Genes & Development

Sex differences in immunity shape cancer risk, autoimmunity, and responses to immunotherapy, yet the sex-chromosome genes that regulate antitumor T cell function remain incompletely defined. Here, we identify the Y chromosome-encoded KDM5D histone demethylase as a male-specific suppressor of CD8+ T cell antitumor immunity. In murine colorectal cancer (CRC) models, male CD8+ T cells displayed reduced cytokine production, proliferation, cytotoxicity, TCR{beta} abundance, and proximal TCR signaling relative to female CD8+ T cells. CRISPR-RNP-mediated KDM5D depletion in male CD8+ T cells enhanced effector function, increased TCR{beta} expression, augmented TCR signaling, and improved tumor control after adoptive transfer. Transcriptomic and functional analyses further linked KDM5D to cholesterol biosynthesis and exhaustion-associated programs, with KDM5D depletion reducing SREBP2/XBP1-associated cholesterol and exhaustion signatures. Correspondingly, human CRC single-cell analyses supported the clinical relevance of this axis, showing enrichment of exhausted and cholesterol-associated CD8+ T cell states in male tumors. Pharmacologic inhibition of cholesterol biosynthesis with lovastatin partially attenuated select exhaustion-associated markers in male CD8+ T cells and delayed tumor growth in vivo. Together, these findings define KDM5D as a sex chromosome-encoded regulator of male CD8+ T cell dysfunction and point to cholesterol-exhaustion programs as a potential therapeutic vulnerability in male CRC.

Recognition of 5' splice sites by the U1 small nuclear ribonucleoprotein commits pre-mRNAs to splicing, yet splice-site complementarity alone cannot predict productive U1 engagement. Whether dynamic pre-mRNA structure regulates this early spliceosome assembly step remains unclear. Here, we identify a 5'UTR-intron base-pairing interaction positioned near the 5' splice site that acts as an inducible structural gate for U1 engagement. In budding yeast, this element is enriched among introns required for adaptation to nutrient depletion, and in vivo DMS-MaPseq shows that starvation remodels its structure. Structure-guided disruption of pairing impairs adaptation, whereas compensatory mutations restoring pairing without restoring sequence rescue the phenotype, establishing RNA fold as the critical determinant. U1 association decreases when the gate is disrupted and recovers when pairing is restored, and increased Nam8 levels can compensate for gate disruption by stabilizing U1 engagement under stress. Thus, dynamic pre-mRNA folding gates U1 recognition, revealing how transcript architecture converts physiological state into selective splice-site choice.

The small ubiquitin-like modifier SUMO regulates key events of meiosis, including pairing and crossing over between homologous chromosomes. Auto-sumoylation of the SUMO E2-conjugating enzyme UBC9 at lysine 14 alters its substrate selectivity in vitro, but the role of this modification in vivo is unknown. Here, we show that UBC9-K14 auto-sumoylation helps coordinate meiotic prophase and is important for maintenance of the ovarian reserve. Ubc9K14R/K14R knock-in mice show a variety of defects in meiotic prophase I, including altered assembly of DNA strand-exchange complexes, delayed and defective homolog synapsis, and unstable crossover recombination complexes. In spermatocytes, these defