1 and IR in regulation of genes in metabolism and other important biological pathways. Full Text

Baldwin, E.T., van Eeuwen, T., Hoyos, D., Zalevsky, A., Tchesnokov, E.P., Sánchez, R., Miller, B.D., Di Stefano, L.H., Ruiz, F.X., Hancock, M., Kera Xibinaku, , Samantha Congreve , Maximiliaan Hennink, et al. (2023). Structures, functions, and adaptations of the human LINE-1 ORF2 protein. Nature. Online Ahead of Print. The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one third of the human genome through a "copy-and-paste" mechanism catalyzed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)(1). ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer(2,3), autoimmunity(4,5), and aging(6,7), making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-EM in multiple conformational states. Our analyses reveal two novel folded domains, extensive contacts to RNA templates, and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry reveal that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signaling via cGAS/STING and resulting in interferon production(6-8). In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which likely explains cytosolic priming. Additional biochemical activities including processivity, DNA-directed polymerization, non-templated base addition, and template switching together allow us to propose an updated L1 insertion model. Finally, our evolutionary analysis reveals structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on L1 evolutionary history, and enable rational drug development targeting L1. 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. (2023). Layer-by-layer interleukin-12 nanoparticles drive a safe and effective response in ovarian tumors. Bioengineering and Translational Medicine 8(2):e10453. 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

Barrachina, F., Ottino, K., Elizagaray, M.L., Gervasi, M.G., Tu, L.J., Markoulaki, S., Spallanzani, R.G., Capen, D., Brown, D., and Battistone, M.A. (2023). Regulatory T cells play a crucial role in maintaining sperm tolerance and male fertility. PNAS 120(37):e2306797120. Regulatory T cells (Tregs) modulate tissue homeostatic processes and immune responses. Understanding tissue-Treg biology will contribute to developing precision-targeting treatment strategies. Here, we show that Tregs maintain the tolerogenic state of the testis and epididymis, where sperm are produced and mature. We found that Treg depletion induces severe autoimmune orchitis and epididymitis, manifested by an exacerbated immune cell infiltration [CD4 T cells, monocytes, and mononuclear phagocytes (MPs)] and the development of antisperm antibodies (ASA). In Treg-depleted mice, MPs increased projections toward the epididymal lumen as well as invading the lumen. ASA-bound sperm enhance sperm agglutination and might facilitate sperm phagocytosis. Tolerance breakdown impaired epididymal epithelial function and altered extracellular vesicle cargo, both of which play crucial roles in the acquisition of sperm fertilizing ability and subsequent embryo development. The affected mice had reduced sperm number and motility and severe fertility defects. Deciphering these immunoregulatory mechanisms may help to design new strategies to treat male infertility, as well as to identify potential targets for immunocontraception. Full Text

Bedree, J.K., Kerns, K., Chen, T.T., Lima, B.P., Liu, G., Ha, P., Shi, J.Y., Pan, H.C., Kim, J.K., Tran, L., Fabian Schulte, et al. (2023). Specific host metabolite and gut microbiome alterations are associated with bone loss during spaceflight. Cell Reports 42(5):112299. Understanding the axis of the human microbiome and physiological homeostasis is an essential task in managing deep-space-travel-associated health risks. The NASA-led Rodent Research 5 mission enabled an ancillary investigation of the gut microbiome, varying exposure to microgravity (flight) relative to ground controls in the context of previously shown bone mineral density (BMD) loss that was observed in these flight groups. We demonstrate elevated abundance of Lactobacillus murinus and Dorea sp. during micro -gravity exposure relative to ground control through whole-genome sequencing and 16S rRNA analyses. Specific functionally assigned gene clusters of L. murinus and Dorea sp. capable of producing metabolites, lactic acid, leucine/isoleucine, and glutathione are enriched. These metabolites are elevated in the micro -gravity-exposed host serum as shown by liquid chromatography-tandem mass spectrometry (LC-MS/ MS) metabolomic analysis. Along with BMD loss, ELISA reveals increases in osteocalcin and reductions in tartrate-resistant acid phosphatase 5b signifying additional loss of bone homeostasis in flight. Full Text

Belkina, A.C., Roe, C.E., Tang, V.A., Back, J.B., Bispo, C., Conway, A., Chakraborty, U., Daniels, K.T., de la Cruz, G., Ferrer-Font, L., et al. (2023). Guidelines for establishing a cytometry laboratory. Cytometry A. Online Ahead of Print. The purpose of this document is to provide guidance for establishing and maintaining growth and development of flow cytometry shared resource laboratories. While the best practices offered in this manuscript are not intended to be universal or exhaustive, they do outline key goals that should be prioritized to achieve operational excellence and meet the needs of the scientific community. Additionally, this document provides information on available technologies and software relevant to shared resource laboratories. This manuscript builds on the work of Barsky et al. 2016 published in Cytometry Part A and incorporates recent advancements in cytometric technology. A flow cytometer is a specialized piece of technology that require special care and consideration in its housing and operations. As with any scientific equipment, a thorough evaluation of the location, space requirements, auxiliary resources, and support is crucial for successful operation. This comprehensive resource has been written by past and present members of the International Society for Advancement of Cytometry (ISAC) Shared Resource Laboratory (SRL) Emerging Leaders Program https://isac-net.org/general/custom.asp?page=SRL-Emerging-Leaders with extensive expertise in managing flow cytometry SRLs from around the world in different settings including academia and industry. It is intended to assist in establishing a new flow cytometry SRL, re-purposing an existing space into such a facility, or adding a flow cytometer to an individual lab in academia or industry. This resource reviews the available cytometry technologies, the operational requirements, and best practices in SRL staffing and management. Full Text

Carlson, R.J., Leiken, M.D., Guna, A., Hacohen, N., and Blainey, P.C. (2023). A genome-wide optical pooled screen reveals regulators of cellular antiviral responses. PNAS 120(16):e2210623120. The infection of mammalian cells by viruses and innate immune responses to infection are spatiotemporally organized processes. Cytosolic RNA sensors trigger nuclear translocation of the transcription factor interferon regulatory factor 3 (IRF3) and consequent induction of host immune responses to RNA viruses. Previous genetic screens for factors involved in viral sensing did not resolve changes in the subcellular localization of host or viral proteins. Here, we increased the throughput of our optical pooled screening technology by over fourfold. This allowed us to carry out a genome-wide CRISPR knockout screen using high-resolution multiparameter imaging of cellular responses to Sendai virus infection coupled with in situ cDNA sequencing by synthesis (SBS) to identify 80,408 single guide RNAs (sgRNAs) in 10,366,390 cells-over an order of magnitude more genomic perturbations than demonstrated previously using an in situ SBS readout. By ranking perturbations using human-designed and deep learning image feature scores, we identified regulators of IRF3 translocation, Sendai virus localization, and peroxisomal biogenesis. Among the hits, we found that ATP13A1, an ER-localized P5A-type ATPase, is essential for viral sensing and is required for targeting of mitochondrial antiviral signaling protein (MAVS) to mitochondrial membranes where MAVS must be localized for effective signaling through retinoic acid-inducible gene I (RIG-I). The ability to carry out genome-wide pooled screens with complex high-resolution image-based phenotyping dramatically expands the scope of functional genomics approaches. Full Text

Chan, A.W., Broncel, M., Yifrach, E., Haseley, N.R., Chakladar, S., Andree, E., Herneisen, A.L., Shortt, E., Treeck, M., and Lourido, S. (2023). Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma. Elife 12 : RP85654. Apicomplexan parasites use Ca(2+)-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca(2+)-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired microneme protein secretion, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle. Full Text

Chauvin, M., Meinsohn, M.C., Dasari, S., May, P., Iyer, S., Nguyen, N.M.P., Oliva, E., Lucchini, Z., Nagykery, N., Kashiwagi, A., Mishra, R., et al. (2023). Cancer-associated mesothelial cells are regulated by the anti-Müllerian hormone axis.Cell Reports Volume 42, Issue 7 : 112730. Cancer-associated mesothelial cells (CAMCs) in the tumor microenvironment are thought to promote growth and immune evasion. We find that, in mouse and human ovarian tumors, cancer cells express anti-Müllerian hormone (AMH) while CAMCs express its receptor AMHR2, suggesting a paracrine axis. Factors secreted by cancer cells induce AMHR2 expression during their reprogramming into CAMCs in mouse and human in vitro models. Overexpression of AMHR2 in the Met5a mesothelial cell line is sufficient to induce expression of immunosuppressive cytokines and growth factors that stimulate ovarian cancer cell growth in an AMH-dependent way. Finally, syngeneic cancer cells implanted in transgenic mice with Amhr2(-/-) CAMCs grow significantly slower than in wild-type hosts. The cytokine profile of Amhr2(-/-) tumor-bearing mice is altered and their tumors express less immune checkpoint markers programmed-cell-death 1 (PD1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4). Taken together, these data suggest that the AMH/AMHR2 axis plays a critical role in regulating the pro-tumoral function of CAMCs in ovarian cancer. Full Text

Clark, I.C., Fontanez, K.M., Meltzer, R.H., Xue, Y., Hayford, C., May-Zhang, A., D'Amato, C., Osman, A., Zhang, J.Q., Hettige, P., Replogle J.M., Jost M., Jonathan S. Weissman, et al. (2023). Microfluidics-free single-cell genomics with templated emulsification. Nature Biotechnology. Online Ahead of Print. Current single-cell RNA-sequencing approaches have limitations that stem from the microfluidic devices or fluid handling steps required for sample processing. We develop a method that does not require specialized microfluidic devices, expertise or hardware. Our approach is based on particle-templated emulsification, which allows single-cell encapsulation and barcoding of cDNA in uniform droplet emulsions with only a vortexer. Particle-templated instant partition sequencing (PIP-seq) accommodates a wide range of emulsification formats, including microwell plates and large-volume conical tubes, enabling thousands of samples or millions of cells to be processed in minutes. We demonstrate that PIP-seq produces high-purity transcriptomes in mouse-human mixing studies, is compatible with multiomics measurements and can accurately characterize cell types in human breast tissue compared to a commercial microfluidic platform. Single-cell transcriptional profiling of mixed phenotype acute leukemia using PIP-seq reveals the emergence of heterogeneity within chemotherapy-resistant cell subsets that were hidden by standard immunophenotyping. PIP-seq is a simple, flexible and scalable next-generation workflow that extends single-cell sequencing to new applications. Full Text

Dall'Agnese, A., and Young, R. (2023). Regulatory architecture of cell identity genes and housekeeping genes.Trends in Cell Biology. Online Ahead of Print. Gene regulation and chromosome architecture are intimately linked. Genes with prominent roles in cell identity are often regulated by clusters of enhancer elements. By contrast, a recent study shows housekeeping genes are often regulated through clustering of promoters. We discuss here new regulatory insights for these two types of genes. Full Text

Dall'Agnese, G., Dall'Agnese, A., Banani, S.F., Codrich, M., Malfatti, M.C., Antoniali, G., and Tell, G. (2023). Role of condensates in modulating DNA repair pathways and its implication for chemoresistance. Journal of Biological Chemistry. Online Ahead of Print. For cells, it is important to repair DNA damage, such as double strand and single strand DNA breaks, because unrepaired DNA can compromise genetic integrity, potentially leading to cell death or cancer. Cells have multiple DNA damage repair pathways that have been the subject of detailed genetic, biochemical, and structural studies. Recently, the scientific community has started to gain evidence that the repair of DNA double strand breaks may occur within biomolecular condensates and that condensates may also contribute to DNA damage through concentrating genotoxic agents used to treat various cancers. Here, we summarize key features of biomolecular condensates and note where they have been implicated in the repair of DNA double strand breaks. We also describe evidence suggesting that condensates may be involved in the repair of other types of DNA damage, including single strand DNA breaks, nucleotide modifications (e.g., mismatch and oxidized bases) and bulky lesions, among others. Finally, we discuss old and new mysteries that could now be addressed considering the properties of condensates, including chemoresistance mechanisms. Full Text

Das, M.R., Chang, Y., Anderson, R., Saunders, R.A., Zhang, N., Tomberlin, C.P., Vale, R.D., and Jain, A. (2023). Repeat-associated non-AUG translation induces cytoplasmic aggregation of CAG repeat-containing RNAs. PNAS 120(3):e2215071120. CAG trinucleotide repeat expansions cause several neurodegenerative diseases, including Huntington's disease and spinocerebellar ataxia. RNAs with expanded CAG repeats contribute to disease in two unusual ways. First, these repeat-containing RNAs may agglomerate in the nucleus as foci that sequester several RNA-binding proteins. Second, these RNAs may undergo aberrant repeat-associated non-AUG (RAN) translation in multiple frames and produce aggregation-prone proteins. The relationship between RAN translation and RNA foci, and their relative contributions to cellular dysfunction, are unclear. Here, we show that CAG repeat-containing RNAs that undergo RAN translation first accumulate at nuclear foci and, over time, are exported to the cytoplasm. In the cytoplasm, these RNAs are initially dispersed but, upon RAN translation, aggregate with the RAN translation products. These RNA-RAN protein agglomerates sequester various RNA-binding proteins and are associated with the disruption of nucleocytoplasmic transport and cell death. In contrast, RNA accumulation at nuclear foci alone does not produce discernable defects in nucleocytoplasmic transport or cell viability. Inhibition of RAN translation prevents cytoplasmic RNA aggregation and alleviates cell toxicity. Our findings demonstrate that RAN translation-induced RNA-protein aggregation correlates with the key pathological hallmarks observed in disease and suggest that cytoplasmic RNA aggregation may be an underappreciated phenomenon in CAG trinucleotide repeat expansion disorders. Full Text

Dejosez, M., Dall'Agnese, A., Ramamoorthy, M., Platt, J., Yin, X., Hogan, M., Brosh, R., Weintraub, A.S., Hnisz, D., Abraham, B.J., Richard A. Young, et al. (2023). Regulatory architecture of housekeeping genes is driven by promoter assemblies. Cell Reports 42(5):112505. Genes that are key to cell identity are generally regulated by cell-type-specific enhancer elements bound by transcription factors, some of which facilitate looping to distant gene promoters. In contrast, genes that encode housekeeping functions, whose regulation is essential for normal cell metabolism and growth, generally lack interactions with distal enhancers. We find that Ronin (Thap11) assembles multiple promoters of housekeeping and metabolic genes to regulate gene expression. This behavior is analogous to how enhancers are brought together with promoters to regulate cell identity genes. Thus, Ronin-dependent promoter assemblies provide a mechanism to explain why housekeeping genes can forgo distal enhancer elements and why Ronin is important for cellular metabolism and growth control. We propose that clustering of regulatory elements is a mechanism common to cell identity and housekeeping genes but is accomplished by different factors binding distinct control elements to establish enhancer-promoter or promoter-promoter interactions, respectively. Full Text

Déjosez, M., Marin, A., Hughes, G.M., Morales, A.E., Godoy-Parejo, C., Gray, J.L., Qin, Y., Singh, A.A., Xu, H., Juste, J., Richard A Young, et al. (2023). Bat pluripotent stem cells reveal unusual entanglement between host and viruses. Cell. In Press, Corrected Proof. Bats are distinctive among mammals due to their ability to fly, use laryngeal echolocation, and tolerate viruses. However, there are currently no reliable cellular models for studying bat biology or their response to viral infections. Here, we created induced pluripotent stem cells (iPSCs) from two species of bats: the wild greater horseshoe bat (Rhinolophus ferrumequinum) and the greater mouse-eared bat (Myotis myotis). The iPSCs from both bat species showed similar characteristics and had a gene expression profile resembling that of cells attacked by viruses. They also had a high number of endogenous viral sequences, particularly retroviruses. These results suggest that bats have evolved mechanisms to tolerate a large load of viral sequences and may have a more intertwined relationship with viruses than previously thought. Further study of bat iPSCs and their differentiated progeny will provide insights into bat biology, virus host relationships, and the molecular basis of bats' special traits. Full Text

Diaz-Ortiz, M.E., Jain, N., Gallagher, M.D., Posavi, M., Unger, T.L., and Chen-Plotkin, A.S. (2023). Testing for Allele-specific Expression from Human Brain Samples. Bio Protocol 13(19):e4832. .Many single nucleotide polymorphisms (SNPs)identified by genome-wide association studies exert their effects on disease risk as expression quantitative trait loci (eQTL) via allele-specific expression (ASE). While databases for probing eQTLs in tissues from normal individuals exist, one may wish to ascertain eQTLs or ASE in specific tissues or disease-states not characterized in these databases. Here, we present a protocol to assess ASE of two possible target genes (GPNMB and KLHL7) of a known genome-wide association study (GWAS) Parkinson's disease (PD) risk locus in postmortem human brain tissue from PD and neurologically normal individuals. This was done using a sequence of RNA isolation, cDNA library generation, enrichment for transcripts of interest using customizable cDNA capture probes, paired-end RNA sequencing, and subsequent analysis. This method provides increased sensitivity relative to traditional bulk RNAseq-based and a blueprint that can be extended to the study of other genes, tissues, and disease states. Key features • Analysis of GPNMB allele-specific expression (ASE) in brain lysates from cognitively normal controls (NC) and Parkinson's disease (PD) individuals. • Builds on the ASE protocol of Mayba et al. (2014) and extends application from cells to human tissue. • Increased sensitivity by enrichment for desired transcript via RNA CaptureSeq (Mercer et al., 2014). • Optimized for human brain lysates from cingulate gyrus, caudate nucleus, and cerebellum. Full Text

Diegmiller, R., Imran Alsous, J., Li, D., Yamashita, Y.M., and Shvartsman, S.Y. (2023). Fusome topology and inheritance during insect gametogenesis. PLoS Computational Biology 19(2):e1010875. From insects to mammals, oocytes and sperm develop within germline cysts comprising cells connected by intercellular bridges (ICBs). In numerous insects, formation of the cyst is accompanied by growth of the fusome-a membranous organelle that permeates the cyst. Fusome composition and function are best understood in Drosophila melanogaster: during oogenesis, the fusome dictates cyst topology and size and facilitates oocyte selection, while during spermatogenesis, the fusome synchronizes the cyst's response to DNA damage. Despite its distinct and sex-specific roles during insect gametogenesis, elucidating fusome growth and inheritance in females and its structure and connectivity in males has remained challenging. Here, we take advantage of advances in three-dimensional (3D) confocal microscopy and computational image processing tools to reconstruct the topology, growth, and distribution of the fusome in both sexes. In females, our experimental findings inform a theoretical model for fusome assembly and inheritance and suggest that oocyte selection proceeds through an 'equivalency with a bias' mechanism. In males, we find that cell divisions can deviate from the maximally branched pattern observed in females, leading to greater topological variability. Our work consolidates existing disjointed experimental observations and contributes a readily generalizable computational approach for quantitative studies of gametogenesis within and across species. Full Text

Edwards, A., Njaci, I., Sarkar, A., Jiang, Z., Kaithakottil, G.G., Moore, C., Cheema, J., Stevenson, C.E.M., Rejzek, M., Novák, P., Colin Y Kim, Jing-Ke Weng, et al. (2023). Genomics and biochemical analyses reveal a metabolon key to β-L-ODAP biosynthesis in Lathyrus sativus. Nature Communications 14 (1) : 876. Grass pea (Lathyrus sativus L.) is a rich source of protein cultivated as an insurance crop in Ethiopia, Eritrea, India, Bangladesh, and Nepal. Its resilience to both drought and flooding makes it a promising crop for ensuring food security in a changing climate. The lack of genetic resources and the crop's association with the disease neurolathyrism have limited the cultivation of grass pea. Here, we present an annotated, long read-based assembly of the 6.5 Gbp L. sativus genome. Using this genome sequence, we have elucidated the biosynthetic pathway leading to the formation of the neurotoxin, β-L-oxalyl-2,3-diaminopropionic acid (β-L-ODAP). The final reaction of the pathway depends on an interaction between L. sativus acyl-activating enzyme 3 (LsAAE3) and a BAHD-acyltransferase (LsBOS) that form a metabolon activated by CoA to produce β-L-ODAP. This provides valuable insight into the best approaches for developing varieties which produce substantially less toxin. Full Text

Engelen, S.E., Ververs, F.A., Markovska, A., Lagerholm, B.C., Kraaijenhof, J.M., Yousif, L.I., Zurke, Y.X., Gulersonmez, C.M., Kooijman, S., Goddard, M., Hidde L. Ploegh , et al. (2023). Lipoproteins act as vehicles for lipid antigen delivery and activation of invariant natural killer T-cells. JCI Insight : e158089. Invariant Natural Killer T (iNKT) cells act at the interface between lipid metabolism and immunity, due to their restriction to lipid antigens presented on CD1d by antigen presenting cells (APC). How foreign lipid antigens are delivered to APC remains elusive. Since lipoproteins routinely bind glycosylceramides structurally similar to lipid antigens, we hypothesized that circulating lipoproteins form complexes with foreign lipid antigens. In this study, we used 2-color fluorescence correlation spectroscopy to show, for the first time, stable complex formation of lipid antigens α-galactosylceramide (αGalCer), Isoglobotrihexosylceramide (iGb3) and OCH, a sphingosine-truncated analogue of αGalCer, with very-low-density (VLDL) and/or low-density (LDL) lipoproteins in vitro and in vivo. We demonstrate LDL receptor (LDLR)-mediated uptake of lipoprotein-αGalCer complexes by APCs, leading to potent complex-mediated activation of iNKT cells in vitro and in vivo. Finally, LDLR-mutant PBMCs of patients with familial hypercholesterolemia showed impaired activation and proliferation of iNKT cells upon stimulation, underscoring the relevance of lipoproteins as a lipid antigen delivery system in humans. Taken together, circulating lipoproteins form complexes with lipid antigens to facilitate their transport and uptake by APCs, leading to enhanced iNKT cell activation. This study thereby reveals a novel mechanism of lipid antigen delivery to APCs, and provides further insight in the immunological capacities of circulating lipoproteins. Full Text

Ensinck, I., Maman, A., Albihlal, W.S., Lassandro, M., Salzano, G., Sideri, T., Howell, S.A., Calvani, E., Patel, H., Bushkin, G., et al. (2023). The yeast RNA methylation complex consists of conserved yet reconfigured components with m6A-dependent and independent roles. Elife 12:RP87860. N6-methyladenosine (m6A), the most abundant mRNA modification, is deposited in mammals/insects/plants by m6A Methyltransferase complexes (MTC) comprising a catalytic subunit and at least five additional proteins. The yeast MTC is critical for meiosis and was known to comprise three proteins, of which two were conserved. We uncover three novel MTC components (Kar4/Ygl036w-Vir1/Dyn2). All MTC subunits, except for Dyn2, are essential for m6A deposition and have corresponding mammalian MTC orthologues. Unlike the mammalian bipartite MTC, the yeast MTC is unipartite, yet multifunctional. The mRNA interacting module, comprising Ime4, Mum2, Vir1, and Kar4, exerts the MTC's m6A-independent function, while Slz1 enables the MTC catalytic function in m6A deposition. Both functions are critical for meiotic progression. Kar4 also has a mechanistically separate role from the MTC during mating. The yeast MTC constituents play distinguishable m6A-dependent, MTC-dependent, and MTC-independent functions, highlighting their complexity and paving the path towards dissecting multi-layered MTC functions in mammals. Full Text

Fallon, T.R., Čalounová, T., Mokrejš, M., Weng, J.K., and Pluskal, T. (2023). transXpress: a Snakemake pipeline for streamlined de novo transcriptome assembly and annotation. BMC Bioinformatics 24(1):133. BACKGROUND: RNA-seq followed by de novo transcriptome assembly has been a transformative technique in biological research of non-model organisms, but the computational processing of RNA-seq data entails many different software tools. The complexity of these de novo transcriptomics workflows therefore presents a major barrier for researchers to adopt best-practice methods and up-to-date versions of software. RESULTS: Here we present a streamlined and universal de novo transcriptome assembly and annotation pipeline, transXpress, implemented in Snakemake. transXpress supports two popular assembly programs, Trinity and rnaSPAdes, and allows parallel execution on heterogeneous cluster computing hardware. CONCLUSIONS: transXpress simplifies the use of best-practice methods and up-to-date software for de novo transcriptome assembly, and produces standardized output files that can be mined using SequenceServer to facilitate rapid discovery of new genes and proteins in non-model organisms. Full Text

Fingerhut, J.M., and Yamashita, Y.M. (2023). Analysis of Gene Expression Patterns and RNA Localization by Fluorescence in Situ Hybridization in Whole Mount Drosophila Testes. Methods in Molecular Biology 2666:15-28. Researchers have used RNA in situ hybridization to detect the presence of RNA in cells and tissues for approximately 50 years. The recent development of a method capable of visualizing a single RNA molecule by utilizing tiled fluorescently labeled oligonucleotide probes that together produce a diffraction-limited spot has greatly increased the resolution of this technique, allowing for the precise determination of subcellular RNA localization and relative abundance. Here, we present our method for single molecule RNA fluorescence in situ hybridization (smFISH) in whole mount Drosophila testes and discuss how we have utilized this method to better understand the expression pattern of the highly unusual Y-linked genes. Full Text

Flamier, A., Bisht, P., Richards, A., Tomasello, D.L., and Jaenisch, R. (2023). Human iPS cell-derived sensory neurons can be infected by SARS-CoV-2. iScience 26(9):107690. COVID-19 has impacted billions of people since 2019 and unfolded a major healthcare crisis. With an increasing number of deaths and the emergence of more transmissible variants, it is crucial to better understand the biology of the disease-causing virus, the SARS-CoV-2. Peripheral neuropathies appeared as a specific COVID-19 symptom occurring at later stages of the disease. In order to understand the impact of SARS-CoV-2 on the peripheral nervous system, we generated human sensory neurons from induced pluripotent stem cells that we infected with the SARS-CoV-2 strain WA1/2020 and the variants delta and omicron. Using single-cell RNA sequencing, we found that human sensory neurons can be infected by SARS-CoV-2 but are unable to produce infectious viruses. Our data indicate that sensory neurons can be infected by the original WA1/2020 strain of SARS-CoV-2 as well as the delta and omicron variants, yet infectability differs between the original strain and the variants. Full Text

Flynn, J.M., and Yamashita, Y.M. (2023). The implications of satellite DNA instability on cellular function and evolution. Seminars in Cell & Developmental Biology. Online Ahead of Print. Abundant tandemly repeated satellite DNA is present in most eukaryotic genomes. Previous limitations including a pervasive view that it was uninteresting junk DNA, combined with challenges in studying it, are starting to dissolve - and recent studies have found important functions for satellite DNAs. The observed rapid evolution and implied instability of satellite DNA now has important significance for their functions and maintenance within the genome. In this review, we discuss the processes that lead to satellite DNA copy number instability, and the importance of mechanisms to manage the potential negative effects of instability. Satellite DNA is vulnerable to challenges during replication and repair, since it forms difficult-to-process secondary structures and its homology within tandem arrays can result in various types of recombination. Satellite DNA instability may be managed by DNA or chromatin-binding proteins ensuring proper nuclear localization and repair, or by proteins that process aberrant structures that satellite DNAs tend to form. We also discuss the pattern of satellite DNA mutations from recent mutation accumulation (MA) studies that have tracked changes in satellite DNA for up to 1000 generations with minimal selection. Finally, we highlight examples of satellite evolution from studies that have characterized satellites across millions of years of Drosophila fruit fly evolution, and discuss possible ways that selection might act on the satellite DNA composition. Full Text

Flynn, J.M., Hu, K.B., and Clark, A.G. (2023). Three recent sex chromosome-to-autosome fusions in a Drosophila virilis strain with high satellite DNA content. Genetics 224(2):iyad062. The karyotype, or number and arrangement of chromosomes, has varying levels of stability across both evolution and disease. Karyotype changes often originate from DNA breaks near the centromeres of chromosomes, which generally contain long arrays of tandem repeats or satellite DNA. Drosophila virilis possesses among the highest relative satellite abundances of studied species, with almost half its genome composed of three related 7 bp satellites. We discovered a strain of D. virilis that we infer recently underwent three independent chromosome fusion events involving the X and Y chromosomes, in addition to one subsequent fission event. Here, we isolate and characterize the four different karyotypes we discovered in this strain which we believe demonstrates remarkable genome instability. We discovered that one of the substrains with an X-autosome fusion has an X-to-Y chromosome nondisjunction rate 20 x higher than the D. virilis reference strain (21% vs 1%). Finally, we found an overall higher rate of DNA breakage in the substrain with higher satellite DNA compared to a genetically similar substrain with less satellite DNA. This suggests that satellite DNA abundance may play a role in the risk of genome instability. Overall, we introduce a novel system consisting of a single strain with four different karyotypes, which we believe will be useful for future studies of genome instability, centromere function, and sex chromosome evolution. Full Text

Gäbelein, C.G., and Lehmann, R. (2023). Mechanical activation of mitochondria in germ cell differentiation. Trends in Cell Biology. Online Ahead of Print. Mitochondria are activated during stem cell differentiation. Recently, Wang et al. found that mechanical stimulation from tissue surrounding differentiating germ cells in the female fly ovary is necessary to sustain intracellular calcium levels, promoting mitochondrial activity. This suggests a molecular link between cell mechanics and developmental metabolic transitions in eukaryotes. Full Text

Galhenage, P., Zhou, Y., Perry, E., Loc, B., Fietz, K., Iyer, S., Reinhardt, F., Da Silva, T., Botchkarev, V., Chen, J., Robert A Weinberg, et al. (2023). Replication stress and defective checkpoints make fallopian tube epithelial cells putative drivers of high-grade serous ovarian cancer. Cell Reports 42(10):113144. Clinical and molecular evidence indicates that high-grade serous ovarian cancer (HGSOC) primarily originates from the fallopian tube, not the ovarian surface. However, the reasons for this preference remain unclear. Our study highlights significant differences between fallopian tube epithelial (FTE) and ovarian surface epithelial (OSE) cells, providing the molecular basis for FTEs as site of origin of HGSOC. FTEs, unlike OSEs, exhibit heightened replication stress (RS), impaired repair of stalled forks, ineffective G2/M checkpoint, and increased tumorigenicity. BRCA1 heterozygosity exacerbates these defects, resulting in RS suppression haploinsufficiency and an aggressive tumor phenotype. Examination of human and mouse sections reveals buildup of the RS marker 53BP1 primarily in the fallopian tubes, particularly at the fimbrial ends. Furthermore, menopausal status influences RS levels. Our study provides a mechanistic rationale for FTE as the site of origin for HGSOC, investigates the impact of BRCA1 heterozygosity, and lays the groundwork for targeting early HGSOC drivers. Full Text

Gao, J., Zhang, R., Zheng, L., Song, L., Ji, M., Li, S., Wang, J., Yang, J., Kang, G., Zhang, P., David Pincus, et al. (2023). Blue light receptor CRY1 regulates HSFA1d nuclear localization to promote plant thermotolerance. Cell Rep 42(9):113117. Temperature increases as light intensity rises, but whether light signals can be directly linked to high temperature response in plants is unclear. Here, we find that light pre-treatment enables plants to survive better under high temperature, designated as light-induced thermotolerance (LIT). With short-term light treatment, plants induce light-signaling pathway genes and heat shock genes. Blue light photoreceptor cryptochrome 1 (CRY1) is required for LIT. We also find that CRY1 physically interacts with the heat shock transcription factor A1d (HsfA1d) and that HsfA1d is involved in thermotolerance under light treatment. Furthermore, CRY1 promotes HsfA1d nuclear localization through importin alpha 1 (IMPα1). Consistent with this, CRY1 shares more than half of the chromatin binding sites with HsfA1d. Mutation of CRY1 (cry1-304) diminishes a large number of HsfA1d binding sites that are shared with CRY1. We present a model where, by coupling light sensing to high-temperature stress, CRY1 confers thermotolerance in plants via HsfA1d. Full Text

Gerton, T.J., Green, A., Campisi, M., Chen, M., Gjeci, I., Mahadevan, N., Lee, C.A.A., Mishra, R., Vo, H.V., Haratani, K., et al. (2023). Development of a Patient-Derived 3D Immuno-Oncology Platform to Potentiate Immunotherapy Responses in Ascites-Derived Circulating Tumor Cells. Cancers (Basel) 15(16):4128. High-grade serous ovarian cancer (HGSOC) is responsible for the majority of gynecology cancer-related deaths. Patients in remission often relapse with more aggressive forms of disease within 2 years post-treatment. Alternative immuno-oncology (IO) strategies, such as immune checkpoint blockade (ICB) targeting the PD-(L)1 signaling axis, have proven inefficient so far. Our aim is to utilize epigenetic modulators to maximize the benefit of personalized IO combinations in ex vivo 3D patient-derived platforms and in vivo syngeneic models. Using patient-derived tumor ascites, we optimized an ex vivo 3D screening platform (PDOTS), which employs autologous immune cells and circulating ascites-derived tumor cells, to rapidly test personalized IO combinations. Most importantly, patient responses to platinum chemotherapy and poly-ADP ribose polymerase inhibitors in 3D platforms recapitulate clinical responses. Furthermore, similar to clinical trial results, responses to ICB in PDOTS tend to be low and positively correlated with the frequency of CD3+ immune cells and EPCAM+/PD-L1+ tumor cells. Thus, the greatest response observed with anti-PD-1/anti-PD-L1 immunotherapy alone is seen in patient-derived HGSOC ascites, which present with high levels of systemic CD3+ and PD-L1+ expression in immune and tumor cells, respectively. In addition, priming with epigenetic adjuvants greatly potentiates ICB in ex vivo 3D testing platforms and in vivo tumor models. We further find that epigenetic priming induces increased tumor secretion of several key cytokines known to augment T and NK cell activation and cytotoxicity, including IL-6, IP-10 (CXCL10), KC (CXCL1), and RANTES (CCL5). Moreover, epigenetic priming alone and in combination with ICB immunotherapy in patient-derived PDOTS induces rapid upregulation of CD69, a reliable early activation of immune markers in both CD4+ and CD8+ T cells. Consequently, this functional precision medicine approach could rapidly identify personalized therapeutic combinations able to potentiate ICB, which is a great advantage, especially given the current clinical difficulty of testing a high number of potential combinations in patients. Full Text

Green, J., Bruno, C.A., Traunmüller, L., Ding, J., Hrvatin, S., Wilson, D.E., Khodadad, T., Samuels, J., Greenberg, M.E., and Harvey, C.D. (2023). A cell-type-specific error-correction signal in the posterior parietal cortex. Nature. Online Ahead of Print. Neurons in the posterior parietal cortex contribute to the execution of goal-directed navigation(1) and other decision-making tasks(2-4). Although molecular studies have catalogued more than 50 cortical cell types(5), it remains unclear what distinct functions they have in this area. Here we identified a molecularly defined subset of somatostatin (Sst) inhibitory neurons that, in the mouse posterior parietal cortex, carry a cell-type-specific error-correction signal for navigation. We obtained repeatable experimental access to these cells using an adeno-associated virus in which gene expression is driven by an enhancer that functions specifically in a subset of Sst cells(6). We found that during goal-directed navigation in a virtual environment, this subset of Sst neurons activates in a synchronous pattern that is distinct from the activity of surrounding neurons, including other Sst neurons. Using in vivo two-photon photostimulation and ex vivo paired patch-clamp recordings, we show that nearby cells of this Sst subtype excite each other through gap junctions, revealing a self-excitation circuit motif that contributes to the synchronous activity of this cell type. These cells selectively activate as mice execute course corrections for deviations in their virtual heading during navigation towards a reward location, for both self-induced and experimentally induced deviations. We propose that this subtype of Sst neurons provides a self-reinforcing and cell-type-specific error-correction signal in the posterior parietal cortex that may help with the execution and learning of accurate goal-directed navigation trajectories. Full Text

Grill, S., Riley, A., Selvaraj, M., and Lehmann, R. (2023). HP6/Umbrea is dispensable for viability and fertility, suggesting essentiality of newly evolved genes is rare. PNAS 120 (39): e2309478120. The newly evolved gene Heterochromatin Protein 6 (HP6), which has been previously classified as essential, challenged the dogma that functions required for viability are only seen in genes with a long evolutionary history. Based on previous RNA-sequencing analysis in Drosophila germ cells, we asked whether HP6 might play a role in germline development. Surprisingly, we found that CRISPR-generated HP6 mutants are viable and fertile. Using previously generated mutants, we identified an independent lethal allele and an RNAi off-target effect that prevented accurate interpretation of HP6 essentiality. By reviewing existing data, we found that the vast majority of young genes that were previously classified as essential were indeed viable when tested with orthologous methods. Together, our data call into question the frequency with which newly evolved genes gain essential functions and suggest that using multiple independent genetic methods is essential when probing the functions of young genes. Full Text

Guna, A., Page, K.R., Replogle, J.M., Esantsi, T.K., Wang, M.L., Weissman, J.S., and Voorhees, R.M. (2023). A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens. BMC Genomics 24(1):651. Mapping genetic interactions is essential for determining gene function and defining novel biological pathways. We report a simple to use CRISPR interference (CRISPRi) based platform, compatible with Fluorescence Activated Cell Sorting (FACS)-based reporter screens, to query epistatic relationships at scale. This is enabled by a flexible dual-sgRNA library design that allows for the simultaneous delivery and selection of a fixed sgRNA and a second randomized guide, comprised of a genome-wide library, with a single transduction. We use this approach to identify epistatic relationships for a defined biological pathway, showing both increased sensitivity and specificity than traditional growth screening approaches. Full Text

Guna, A., Hazu, M., Tomaleri, G.P., and Voorhees, R.M. (2023). A TAle of Two Pathways: Tail-Anchored Protein Insertion at the Endoplasmic Reticulum.Cold Spring Harbor Perspectives in Biology 15(3):a041252. 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

Han, C.Z., Li, R.Z., Hansen, E., Trescott, S., Fixsen, B.R., Nguyen, C.T., Mora, C.M., Spann, N.J., Bennett, H.R., Poirion, O., Jennifer F Hughes, David C Page, et al. (2023). Human microglia maturation is underpinned by specific gene regulatory networks. Immunity. Online Ahead of Print. Microglia phenotypes are highly regulated by the brain environment, but the transcriptional networks that specify the maturation of human microglia are poorly understood. Here, we characterized stage-specific transcriptomes and epigenetic landscapes of fetal and postnatal human microglia and acquired corresponding data in induced pluripotent stem cell (iPSC)-derived microglia, in cerebral organoids, and following engraftment into humanized mice. Parallel development of computational approaches that considered transcription factor (TF) co-occurrence and enhancer activity allowed prediction of shared and state-specific gene regulatory networks associated with fetal and postnatal microglia. Additionally, many features of the human fetal-to-postnatal transition were recapitulated in a time-dependent manner following the engraftment of iPSC cells into humanized mice. These data and accompanying computational approaches will facilitate further efforts to elucidate mechanisms by which human microglia acquire stage- and disease-specific phenotypes. Full Text

Hemenway E.A., and Gehring, M. (2023). Epigenetic Regulation During Plant Development and the Capacity for Epigenetic Memory. Annual Review of Plant Biology 74, 87-109. 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

Hoang A.T., and Corradin, O. (2023). Epigenetic alterations identify a confluence of genetic vulnerabilities tied to opioid overdose. Neuropsychopharmacology. Online Ahead of Print. The most severe outcome of OUD, opioid overdose, has become the leading cause of accidental death in the US . Epigenetic studies have the potential to further understanding of this complex trait by directly assaying patient tissues. These studies can reveal a snapshot of the combined genetic and environmental influences on gene regulation that occurs throughout the course of disease. Full Text

Hochedlinger, K., Wagner, E.F., and Sabapathy, K. (2023). Correction: Differential effects of JNK1 and JNK2 on signal specific induction of apoptosis. Oncogene. Online Ahead of Print. These corrections do not change any of the conclusions of our manuscript (Oncogene. 2002 Apr 4;21(15):2441-5). Full Text

Inglis, A.J., Guna, A., Gálvez-Merchán, Á., Pal, A., Esantsi, T.K., Keys, H.R., Frenkel, E.M., Oania, R., Weissman, J.S., and Voorhees, R.M. (2023). Coupled protein quality control during nonsense-mediated mRNA decay. Journal of Cell Science 136(10):jcs261216. Translation of mRNAs containing premature termination codons (PTCs) results in truncated protein products with deleterious effects. Nonsense-mediated decay (NMD) is a surveillance pathway responsible for detecting PTC containing transcripts. Although the molecular mechanisms governing mRNA degradation have been extensively studied, the fate of the nascent protein product remains largely uncharacterized. Here, we use a fluorescent reporter system in mammalian cells to reveal a selective degradation pathway specifically targeting the protein product of an NMD mRNA. We show that this process is post-translational and dependent on the ubiquitin proteasome system. To systematically uncover factors involved in NMD-linked protein quality control, we conducted genome-wide flow cytometry-based screens. Our screens recovered known NMD factors but suggested that protein degradation did not depend on the canonical ribosome-quality control (RQC) pathway. A subsequent arrayed screen demonstrated that protein and mRNA branches of NMD rely on a shared recognition event. Our results establish the existence of a targeted pathway for nascent protein degradation from PTC containing mRNAs, and provide a reference for the field to identify and characterize required factors. Full Text

Insco, M.L., Abraham, B.J., Dubbury, S.J., Kaltheuner, I.H., Dust, S., Wu, C., Chen, K.Y., Liu, D., Bellaousov, S., Cox, A.M., Richard A. Young, et al. (2023). Oncogenic CDK13 mutations impede nuclear RNA surveillance. Science 380(6642):eabn7625. RNA surveillance pathways detect and degrade defective transcripts to ensure RNA fidelity. We found that disrupted nuclear RNA surveillance is oncogenic. Cyclin-dependent kinase 13 (CDK13) is mutated in melanoma, and patient-mutated CDK13 accelerates zebrafish melanoma. CDK13 mutation causes aberrant RNA stabilization. CDK13 is required for ZC3H14 phosphorylation, which is necessary and sufficient to promote nuclear RNA degradation. Mutant CDK13 fails to activate nuclear RNA surveillance, causing aberrant protein-coding transcripts to be stabilized and translated. Forced aberrant RNA expression accelerates melanoma in zebrafish. We found recurrent mutations in genes encoding nuclear RNA surveillance components in many malignancies, establishing nuclear RNA surveillance as a tumor-suppressive pathway. Activating nuclear RNA surveillance is crucial to avoid accumulation of aberrant RNAs and their ensuing consequences in development and disease. Full Text

Jo, A., Green, A., Medina, J.E., Iyer, S., Ohman, A.W., McCarthy, E.T., Reinhardt, F., Gerton, T., Demehin, D., Mishra, R., Robert A. Weinberg, et al. (2023). Inaugurating High-Throughput Profiling of Extracellular Vesicles for Earlier Ovarian Cancer Detection. Advanced Science (Weinheim) : e2301930. Detecting early cancer through liquid biopsy is challenging due to the lack of specific biomarkers for early lesions and potentially low levels of these markers. The current study systematically develops an extracellular-vesicle (EV)-based test for early detection, specifically focusing on high-grade serous ovarian carcinoma (HGSOC). The marker selection is based on emerging insights into HGSOC pathogenesis, notably that it arises from precursor lesions within the fallopian tube. This work thus establishes murine fallopian tube (mFT) cells with oncogenic mutations and performs proteomic analyses on mFT-derived EVs. The identified markers are then evaluated with an orthotopic HGSOC animal model. In serially-drawn blood of tumor-bearing mice, mFT-EV markers increase with tumor initiation, supporting their potential use in early cancer detection. A pilot clinical study (n = 51) further narrows EV markers to five candidates, EpCAM, CD24, VCAN, HE4, and TNC. The combined expression of these markers distinguishes HGSOC from non-cancer with 89% sensitivity and 93% specificity. The same markers are also effective in classifying three groups (non-cancer, early-stage HGSOC, and late-stage HGSOC). The developed approach, for the first time inaugurated in fallopian tube-derived EVs, could be a minimally invasive tool to monitor women at high risk of ovarian cancer for timely intervention. Full Text

Jones, M.G., Yang, D., and Weissman, J.S. (2023). New Tools for Lineage Tracing in Cancer In Vivo.Annual Review of Cancer Biology 7: 111-129. During tumor evolution, cancer cells can acquire the ability to proliferate, invade neighboring tissues, evade the immune system, and spread systemically. Tracking this process remains challenging, as many key events occur stochastically and over long times, which could be addressed by studying the phylogenetic relationships among cancer cells. Several lineage tracing approaches have been developed and employed in many tumor models and contexts, providing critical insights into tumor evolution. Recent advances in single-cell lineage tracing have greatly expanded the resolution, scale, and readout of lineage tracing toolkits. In this review, we provide an overview of static lineage tracing methods, and then focus on evolving lineage tracing technologies that enable reconstruction of tumor phylogenies at unprecedented resolution. We also discuss in vivo applications of these technologies to profile subclonal dynamics, quantify tumor plasticity, and track metastasis. Finally, we highlight outstanding questions and emerging technologies for building comprehensive cancer evolution roadmaps. Full Text

Karg, M.M., Lu, Y.R., Refaian, N., Cameron, J., Hoffmann, E., Hoppe, C., Shirahama, S., Shah, M., Krasniqi, D., Krishnan, A., et al. (2023). Sustained Vision Recovery by OSK Gene Therapy in a Mouse Model of Glaucoma. Cell Reprogramming. Online Ahead of Print. Glaucoma, a chronic neurodegenerative disease, is a leading cause of age-related blindness worldwide and characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons. Previously, we developed a novel epigenetic rejuvenation therapy, based on the expression of the three transcription factors Oct4, Sox2, and Klf4 (OSK), which safely rejuvenates RGCs without altering cell identity in glaucomatous and old mice after 1 month of treatment. In the current year-long study, mice with continuous or cyclic OSK expression induced after glaucoma-induced vision damage had occurred were tracked for efficacy, duration, and safety. Surprisingly, only 2 months of OSK fully restored impaired vision, with a restoration of vision for 11 months with prolonged expression. In RGCs, transcription from the doxycycline (DOX)-inducible Tet-On AAV system, returned to baseline 4 weeks after DOX withdrawal. Significant vision improvements remained for 1 month post switching off OSK, after which the vision benefit gradually diminished but remained better than baseline. Notably, no adverse effects on retinal structure or body weight were observed in glaucomatous mice with OSK continuously expressed for 21 months providing compelling evidence of efficacy and safety. This work highlights the tremendous therapeutic potential of rejuvenating gene therapies using OSK, not only for glaucoma but also for other ocular and systemic injuries and age-related diseases. Full Text

Kasprzyk, P.G., Tremaine, L., Fahmi, O.A., and Weng, J.K. (2023). In Vitro Evaluation of the Potential for Drug Interactions by Salidroside. Nutrients 15(17): 3723. Several studies utilizing Rhodiola rosea, which contains a complex mixture of phytochemicals, reported some positive drug-drug interaction (DDI) findings based on in vitro CYP450's enzyme inhibition, MAO-A and MAO-B inhibition, and preclinical pharmacokinetic studies in either rats or rabbits. However, variation in and multiplicity of constituents present in Rhodiola products is a cause for concern for accurately evaluating drug-drug interaction (DDI) risk. In this report, we examined the effects of bioengineered, nature-identical salidroside on the inhibition potential of salidroside on CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 utilizing human liver microsomes, the induction potential of salidroside on CYP1A2, CYP2B6 and CYP3A4 in cryopreserved human hepatocytes, the inhibitory potential of salidroside against recombinant human MAO-A and MAO-B, and the OATP human uptake transport inhibitory potential of salidroside using transfected HEK293-OATP1B1 and OATP1B3 cells. The results demonstrate that the bioengineered salidroside at a concentration exceeding the predicted plasma concentrations of <2 µM (based on 60 mg PO) shows no risk for drug-drug interaction due to CYP450, MAO enzymes, or OATP drug transport proteins. Our current studies further support the safe use of salidroside in combination with other drugs cleared by CYP or MAO metabolism or OATP-mediated disposition. Full Text

Kilgore, H.R., Mikhael, P.G., Overholt, K.J., Boija, A., Hannett, N.M., Van Dongen, C., Lee, T.I., Chang, Y.T., Barzilay, R., and Young, R.A. (2023). Author Correction: Distinct chemical environments in biomolecular condensates. Nature Chemical Biology Published: 25 October 2023. In the version of the article initially published, there was an error in the “F1 score” bar graph in Fig. 4e, where the bars representing HP1α and “Random” were considerably lower than their intended values of 0.8 and 0.5, respectively. This has now been corrected in the HTML and PDF versions of the article. Full Text

Kilgore, H.R., Mikhael, P.G., Overholt, K.J., Boija, A., Hannett, N.M., Van Dongen, C., Lee, T.I., Chang, Y.T., Barzilay, R., and Young, R.A. (2023). Distinct chemical environments in biomolecular condensates.Nature Chemical Biology. Online Ahead of Print. Diverse mechanisms have been described for selective enrichment of biomolecules in membrane-bound organelles, but less is known about mechanisms by which molecules are selectively incorporated into biomolecular assemblies such as condensates that lack surrounding membranes. The chemical environments within condensates may differ from those outside these bodies, and if these differed among various types of condensate, then the different solvation environments would provide a mechanism for selective distribution among these intracellular bodies. Here we use small molecule probes to show that different condensates have distinct chemical solvating properties and that selective partitioning of probes in condensates can be predicted with deep learning approaches. Our results demonstrate that different condensates harbor distinct chemical environments that influence the distribution of molecules, show that clues to condensate chemical grammar can be ascertained by machine learning and suggest approaches to facilitate development of small molecule therapeutics with optimal subcellular distribution and therapeutic benefit. Full Text

Kilwein, M.D., Johnson, M.R., Thomalla, J.M., Mahowald, A., and Welte, M.A. (2023). Drosophila embryos spatially sort their nutrient stores to facilitate their utilization. Development. Online Ahead of Print. Animal embryos are provisioned by their mothers with a diverse nutrient supply critical for development. In Drosophila, the three most abundant nutrients (triglycerides, proteins, and glycogen) are sequestered in distinct storage structures, lipid droplets (LDs), yolk vesicles (YVs) and glycogen granules (GGs). Using transmission electron microscopy as well as live and fixed-sample fluorescence imaging, we find that all three storage structures are dispersed throughout the egg but are then spatially allocated to distinct tissues by gastrulation: LDs largely to the peripheral epithelium, YVs and GGs to the central yolk cell. To confound the embryo's ability to sort its nutrients, we employ mutants in Jabba and Mauve to generate LD:GG or LD:YV compound structures. In these mutants, LDs are missorted to the yolk cell and their turnover is delayed. Our observations demonstrate dramatic spatial nutrient sorting in early embryos and provide the first evidence for its functional importance. Full Text

Kim, C.Y., Mitchell, A.J., Kastner, D.W., Albright, C.E., Gutierrez, M.A., Glinkerman, C.M., Kulik, H.J., and Weng, J.K. (2023). Emergence of a proton exchange-based isomerization and lactonization mechanism in the plant coumarin synthase COSY. Nature Communications 14(1):597. Plants contain rapidly evolving specialized enzymes that support the biosynthesis of functionally diverse natural products. In coumarin biosynthesis, a BAHD acyltransferase-family enzyme COSY was recently discovered to accelerate coumarin formation as the only known BAHD enzyme to catalyze an intramolecular acyl transfer reaction. Here we investigate the structural and mechanistic basis for COSY's coumarin synthase activity. Our structural analyses reveal an unconventional active-site configuration adapted to COSY's specialized activity. Through mutagenesis studies and deuterium exchange experiments, we identify a unique proton exchange mechanism at the α-carbon of the o-hydroxylated trans-hydroxycinnamoyl-CoA substrates during the catalytic cycle of COSY. Quantum mechanical cluster modeling and molecular dynamics further support this key mechanism for lowering the activation energy of the rate-limiting trans-to-cis isomerization step in coumarin production. This study unveils an unconventional catalytic mechanism mediated by a BAHD-family enzyme, and sheds light on COSY's evolutionary origin and its recruitment to coumarin biosynthesis in eudicots. Full Text

Kim, J.J., Steinson, E.R., Lau, M.S., de Rooij, D.G., Page, D.C., and Kingston, R.E. (2023). Cell type-specific role of CBX2 and its disordered region in spermatogenesis. Genes and Development Online Ahead of Print. Polycomb group (PcG) proteins maintain the repressed state of lineage-inappropriate genes and are therefore essential for embryonic development and adult tissue homeostasis. One critical function of PcG complexes is modulating chromatin structure. Canonical Polycomb repressive complex 1 (cPRC1), particularly its component CBX2, can compact chromatin and phase-separate in vitro. These activities are hypothesized to be critical for forming a repressed physical environment in cells. While much has been learned by studying these PcG activities in cell culture models, it is largely unexplored how cPRC1 regulates adult stem cells and their subsequent differentiation in living animals. Here, we show in vivo evidence of a critical nonenzymatic repressive function of cPRC1 component CBX2 in the male germline. CBX2 is up-regulated as spermatogonial stem cells differentiate and is required to repress genes that were active in stem cells. CBX2 forms condensates (similar to previously described Polycomb bodies) that colocalize with target genes bound by CBX2 in differentiating spermatogonia. Single-cell analyses of mosaic Cbx2 mutant testes show that CBX2 is specifically required to produce differentiating A1 spermatogonia. Furthermore, the region of CBX2 responsible for compaction and phase separation is needed for the long-term maintenance of male germ cells in the animal. These results emphasize that the regulation of chromatin structure by CBX2 at a specific stage of spermatogenesis is critical, which distinguishes this from a mechanism that is reliant on histone modification. Full Text

Koblan, L.W., Arbab, M., Shen, M.W., Hussmann, J.A., Anzalone, A.V., Doman, J.L., Newby, G.A., Yang, D., Mok, B., Replogle, J.M., Jonathan S. Weissman, et al. (2023). Author Correction: Efficient C•G-to-G•C base editors developed using CRISPRi screens, target-library analysis, and machine learning. Nature Biotechnology. Published: 18 October 2023. In the version of the article initially published, in the “High-throughput DNA sequencing of genomic DNA samples” section of the Methods, the formula used to calculate product purity was incorrect, and has been updated to read “C•G-to-G•C editing yield ÷ (C•G-to-G•C editing yield + C•G-to-T•A yield + C•G-to-A•T yield + indels)” in the HTML and PDF versions of the article. Full Text

Krishnan, B., Sanidas, I., and Dyson, N.J. (2023). Seeing is believing: the impact of RB on nuclear organization. Cell Cycle. Online Ahead of Print. The retinoblastoma tumor suppressor (RB) prevents G1 to S cell cycle transition by inhibiting E2F activity. This function requires that RB remains un- or underphosphorylated (the so-called active forms of RB). Recently, we showed that active forms of RB cause widespread changes in nuclear architecture that are visible under a microscope. These phenotypes did not correlate with cell cycle arrest or repression of the E2F transcriptional program, but appeared later, and were associated with the appearance of autophagy or in IMR-90 cells with senescence markers. In this perspective, we describe the relative timing of these RB-induced events and discuss the mechanisms that may underlie RB-induced chromatin dispersion. We consider the relationship between RB-induced dispersion, autophagy, and senescence and the potential connection between dispersion and cell cycle exit. Full Text

Lasry R., Maoz, N., Cheng, A.W., Yom Tov, N., Kulenkampff, E., Azagury, M., Yang, H., Ople, C., Markoulaki, S., Faddah, D.A.Rudolf Jaenisch, et al. (2023). Complex haploinsufficiency in pluripotent cells yields somatic cells with DNA methylation abnormalities and pluripotency induction defects. Stem Cell Reports. Online Ahead of Print. A complete knockout of a single key pluripotency gene may drastically affect embryonic stem cell function and epigenetic reprogramming. In contrast, elimination of only one allele of a single pluripotency gene is mostly considered harmless to the cell. To understand whether complex haploinsufficiency exists in pluripotent cells, we simultaneously eliminated a single allele in different combinations of two pluripotency genes (i.e., Nanog(+/-);Sall4(+/-), Nanog(+/-);Utf1(+/-), Nanog(+/-);Esrrb(+/-) and Sox2(+/-);Sall4(+/-)). Although these double heterozygous mutant lines similarly contribute to chimeras, fibroblasts derived from these systems show a significant decrease in their ability to induce pluripotency. Tracing the stochastic expression of Sall4 and Nanog at early phases of reprogramming could not explain the seen delay or blockage. Further exploration identifies abnormal methylation around pluripotent and developmental genes in the double heterozygous mutant fibroblasts, which could be rescued by hypomethylating agent or high OSKM levels. This study emphasizes the importance of maintaining two intact alleles for pluripotency induction. Full Text

Licon, M.H., Giuliano, C.J., Chan, A.W. Chakladar, S., Eberhard, J.N., Shallberg, L.A., Chandrasekaran, S., Waldman, B.S.., Koshy, A.A., Hunter, C.A., Sebastian Lourido, et al. (2023). A positive feedback loop controls Toxoplasma chronic differentiation. Nature Microbiology. Online Ahead of Print. Successful infection strategies must balance pathogen amplification and persistence. In the obligate intracellular parasite Toxoplasma gondii this is accomplished through differentiation into dedicated cyst-forming chronic stages that avoid clearance by the host immune system. The transcription factor BFD1 is both necessary and sufficient for stage conversion; however, its regulation is not understood. In this study we examine five factors that are transcriptionally activated by BFD1. One of these is a cytosolic RNA-binding protein of the CCCH-type zinc-finger family, which we name bradyzoite formation deficient 2 (BFD2). Parasites lacking BFD2 fail to induce BFD1 and are consequently unable to fully differentiate in culture or in mice. BFD2 interacts with the BFD1 transcript under stress, and deletion of BFD2 reduces BFD1 protein levels but not messenger RNA abundance. The reciprocal effects on BFD2 transcription and BFD1 translation outline a positive feedback loop that enforces the chronic-stage gene-expression programme. Thus, our findings help explain how parasites both initiate and commit to chronic differentiation. This work provides new mechanistic insight into the regulation of T. gondii persistence, and can be exploited in the design of strategies to prevent and treat these key reservoirs of human infection. Full Text

Licon, M.H., Giuliano, C.J., Chan, A.W. Chakladar, S., Eberhard, J.N., Shallberg, L.A., Chandrasekaran, S., Waldman, B.S.., Koshy, A.A., Hunter, C.A., Sebastian Lourido, et al.(2023). Author Correction: A positive feedback loop controls Toxoplasma chronic differentiation. Nature Microbiology. Online Ahead of Print. Full Text

Linder, S.J., Bernasocchi, T., Martínez-Pastor, B., Sullivan, K.D., Galbraith, M.D., Lewis, C.A., Ferrer, C.M., Boon, R., Silveira, G.G., Cho, H.M., et al. (2023). Inhibition of the proline metabolism rate-limiting enzyme P5CS allows proliferation of glutamine-restricted cancer cells. Nature Metabolism. Online Ahead of Print. Glutamine is a critical metabolite for rapidly proliferating cells as it is used for the synthesis of key metabolites necessary for cell growth and proliferation. Glutamine metabolism has been proposed as a therapeutic target in cancer and several chemical inhibitors are in development or in clinical trials. How cells subsist when glutamine is limiting is poorly understood. Here, using an unbiased screen, we identify ALDH18A1, which encodes P5CS, the rate-limiting enzyme in the proline biosynthetic pathway, as a gene that cells can downregulate in response to glutamine starvation. Notably, P5CS downregulation promotes de novo glutamine synthesis, highlighting a previously unrecognized metabolic plasticity of cancer cells. The glutamate conserved from reducing proline synthesis allows cells to produce the key metabolites necessary for cell survival and proliferation under glutamine-restricted conditions. Our findings reveal an adaptive pathway that cancer cells acquire under nutrient stress, identifying proline biosynthesis as a previously unrecognized major consumer of glutamate, a pathway that could be exploited for developing effective metabolism-driven anticancer therapies. Full Text

Lofrano-Porto, A., Pereira, S.A., Dauber, A., Bloom, J.C., Fontes, A.N., Asimow, N., de Moraes, O.L., Araujo, P.A.T., Abreu, A.P., Guo, M.H., et al. (2023). OSR1 disruption contributes to uterine factor infertility via impaired Müllerian duct development and endometrial receptivity. Journal of Clinical Investigation : 161701. Three sisters, born from consanguineous parents, manifested a unique Mullerian anomaly characterized by uterine hypoplasia with thin estrogen-unresponsive endometrium, primary amenorrhea, but spontaneous tubal pregnancies. Through whole-exome sequencing followed by comprehensive genetic analysis, a missense variant was identified in the OSR1 gene. We therefore investigated OSR1/OSR1 expression in postpubertal human uteri, and the prenatal and postnatal expression pattern of Osr1/Osr1 in murine developing Mullerian ducts (MDs) and endometrium, respectively. We then investigated whether Osr1 deletion would affect MD development, using wild-type and genetically engineered mice. Human uterine OSR1/OSR1 expression was found primarily in the endometrium. Mouse Osr1 was expressed prenatally in MDs and Wolffian ducts (WDs), from rostral to caudal segments, in E13.5 embryos. MDs and WDs were absent on the left side and MDs were rostrally truncated on the right side of E13.5 Osr1-/- embryos. Postnatally, Osr1 was expressed in mouse uteri throughout lifespan, peaking at postnatal days 14 and 28. Osr1 protein was present primarily in uterine luminal and glandular epithelial cells and in the epithelial cells of mouse oviducts. Through this translational approach, we demonstrated that OSR1/Osr1 is important for MD development and endometrial receptivity and may be implicated in uterine factor infertility. Full Text

Lu, Y.R., Tian, X., and Sinclair, D.A. (2023). The Information Theory of Aging. Nature Aging 3(12):1486-1499. Information storage and retrieval is essential for all life. In biology, information is primarily stored in two distinct ways: the genome, comprising nucleic acids, acts as a foundational blueprint and the epigenome, consisting of chemical modifications to DNA and histone proteins, regulates gene expression patterns and endows cells with specific identities and functions. Unlike the stable, digital nature of genetic information, epigenetic information is stored in a digital-analog format, susceptible to alterations induced by diverse environmental signals and cellular damage. The Information Theory of Aging (ITOA) states that the aging process is driven by the progressive loss of youthful epigenetic information, the retrieval of which via epigenetic reprogramming can improve the function of damaged and aged tissues by catalyzing age reversal. Full Text

Mangalhara, K.C., Varanasi, S.K., Johnson, M.A., Burns, M.J., Rojas, G.R., Esparza Moltó, P.B., Sainz, A.G., Tadepalle, N., Abbott, K.L., Mendiratta, G., Tenzin Kunchok , et al. (2023). Manipulating mitochondrial electron flow enhances tumor immunogenicity. Science 381 (6664): 1316-1323. Although tumor growth requires the mitochondrial electron transport chain (ETC), the relative contribution of complex I (CI) and complex II (CII), the gatekeepers for initiating electron flow, remains unclear. In this work, we report that the loss of CII, but not that of CI, reduces melanoma tumor growth by increasing antigen presentation and T cell-mediated killing. This is driven by succinate-mediated transcriptional and epigenetic activation of major histocompatibility complex-antigen processing and presentation (MHC-APP) genes independent of interferon signaling. Furthermore, knockout of methylation-controlled J protein (MCJ), to promote electron entry preferentially through CI, provides proof of concept of ETC rewiring to achieve antitumor responses without side effects associated with an overall reduction in mitochondrial respiration in noncancer cells. Our results may hold therapeutic potential for tumors that have reduced MHC-APP expression, a common mechanism of cancer immunoevasion. Full Text

Maoz, A., and Weiskopf, K. (2023). Phagocytic cooperativity by tumour macrophages. Nature Biomedical Engineering. Online Ahead of Print. Disrupting the CD47–SIRPα checkpoint in tumour macrophages and delivering a tumour-opsonizing monoclonal antibody maximizes the macrophages’ cooperative phagocytic potency. Full Text

Martin-Rufino, J.D., Castano, N., Pang, M., Grody, E.I., Joubran, S., Caulier, A., Wahlster, L., Li, T., Qiu, X., Riera-Escandell, A.M., et al. (2023). Massively parallel base editing to map variant effects in human hematopoiesis. Cell.Online Ahead of Print. Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow for rich phenotyping through single-cell RNA sequencing readouts and separately for characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases. Full Text

Nelson, J.O., Kumon, T., and Yamashita, Y.M. (2023). rDNA magnification is a unique feature of germline stem cells. PNAS 120(47):e2314440120. Ribosomal DNA (rDNA) encodes ribosomal RNA and exists as tandem repeats of hundreds of copies in the eukaryotic genome to meet the high demand of ribosome biogenesis. Tandemly repeated DNA elements are inherently unstable; thus, mechanisms must exist to maintain rDNA copy number (CN), in particular in the germline that continues through generations. A phenomenon called rDNA magnification was discovered over 50 y ago in Drosophila as a process that recovers the rDNA CN on chromosomes that harbor minimal CN. Our recent studies indicated that rDNA magnification is the mechanism to maintain rDNA CN under physiological conditions to counteract spontaneous CN loss that occurs during aging. Our previous studies that explored the mechanism of rDNA magnification implied that asymmetric division of germline stem cells (GSCs) may be particularly suited to achieve rDNA magnification. However, it remains elusive whether GSCs are the unique cell type that undergoes rDNA magnification or differentiating germ cells are also capable of magnification. In this study, we provide empirical evidence that suggests that rDNA magnification operates uniquely in GSCs, but not in differentiating germ cells. We further provide computer simulation that suggests that rDNA magnification is only achievable through asymmetric GSC divisions. We propose that despite known plasticity and transcriptomic similarity between GSCs and differentiating germ cells, GSCs' unique ability to divide asymmetrically serves a critical role of maintaining rDNA CN through generations, supporting germline immortality. Full Text

Nelson, J.O., Slicko, A., and Yamashita, Y.M. (2023). The retrotransposon R2 maintains Drosophila ribosomal DNA repeats. PNAS 120 (23) e2221613120. Ribosomal DNA (rDNA) loci contain hundreds of tandemly repeated copies of ribosomal RNA genes needed to support cellular viability. This repetitiveness makes it highly susceptible to copy number (CN) loss due to intrachromatid recombination between rDNA copies, threatening multigenerational maintenance of rDNA. How this threat is counteracted to avoid extinction of the lineage has remained unclear. Here, we show that the rDNA-specific retrotransposon R2 is essential for restorative rDNA CN expansion to maintain rDNA loci in the Drosophila male germline. The depletion of R2 led to defective rDNA CN maintenance, causing a decline in fecundity over generations and eventual extinction. We find that double-stranded DNA breaks created by the R2 endonuclease, a feature of R2's rDNA-specific retrotransposition, initiate the process of rDNA CN recovery, which relies on homology-dependent repair of the DNA break at rDNA copies. This study reveals that an active retrotransposon provides an essential function for its host, contrary to transposable elements' reputation as entirely selfish. These findings suggest that benefiting host fitness can be an effective selective advantage for transposable elements to offset their threat to the host, which may contribute to retrotransposons' widespread success throughout taxa. Full Text

Oksuz O., Henninger, J.E.,, Warneford-Thomson, R., Zheng, M.M., Erb, H., Vancura, A., Overholt, K.J., Hawken, S.W., Banani, S.F., Lauman, R., Nancy M. Hannett , Tong I. Lee, Richard A. Young, et al. (2023). Transcription factors interact with RNA to regulate genes. Molecular Cell. Online Ahead of Print. Transcription factors (TFs) orchestrate the gene expression programs that define each cell's identity. The canonical TF accomplishes this with two domains, one that binds specific DNA sequences and the other that binds protein coactivators or corepressors. We find that at least half of TFs also bind RNA, doing so through a previously unrecognized domain with sequence and functional features analogous to the arginine-rich motif of the HIV transcriptional activator Tat. RNA binding contributes to TF function by promoting the dynamic association between DNA, RNA, and TF on chromatin. TF-RNA interactions are a conserved feature important for vertebrate development and disrupted in disease. We propose that the ability to bind DNA, RNA, and protein is a general property of many TFs and is fundamental to their gene regulatory function. Full Text

Overson, R.P., Johnson, M.G., Bechen, L.L., Kinosian, S.P., Douglas, N.A., Fant, J.B., Hoch, P.C., Levin, R.A., Moore, M.J., Raguso, R.A., et al. (2023). A phylogeny of the evening primrose family (Onagraceae) using a target enrichment approach with 303 nuclear loci. BMC Ecology & Evolution 23(1):66. BACKGROUND: The evening primrose family (Onagraceae) includes 664 species (803 taxa) with a center of diversity in the Americas, especially western North America. Ongoing research in Onagraceae includes exploring striking variation in floral morphology, scent composition, and breeding system, as well as the role of these traits in driving diversity among plants and their interacting pollinators and herbivores. However, these efforts are limited by the lack of a comprehensive, well-resolved phylogeny. Previous phylogenetic studies based on a few loci strongly support the monophyly of the family and the sister relationship of the two largest tribes but fail to resolve several key relationships. RESULTS: We used a target enrichment approach to reconstruct the phylogeny of Onagraceae using 303 highly conserved, low-copy nuclear loci. We present a phylogeny for Onagraceae with 169 individuals representing 152 taxa sampled across the family, including extensive sampling within the largest tribe, Onagreae. Deep splits within the family are strongly supported, whereas relationships among closely related genera and species are characterized by extensive conflict among individual gene trees. CONCLUSIONS: This phylogenetic resource will augment current research projects focused throughout the family in genomics, ecology, coevolutionary dynamics, biogeography, and the evolution of characters driving diversification in the family. Full Text

Park, C., Owusu-Boaitey, K.E., Valdes, G.M., and Reddien, P.W. (2023). Fate specification is spatially intermingled across planarian stem cells. Nature Communications 14(1):7422. Regeneration requires mechanisms for producing a wide array of cell types. Neoblasts are stem cells in the planarian Schmidtea mediterranea that undergo fate specification to produce over 125 adult cell types. Fate specification in neoblasts can be regulated through expression of fate-specific transcription factors. We utilize multiplexed error-robust fluorescence in situ hybridization (MERFISH) and whole-mount FISH to characterize fate choice distribution of stem cells within planarians. Fate choices are often made distant from target tissues and in a highly intermingled manner, with neighboring neoblasts frequently making divergent fate choices for tissues of different location and function. We propose that pattern formation is driven primarily by the migratory assortment of progenitors from mixed and spatially distributed fate-specified stem cells and that fate choice involves stem-cell intrinsic processes. Full Text

Park, J.I., Bell, G.W., and Yamashita, Y.M.(2023). Derepression of Y-linked multicopy protamine-like genes interferes with sperm nuclear compaction in D. melanogaster. PNAS 120(16) : e2220576120. Across species, sperm maturation involves the dramatic reconfiguration of chromatin into highly compact nuclei that enhance hydrodynamic ability and ensure paternal genomic integrity. This process is mediated by the replacement of histones by sperm nuclear basic proteins, also referred to as protamines. In humans, a carefully balanced dosage between two known protamine genes is required for optimal fertility. However, it remains unknown how their proper balance is regulated and how defects in balance may lead to compromised fertility. Here, we show that a nucleolar protein, modulo, a homolog of nucleolin, mediates the histone-to-protamine transition during Drosophila spermatogenesis. We find that modulo mutants display nuclear compaction defects during late spermatogenesis due to decreased expression of autosomal protamine genes (including Mst77F) and derepression of Y-linked multicopy Mst77F homologs (Mst77Y), leading to the mutant's known sterility. Overexpression of Mst77Y in a wild-type background is sufficient to cause nuclear compaction defects, similar to modulo mutant, indicating that Mst77Y is a dominant-negative variant interfering with the process of histone-to-protamine transition. Interestingly, ectopic overexpression of Mst77Y caused decompaction of X-bearing spermatids nuclei more frequently than Y-bearing spermatid nuclei, although this did not greatly affect the sex ratio of offspring. We further show that modulo regulates these protamine genes at the step of transcript polyadenylation. We conclude that the regulation of protamines mediated by modulo, ensuring the expression of functional ones while repressing dominant-negative ones, is critical for male fertility. Full Text

Perillo, M., Swartz, S.Z., Pieplow, C., and Wessel, G.M. (2023). Molecular mechanisms of tubulogenesis revealed in the sea star hydro-vascular organ. Nature Communications 14(1):2402. A fundamental goal in the organogenesis field is to understand how cells organize into tubular shapes. Toward this aim, we have established the hydro-vascular organ in the sea star Patiria miniata as a model for tubulogenesis. In this animal, bilateral tubes grow out from the tip of the developing gut, and precisely extend to specific sites in the larva. This growth involves cell migration coupled with mitosis in distinct zones. Cell proliferation requires FGF signaling, whereas the three-dimensional orientation of the organ depends on Wnt signaling. Specification and maintenance of tube cell fate requires Delta/Notch signaling. Moreover, we identify target genes of the FGF pathway that contribute to tube morphology, revealing molecular mechanisms for tube outgrowth. Finally, we report that FGF activates the Six1/2 transcription factor, which serves as an evolutionarily ancient regulator of branching morphogenesis. This study uncovers distinct mechanisms of tubulogenesis in vivo and we propose that cellular dynamics in the sea star hydro-vascular organ represents a key comparison for understanding the evolution of vertebrate organs. Full Text

Phadnis, V.V., Snider, J., Varadharajan, V., Ramachandiran, I., Deik, A.A., Lai, Z.W., Kunchok, T., Eaton, E.N., Sebastiany, C., Lyakisheva, A. , Kyle D. Vaccaro , Juliet Allen , Kipp Weiskopf , Robert A. Weinberg , Whitney S. Henry , et al. (2023). MMD collaborates with ACSL4 and MBOAT7 to promote polyunsaturated phosphatidylinositol remodeling and susceptibility to ferroptosis. Cell Reports 42(9): 113023. Ferroptosis is a form of regulated cell death with roles in degenerative diseases and cancer. Excessive iron-catalyzed peroxidation of membrane phospholipids, especially those containing the polyunsaturated fatty acid arachidonic acid (AA), is central in driving ferroptosis. Here, we reveal that an understudied Golgi-resident scaffold protein, MMD, promotes susceptibility to ferroptosis in ovarian and renal carcinoma cells in an ACSL4- and MBOAT7-dependent manner. Mechanistically, MMD physically interacts with both ACSL4 and MBOAT7, two enzymes that catalyze sequential steps to incorporate AA in phosphatidylinositol (PI) lipids. Thus, MMD increases the flux of AA into PI, resulting in heightened cellular levels of AA-PI and other AA-containing phospholipid species. This molecular mechanism points to a pro-ferroptotic role for MBOAT7 and AA-PI, with potential therapeutic implications, and reveals that MMD is an important regulator of cellular lipid metabolism. Full Text

Qian, J., Guan, X., Xie, B., Xu, C., Niu, J., Tang, X., Li, C.H., Colecraft, H.M., Jaenisch, R., and Liu, X.S. (2023). Multiplex epigenome editing of MECP2 to rescue Rett syndrome neurons. Science Translational Medicine 15(679):eadd4666. Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by loss-of-function heterozygous mutations of methyl CpG-binding protein 2 (MECP2) on the X chromosome in young females. Reactivation of the silent wild-type MECP2 allele from the inactive X chromosome (Xi) represents a promising therapeutic opportunity for female patients with RTT. Here, we applied a multiplex epigenome editing approach to reactivate MECP2 from Xi in RTT human embryonic stem cells (hESCs) and derived neurons. Demethylation of the MECP2 promoter by dCas9-Tet1 with target single-guide RNA reactivated MECP2 from Xi in RTT hESCs without detectable off-target effects at the transcriptional level. Neurons derived from methylation-edited RTT hESCs maintained MECP2 reactivation and reversed the smaller soma size and electrophysiological abnormalities, two hallmarks of RTT. In RTT neurons, insulation of the methylation-edited MECP2 locus by dCpf1-CTCF (a catalytically dead Cpf1 fused with CCCTC-binding factor) with target CRISPR RNA enhanced MECP2 reactivation and rescued RTT-related neuronal defects, providing a proof-of-concept study for epigenome editing to treat RTT and potentially other dominant X-linked diseases. Full Text

Raz, A.A., Vida, G.S., Stern, S.R., Mahadevaraju, S., Fingerhut, J.M., Viveiros, J.M., Pal, S., Grey, J.R., Grace, M.R., Berry, C.W., et al. (2023). Emergent dynamics of adult stem cell lineages from single nucleus and single cell RNA-Seq of Drosophila testes. Elife 12:e82201. Proper differentiation of sperm from germline stem cells, essential for production of the next generation, requires dramatic changes in gene expression that drive remodeling of almost all cellular components, from chromatin to organelles to cell shape itself. Here, we provide a single nucleus and single cell RNA-seq resource covering all of spermatogenesis in Drosophila starting from in-depth analysis of adult testis single nucleus RNA-seq (snRNA-seq) data from the Fly Cell Atlas (FCA) study. With over 44,000 nuclei and 6000 cells analyzed, the data provide identification of rare cell types, mapping of intermediate steps in differentiation, and the potential to identify new factors impacting fertility or controlling differentiation of germline and supporting somatic cells. We justify assignment of key germline and somatic cell types using combinations of known markers, in situ hybridization, and analysis of extant protein traps. Comparison of single cell and single nucleus datasets proved particularly revealing of dynamic developmental transitions in germline differentiation. To complement the web-based portals for data analysis hosted by the FCA, we provide datasets compatible with commonly used software such as Seurat and Monocle. The foundation provided here will enable communities studying spermatogenesis to interrogate the datasets to identify candidate genes to test for function in vivo. Full Text

Reichart, D., Newby, G.A., Wakimoto, H., Lun, M., Gorham, J.M., Curran, J.J., Raguram, A., DeLaughter, D.M., Conner, D.A., Marsiglia, J.D.C., David C Page , et al. (2023). Efficient in vivo genome editing prevents hypertrophic cardiomyopathy in mice. Nature Medicine 29(2):412-421. Dominant missense pathogenic variants in cardiac myosin heavy chain cause hypertrophic cardiomyopathy (HCM), a currently incurable disorder that increases risk for stroke, heart failure and sudden cardiac death. In this study, we assessed two different genetic therapies-an adenine base editor (ABE8e) and a potent Cas9 nuclease delivered by AAV9-to prevent disease in mice carrying the heterozygous HCM pathogenic variant myosin R403Q. One dose of dual-AAV9 vectors, each carrying one half of RNA-guided ABE8e, corrected the pathogenic variant in ≥70% of ventricular cardiomyocytes and maintained durable, normal cardiac structure and function. An additional dose provided more editing in the atria but also increased bystander editing. AAV9 delivery of RNA-guided Cas9 nuclease effectively inactivated the pathogenic allele, albeit with dose-dependent toxicities, necessitating a narrow therapeutic window to maintain health. These preclinical studies demonstrate considerable potential for single-dose genetic therapies to correct or silence pathogenic variants and prevent the development of HCM. Full Text

Ren, J., Zhou, H., Zeng, H., Wang, C.K., Huang, J., Qiu, Xiaojie, Sui, X., Li, Q., Wu, X., Lin, Z., et al. (2023). Spatiotemporally resolved transcriptomics reveals the subcellular RNA kinetic landscape. Nature Methods. Online Ahead of Print. Spatiotemporal regulation of the cellular transcriptome is crucial for proper protein expression and cellular function. However, the intricate subcellular dynamics of RNA remain obscured due to the limitations of existing transcriptomics methods. Here, we report TEMPOmap-a method that uncovers subcellular RNA profiles across time and space at the single-cell level. TEMPOmap integrates pulse-chase metabolic labeling with highly multiplexed three-dimensional in situ sequencing to simultaneously profile the age and location of individual RNA molecules. Using TEMPOmap, we constructed the subcellular RNA kinetic landscape in various human cells from transcription and translocation to degradation. Clustering analysis of RNA kinetic parameters across single cells revealed 'kinetic gene clusters' whose expression patterns were shaped by multistep kinetic sculpting. Importantly, these kinetic gene clusters are functionally segregated, suggesting that subcellular RNA kinetics are differentially regulated in a cell-state- and cell-type-dependent manner. Spatiotemporally resolved transcriptomics provides a gateway to uncovering new spatiotemporal gene regulation principles. Full Text

San Roman, A.K., Skaletsky, H., Godfrey, A.K., Bokil, N.V., Teitz, L., Singh, I., Blanton, L.V., Bellott, D.W., Pyntikova, T., Lange, J., Koutseva, N., Hughes, J.F., Brown, L., Phou, S., David C. Page, et al. The human Y and inactive X chromosomes similarly modulate autosomal gene expression. Cell Genomics. Somatic cells of human males and females have 45 chromosomes in common, including the "active" X chromosome. In males the 46(th) chromosome is a Y; in females it is an "inactive" X (Xi). Through linear modeling of autosomal gene expression in cells from individuals with zero to three Xi and zero to four Y chromosomes, we found that Xi and Y impact autosomal expression broadly and with remarkably similar effects. Studying sex-chromosome structural anomalies, promoters of Xi- and Y-responsive genes, and CRISPR inhibition, we traced part of this shared effect to homologous transcription factors - ZFX and ZFY - encoded by Chr X and Y. This demonstrates sex-shared mechanisms by which Xi and Y modulate autosomal expression. Combined with earlier analyses of sex-linked gene expression, our studies show that 21% of all genes expressed in lymphoblastoid cells or fibroblasts change expression significantly in response to Xi or Y chromosomes. Full Text

San Roman, Adrianna K., Alexander K. Godfrey, Helen Skaletsky, Daniel W. Bellott, Abigail F. Groff, Hannah L. Harris, Laura V. Blanton, Jennifer F. Hughes, Laura Brown, Sidaly Phou, David C. Page, et al.(2023). The human inactive X chromosome modulates expression of the active X chromosome. Cell Genomics 3(2) : 100259. The “inactive” X chromosome (Xi) has been assumed to have little impact, in trans, on the “active” X (Xa). To test this, we quantified Xi and Xa gene expression in individuals with one Xa and zero to three Xis. Our linear modeling revealed modular Xi and Xa transcriptomes and significant Xi-driven expression changes for 38% (162/423) of expressed X chromosome genes. By integrating allele-specific analyses, we found that modulation of Xa transcript levels by Xi contributes to many of these Xi-driven changes (≥121 genes). By incorporating metrics of evolutionary constraint, we identified 10 X chromosome genes most likely to drive sex differences in common disease and sex chromosome aneuploidy syndromes. We conclude that human X chromosomes are regulated both in cis, through Xi-wide transcriptional attenuation, and in trans, through positive or negative modulation of individual Xa genes by Xi. The sum of these cis and trans effects differs widely among genes. Full Text

Schmid, R., Heuckeroth, S., Korf, A., Smirnov, A., Myers, O., Dyrlund, T.S., Bushuiev, R., Murray, K.J., Hoffmann, N., Lu, M., Jing-Ke Weng, et al. (2023). Integrative analysis of multimodal mass spectrometry data in MZmine 3. Nature Biotechnology. Online Ahead of Print. Innovation in mass spectrometry (MS) and the rapidly increasing throughput and sensitivity of MS instrumentation require adaptations and innovations in data processing tools. Here, we introduce MZmine 3, a scalable MS data analysis platform that supports hybrid datasets from various instrumental setups, including liquid and gas chromatography (LC and GC)–MS, ion mobility spectrometry (IMS)–MS and MS imaging. In particular, the integration of IMS–MS imaging and LC–IMS–MS datasets provides opportunities for spatial metabolomics analyses with increased annotation confidence. Full Text

Schwarz, D., and Lourido, S. (2023). The multifaceted roles of Myb domain-containing proteins in apicomplexan parasites. Current Opinion in Microbiology 76 : 102395. Apicomplexan parasites are a large and diverse clade of protists responsible for significant diseases of humans and animals. Central to the ability of these parasites to colonize their host and evade immune responses is an expanded repertoire of gene-expression programs that requires the coordinated action of complex transcriptional networks. DNA-binding proteins and chromatin regulators are essential orchestrators of apicomplexan gene expression that often act in concert. Although apicomplexan genomes encode various families of putative DNA-binding proteins, most remain functionally and mechanistically unexplored. This review highlights the versatile role of myeloblastosis (Myb) domain-containing proteins in apicomplexan parasites as transcription factors and chromatin regulators. We explore the diversity of Myb domain structure and use phylogenetic analysis to identify common features across the phylum. This provides a framework to discuss functional heterogeneity and regulation of Myb domain-containing proteins particularly emphasizing their role in parasite differentiation. Full Text

Shao, L., Fingerhut, J.M., Falk, B.L., Han, H., Maldonado, G., Qiao, Y., Lee, V., Hall, E., Chen, L., Polevoy, G., et al. (2023). Eukaryotic translation initiation factor eIF4E-5 is required for spermiogenesis in Drosophila melanogaster. Development. Online Ahead of Print. Drosophila sperm development is characterized by extensive post-transcriptional regulation whereby thousands of transcripts are preserved for translation during later stages. A key step in translation initiation is the binding of eukaryotic initiation factor 4E (eIF4E) to the 5' mRNA cap. In addition to canonical eIF4E-1, Drosophila has multiple eIF4E paralogs, including four (eIF4E-3, -4, -5, and -7) that are highly expressed in the testis. Among these, only eIF4E-3 has been characterized genetically. Here, using CRISPR/Cas9 mutagenesis, we determined that eIF4E-5 is essential for male fertility. eIF4E-5 protein localizes to the distal ends of elongated spermatid cysts, and eIF4E-5 mutants exhibit defects during post-meiotic stages, including a mild defect in spermatid cyst polarization. eIF4E-5 mutants also have a fully penetrant defect in individualization, resulting in failure to produce mature sperm. Indeed, our data indicate that eIF4E-5 regulates non-apoptotic caspase activity during individualization by promoting local accumulation of the E3 ubiquitin ligase inhibitor Soti. Our results further extend the diversity of non-canonical eIF4Es that carry out distinct spatiotemporal roles during spermatogenesis. Full Text

She R., Fair, T., Schaefer, N.K., Saunders, R.A., Pavlovic, B.J., Weissman, J.S., and Pollen, A.A. (2023). Comparative landscape of genetic dependencies in human and chimpanzee stem cells. Cell. Online Ahead of Print. Comparative studies of great apes provide a window into our evolutionary past, but the extent and identity of cellular differences that emerged during hominin evolution remain largely unexplored. We established a comparative loss-of-function approach to evaluate whether human cells exhibit distinct genetic dependencies. By performing genome-wide CRISPR interference screens in human and chimpanzee pluripotent stem cells, we identified 75 genes with species-specific effects on cellular proliferation. These genes comprised coherent processes, including cell-cycle progression and lysosomal signaling, which we determined to be human-derived by comparison with orangutan cells. Human-specific robustness to CDK2 and CCNE1 depletion persisted in neural progenitor cells and cerebral organoids, supporting the G1-phase length hypothesis as a potential evolutionary mechanism in human brain expansion. Our findings demonstrate that evolutionary changes in human cells reshaped the landscape of essential genes and establish a platform for systematically uncovering latent cellular and molecular differences between species. Full Text

She, R., Luo, J., and Weissman, J.S. (2023). Translational fidelity screens in mammalian cells reveal eIF3 and eIF4G2 as regulators of start codon selectivity. Nucleic Acids Research. Online Ahead of Print. The translation initiation machinery and the ribosome orchestrate a highly dynamic scanning process to distinguish proper start codons from surrounding nucleotide sequences. Here, we performed genome-wide CRISPRi screens in human K562 cells to systematically identify modulators of the frequency of translation initiation at near-cognate start codons. We observed that depletion of any eIF3 core subunit promoted near-cognate start codon usage, though sensitivity thresholds of each subunit to sgRNA-mediated depletion varied considerably. Double sgRNA depletion experiments suggested that enhanced near-cognate usage in eIF3D depleted cells required canonical eIF4E cap-binding and was not driven by eIF2A or eIF2D-dependent leucine tRNA initiation. We further characterized the effects of eIF3D depletion and found that the N-terminus of eIF3D was strictly required for accurate start codon selection, whereas disruption of the cap-binding properties of eIF3D had no effect. Lastly, depletion of eIF3D activated TNFα signaling via NF-κB and the interferon gamma response. Similar transcriptional profiles were observed upon knockdown of eIF1A and eIF4G2, which also promoted near-cognate start codon usage, suggesting that enhanced near-cognate usage could potentially contribute to NF-κB activation. Our study thus provides new avenues to study the mechanisms and consequences of alternative start codon usage. Full Text

Shortt, E., Hackett, C.G., Stadler, R.V., Kent, R.S., Herneisen, A.L., Ward, G.E., and Lourido, S. (2023). CDPK2A and CDPK1 form a signaling module upstream of Toxoplasma motility. mBio : e0135823. Online Ahead of Print. In apicomplexan parasites, the transition between replication and dissemination is regulated by fluctuations in cytosolic calcium concentrations, transduced in part by calcium-dependent protein kinases (CDPKs). We examined the role of CDPK2A in the lytic cycle of Toxoplasma, analyzing its role in the regulation of cellular processes associated with parasite motility. We used chemical-genetic approaches and conditional depletion to determine that CDPK2A contributes to the initiation of parasite motility through microneme discharge. We demonstrate that the N-terminal extension of CDPK2A is necessary for the protein's function. Conditional depletion revealed an epistatic interaction between CDPK2A and CDPK1, suggesting that the two kinases work together to mediate motility in response to certain stimuli. This signaling module appears distinct from that of CDPK3 and protein kinase G, which also control egress. CDPK2A is revealed as an important regulator of the Toxoplasma kinetic phase, linked to other kinases that govern this critical transition. Our work uncovers extensive interconnectedness between the signaling pathways that regulate parasite motility. IMPORTANCE This work uncovers interactions between various signaling pathways that govern Toxoplasma gondii egress. Specifically, we compare the function of three canonical calcium-dependent protein kinases (CDPKs) using chemical-genetic and conditional-depletion approaches. We describe the function of a previously uncharacterized CDPK, CDPK2A, in the Toxoplasma lytic cycle, demonstrating that it contributes to parasite fitness through regulation of microneme discharge, gliding motility, and egress from infected host cells. Comparison of analog-sensitive kinase alleles and conditionally depleted alleles uncovered epistasis between CDPK2A and CDPK1, implying a partial functional redundancy. Understanding the topology of signaling pathways underlying key events in the parasite life cycle can aid in efforts targeting kinases for anti-parasitic therapies. Full Text

Sun, C., Seranova, E., Cohen, M.A., Chipara, M., Roberts, J., Astuti, D., Palhegyi, A.M., Acharjee, A., Sedlackova, L., Kataura, T., Shupei Zhang, Rudolf Jaenisch, et al. (2023). NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency.Cell Reports 42(5) : 112372. Autophagy is a homeostatic process critical for cellular survival, and its malfunction is implicated in human diseases including neurodegeneration. Loss of autophagy contributes to cytotoxicity and tissue degeneration, but the mechanistic understanding of this phenomenon remains elusive. Here, we generated autophagy-deficient (ATG5(-/-)) human embryonic stem cells (hESCs), from which we established a human neuronal platform to investigate how loss of autophagy affects neuronal survival. ATG5(-/-) neurons exhibit basal cytotoxicity accompanied by metabolic defects. Depletion of nicotinamide adenine dinucleotide (NAD) due to hyperactivation of NAD-consuming enzymes is found to trigger cell death via mitochondrial depolarization in ATG5(-/-) neurons. Boosting intracellular NAD levels improves cell viability by restoring mitochondrial bioenergetics and proteostasis in ATG5(-/-) neurons. Our findings elucidate a mechanistic link between autophagy deficiency and neuronal cell death that can be targeted for therapeutic interventions in neurodegenerative and lysosomal storage diseases associated with autophagic defect. Full Text

Sunshine, S., Puschnik, A.S., Replogle, J.M., Laurie, M.T., Liu, J., Zha, B.S., Nuñez, J.K., Byrum, J.R., McMorrow, A.H., Frieman, M.B., Xiaojie Qiu, Jonathan S. Weissman, et al. (2023). Systematic functional interrogation of SARS-CoV-2 host factors using Perturb-seq. Nature Communications 14(1):6245. Genomic and proteomic screens have identified numerous host factors of SARS-CoV-2, but efficient delineation of their molecular roles during infection remains a challenge. Here we use Perturb-seq, combining genetic perturbations with a single-cell readout, to investigate how inactivation of host factors changes the course of SARS-CoV-2 infection and the host response in human lung epithelial cells. Our high-dimensional data resolve complex phenotypes such as shifts in the stages of infection and modulations of the interferon response. However, only a small percentage of host factors showed such phenotypes upon perturbation. We further identified the NF-κB inhibitor IκBα (NFKBIA), as well as the translation factors EIF4E2 and EIF4H as strong host dependency factors acting early in infection. Overall, our study provides massively parallel functional characterization of host factors of SARS-CoV-2 and quantitatively defines their roles both in virus-infected and bystander cells. Full Text

Tringides, C.M., Boulingre, M., Khalil, A., Lungjangwa, T., Jaenisch, R. , and Mooney, D.J. (2023). Tunable Conductive Hydrogel Scaffolds for Neural Cell Differentiation. Advanced Healthcare Materials 12(7):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. It is 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 are formed. Neural progenitor cells (NPCs) incorporated in the scaffolds form neurite networks which span the material in 3D and differentiate into astrocytes and myelinating oligodendrocytes. Viscoelastic and more conductive scaffolds produce more dense neurite networks, with an increased percentage of astrocytes and higher myelination. Application of exogenous electrical stimulation to the scaffolds increases the percentage of astrocytes and the supporting cells localize differently with the surrounding neurons. The tunable biomaterial scaffolds can support neural cocultures 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

Tsang, M.J., and Cheeseman, I.M. (2023). Alternative CDC20 translational isoforms tune mitotic arrest duration. Nature. Online Ahead of Print. Mitotic defects activate the spindle-assembly checkpoint, which inhibits the anaphase-promoting complex co-activator CDC20 to induce a prolonged cell cycle arrest(1,2). Once errors are corrected, the spindle-assembly checkpoint is silenced, allowing anaphase onset to occur. However, in the presence of persistent unresolvable errors, cells can undergo 'mitotic slippage', exiting mitosis into a tetraploid G1 state and escaping the cell death that results from a prolonged arrest. The molecular logic that enables cells to balance these duelling mitotic arrest and slippage behaviours remains unclear. Here we demonstrate that human cells modulate the duration of their mitotic arrest through the presence of conserved, alternative CDC20 translational isoforms. Downstream translation initiation results in a truncated CDC20 isoform that is resistant to spindle-assembly-checkpoint-mediated inhibition and promotes mitotic exit even in the presence of mitotic perturbations. Our study supports a model in which the relative levels of CDC20 translational isoforms control the duration of mitotic arrest. During a prolonged mitotic arrest, new protein synthesis and differential CDC20 isoform turnover create a timer, with mitotic exit occurring once the truncated Met43 isoform achieves sufficient levels. Targeted molecular changes or naturally occurring cancer mutations that alter CDC20 isoform ratios or its translational control modulate mitotic arrest duration and anti-mitotic drug sensitivity, with potential implications for the diagnosis and treatment of human cancers. Full Text

Valleau, D., Sidik, S.M., Godoy, L.C., Acevedo-Sánchez, Y., Pasaje, C.F.A., Huynh, M.H., Carruthers, V.B., Niles, J.C., and Lourido, S. (2023). A conserved complex of microneme proteins mediates rhoptry discharge in Toxoplasma. Embo Journal : e113155. Online Ahead of Print. Apicomplexan parasites discharge specialized organelles called rhoptries upon host cell contact to mediate invasion. The events that drive rhoptry discharge are poorly understood, yet essential to sustain the apicomplexan parasitic life cycle. Rhoptry discharge appears to depend on proteins secreted from another set of organelles called micronemes, which vary in function from allowing host cell binding to facilitation of gliding motility. Here we examine the function of the microneme protein CLAMP, which we previously found to be necessary for Toxoplasma gondii host cell invasion, and demonstrate its essential role in rhoptry discharge. CLAMP forms a distinct complex with two other microneme proteins, the invasion-associated SPATR, and a previously uncharacterized protein we name CLAMP-linked invasion protein (CLIP). CLAMP deficiency does not impact parasite adhesion or microneme protein secretion; however, knockdown of any member of the CLAMP complex affects rhoptry discharge. Phylogenetic analysis suggests orthologs of the essential complex components, CLAMP and CLIP, are ubiquitous across apicomplexans. SPATR appears to act as an accessory factor in Toxoplasma, but despite incomplete conservation is also essential for invasion during Plasmodium falciparum blood stages. Together, our results reveal a new protein complex that mediates rhoptry discharge following host-cell contact. Full Text

Venkei, Z.G., Gainetdinov, I., Bagci, A., Starostik, M.R., Choi, C.P., Fingerhut, J.M. , Chen, P., Balsara, C., Whitfield, T.W., Bell, G.W. , Yukiko M Yamashita , et al. (2023). A maternally programmed intergenerational mechanism enables male offspring to make piRNAs from Y-linked precursor RNAs in Drosophila. Nature Cell Biology. Online Ahead of Print. In animals, PIWI-interacting RNAs (piRNAs) direct PIWI proteins to silence complementary targets such as transposons. In Drosophila and other species with a maternally specified germline, piRNAs deposited in the egg initiate piRNA biogenesis in the progeny. However, Y chromosome loci cannot participate in such a chain of intergenerational inheritance. How then can the biogenesis of Y-linked piRNAs be initiated? Here, using Suppressor of Stellate (Su(Ste)), a Y-linked Drosophila melanogaster piRNA locus as a model, we show that Su(Ste) piRNAs are made in the early male germline via 5'-to-3' phased piRNA biogenesis initiated by maternally deposited 1360/Hoppel transposon piRNAs. Notably, deposition of Su(Ste) piRNAs from XXY mothers obviates the need for phased piRNA biogenesis in sons. Together, our study uncovers a developmentally programmed, intergenerational mechanism that allows fly mothers to protect their sons using a Y-linked piRNA locus. Full Text

Wang, P.Y., and Bartel, D.P. (2023). A statistical approach for identifying primary substrates of ZSWIM8-mediated microRNA degradation in small-RNA sequencing data. BMC Bioinformatics 24 (1): 195. One strategy for identifying targets of a regulatory factor is to perturb the factor and use high-throughput RNA sequencing to examine the consequences. However, distinguishing direct targets from secondary effects and experimental noise can be challenging when confounding signal is present in the background at varying levels. RESULTS: Here, we present a statistical modeling strategy to identify microRNAs that are primary substrates of target-directed miRNA degradation (TDMD) mediated by ZSWIM8. This method uses a bi-beta-uniform mixture (BBUM) model to separate primary from background signal components, leveraging the expectation that primary signal is restricted to upregulation and not downregulation upon loss of ZSWIM8. The BBUM model strategy retained the apparent sensitivity and specificity of the previous ad hoc approach but was more robust against outliers, achieved a more consistent stringency, and could be performed using a single cutoff of false discovery rate (FDR). CONCLUSIONS: We developed the BBUM model, a robust statistical modeling strategy to account for background secondary signal in differential expression data. It performed well for identifying primary substrates of TDMD and should be useful for other applications in which the primary regulatory targets are only upregulated or only downregulated. The BBUM model, FDR-correction algorithm, and significance-testing methods are available as an R package at https://github.com/wyppeter/bbum. Full Text

Wei, W., Riley, N.M., Lyu, X., Shen, X., Guo, J., Raun, S.H., Zhao, M., Moya-Garzon, M.D., Basu, H., Sheng-Hwa Tung, A., Wentao Huang, et al. (2023). Organism-wide, cell-type-specific secretome mapping of exercise training in mice. Cell Metabolism. Online Ahead of Print. There is a significant interest in identifying blood-borne factors that mediate tissue crosstalk and function as molecular effectors of physical activity. Although past studies have focused on an individual molecule or cell type, the organism-wide secretome response to physical activity has not been evaluated. Here, we use a cell-type-specific proteomic approach to generate a 21-cell-type, 10-tissue map of exercise training-regulated secretomes in mice. Our dataset identifies >200 exercise training-regulated cell-type-secreted protein pairs, the majority of which have not been previously reported. Pdgfra-cre-labeled secretomes were the most responsive to exercise training. Finally, we show anti-obesity, anti-diabetic, and exercise performance-enhancing activities for proteoforms of intracellular carboxylesterases whose secretion from the liver is induced by exercise training. Full Text

Weichert-Leahey, N., Shi, H., Tao, T., Oldridge, D.A., Durbin, A.D., Abraham, B.J., Zimmerman, M.W., Zhu, S., Wood, A.C., Reyon, D., Richard A. Young, et al. (2023). Genetic predisposition to neuroblastoma results from a regulatory polymorphism that promotes the adrenergic cell state. Journal of Clinical Investigation. 133(10):e166919. Childhood neuroblastomas exhibit plasticity between an undifferentiated neural crest-like mesenchymal cell state and a more differentiated sympathetic adrenergic cell state. These cell states are governed by autoregulatory transcriptional loops called core regulatory circuitries (CRCs), which drive the early development of sympathetic neuronal progenitors from migratory neural crest cells during embryogenesis. The adrenergic cell identity of neuroblastoma requires LMO1 as a transcriptional cofactor. Both LMO1 expression levels and the risk of developing neuroblastoma in children are associated with a single nucleotide polymorphism, G/T, that affects a GATA motif in the first intron of LMO1. Here, we showed that WT zebrafish with the GATA genotype developed adrenergic neuroblastoma, while knock-in of the protective TATA allele at this locus reduced the penetrance of MYCN-driven tumors, which were restricted to the mesenchymal cell state. Whole genome sequencing of childhood neuroblastomas demonstrated that TATA/TATA tumors also exhibited a mesenchymal cell state and were low risk at diagnosis. Thus, conversion of the regulatory GATA to a TATA allele in the first intron of LMO1 reduced the neuroblastoma-initiation rate by preventing formation of the adrenergic cell state. This mechanism was conserved over 400 million years of evolution, separating zebrafish and humans. Full Text

Williams, R.M., Bloom, J.C., Robertus, C.M., Recknagel, A.K., Putnam, D., Schimenti, J.C., and Zipfel, W.R. (2023). Practical strategies for robust and inexpensive imaging of aqueous-cleared tissues. Journal of Microscopy. Online Ahead of Print. Lightsheet microscopy offers an ideal method for imaging of large (mm-cm scale) biological tissues rendered transparent via optical clearing protocols. However the diversity of clearing technologies and tissue types, and how these are adapted to the microscope can make tissue mounting complicated and somewhat irreproducible. Tissue preparation for imaging can involve glues and or equilibration in a variety of expensive and/or proprietary formulations. Here we present practical advice for mounting and capping cleared tissues in optical cuvettes for macroscopic imaging, providing a standardized 3D cell that can be imaged routinely and relatively inexpensively. We show that acrylic cuvettes cause minimal spherical aberration with objective numerical apertures less than 0.65. Furthermore we describe methods for aligning and assessing the light sheets, discriminating fluorescence from autofluorescence, identifying chromatic artifacts due to differential scattering and removing streak artifacts such that they do not confound downstream 3D object segmentation analyses, with mouse embryo, liver and heart imaging as demonstrated examples. Full Text

Yamashita, Y.M. (2023). Asymmetric Stem Cell Division and Germline Immortality. Annual Review of Genetics Online Ahead of Print. Germ cells are the only cell type that is capable of transmitting genetic information to the next generation, which has enabled the continuation of multicellular life for the last 1.5 billion years. Surprisingly little is known about the mechanisms supporting the germline's remarkable ability to continue in this eternal cycle, termed germline immortality. Even unicellular organisms age at a cellular level, demonstrating that cellular aging is inevitable. Extensive studies in yeast have established the framework of how asymmetric cell division and gametogenesis may contribute to the resetting of cellular age. This review examines the mechanisms of germline immortality-how germline cells reset the aging of cells-drawing a parallel between yeast and multicellular organisms. Expected final online publication date for the Annual Review of Genetics, Volume 57 is November 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates. Full Text

Yamashita, Y.M. (2023). Why Ys are not necessarily toxic. Nature Ecology and Evolution. Online Ahead of Print. Sex differences in physiology and longevity are widely observed. A study that manipulates heterochromatin content in Drosophila Y chromosomes shows no association between the length of the Y chromosome and longevity, thus challenging the hypothesis that Y chromosome-derived heterochromatin causes Y chromosome-bearing animals to live shorter lives. Full Text

Yao, Y., Fong Ng, J., Park, W.D., Samur, M.K., Morelli, E., Encinas, J., Chyra, Z., Xu, Y., Derebail, S., Epstein, C.B., Richard A.Young , et al. (2023). CDK7 controls E2F- and MYC-driven proliferative and metabolic vulnerabilities in multiple myeloma. Blood. Online Ahead of Print. Therapeutic targeting of CDK7 has proven beneficial in pre-clinical studies, yet the off-target effects of currently available CDK7 inhibitors make it difficult to pinpoint the exact mechanisms behind MM cell death mediated by CDK7 inhibition. Here, we show that CDK7 expression positively correlates with E2F and MYC transcriptional programs in multiple myeloma (MM) patient cells; and its selective targeting counteracts E2F activity via perturbation of the CDKs/Rb axis and impairs MYC-regulated metabolic gene signatures translating into defects in glycolysis and reduced levels of lactate production in MM cells. CDK7 inhibition using the covalent small molecule inhibitor YKL-5-124 elicits a strong therapeutic response with minimal effects on normal cells, and causes in vivo tumor regression increasing survival in several MM mouse models including a genetically engineered mouse model of MYC-dependent MM. Through its role as a critical cofactor and regulator of MYC and E2F activity, CDK7 is therefore a master regulator of oncogenic cellular programs supporting MM growth and survival, and a valuable therapeutic target providing rationale for development of YKL-5-124 for clinical use. Full Text

Zhang J., Askenase, P., Jaenisch, R., and Crumpacker, C.S. (2023). Approaches to pandemic prevention - the chromatin vaccine. Frontiers in Immunology 14 : 1324084. Developing effective vaccines against viral infections have significant impacts on development, prosperity and well-being of human populations. Thus, successful vaccines such as smallpox and polio vaccines, have promoted global societal well-being. In contrast, ineffective vaccines may fuel arguments that retard scientific progress. We aim to stimulate a multilevel discussion on how to develop effective vaccines against recent and future pandemics by focusing on acquired immunodeficiency syndrome (AIDS), coronavirus disease (COVID) and other viral infections. We appeal to harnessing recent achievements in this field specifically towards a cure for current pandemics and prevention of the next pandemics. Among these, we propose to apply the HIV DNA in chromatin format - an end product of aborted HIV integration in episomal forms, i.e., the chromatin vaccines (cVacc), to elicit the epigenetic silencing and memory that prevent viral replication and infection. Full Text

Zhang, L., and Jaenisch, R., et al (2023). LINE1-Mediated Reverse Transcription and Genomic Integration of SARS-CoV-2 mRNA Detected in Virus-Infected but Not in Viral mRNA-Transfected Cells. Viruses 15 (3), 629. SARS-CoV-2 sequences can be reverse-transcribed and integrated into the genomes of virus-infected cells by a LINE1-mediated retrotransposition mechanism. Whole-genome sequencing (WGS) methods detected retrotransposed SARS-CoV-2 subgenomic sequences in virus-infected cells overexpressing LINE1, while an enrichment method (TagMap) identified retrotranspositions in cells that did not overexpress LINE1. LINE1 overexpression increased retrotranspositions about 1000-fold as compared to non-overexpressing cells. Nanopore WGS can directly recover retrotransposed viral and flanking host sequences, but its sensitivity depends on the depth of sequencing (a typical 20-fold sequencing depth would only examine 10 diploid cell equivalents). In contrast, TagMap enriches the host–virus junctions and can interrogate up to 20,000 cells and is able to detect rare viral retrotranspositions in LINE1 non-overexpressing cells. Although Nanopore WGS is 10–20-fold more sensitive per tested cell, TagMap can interrogate 1000–2000-fold more cells and, therefore, can identify infrequent retrotranspositions. When comparing SARS-CoV-2 infection and viral nucleocapsid mRNA transfection by TagMap, retrotransposed SARS-CoV-2 sequences were only detected in infected but not in transfected cells. Retrotransposition in virus-infected cells, in contrast to transfected cells, may be facilitated because virus infection, in contrast to viral RNA transfection, results in significantly higher viral RNA levels and stimulates LINE1 expression by causing cellular stress. Full Text

Zheng, E.J., Valeri, J.A., Andrews, I.W., Krishnan, A. Bandyopadhyay, P., Anahtar, M.N., Herneisen, A., Schulte, F., Linnehan, B., Wong, F., Sebastian Lourido, et al. (2023). Discovery of antibiotics that selectively kill metabolically dormant bacteria. Cell Chemical Biology. Online Ahead of Print. There is a need to discover and develop non-toxic antibiotics that are effective against metabolically dormant bacteria, which underlie chronic infections and promote antibiotic resistance. Traditional antibiotic discovery has historically favored compounds effective against actively metabolizing cells, a property that is not predictive of efficacy in metabolically inactive contexts. Here, we combine a stationary-phase screening method with deep learning-powered virtual screens and toxicity filtering to discover compounds with lethality against metabolically dormant bacteria and favorable toxicity profiles. The most potent and structurally distinct compound without any obvious mechanistic liability was semapimod, an anti-inflammatory drug effective against stationary-phase E. coli and A. baumannii. Integrating microbiological assays, biochemical measurements, and single-cell microscopy, we show that semapimod selectively disrupts and permeabilizes the bacterial outer membrane by binding lipopolysaccharide. This work illustrates the value of harnessing non-traditional screening methods and deep learning models to identify non-toxic antibacterial compounds that are effective in infection-relevant contexts. Full Text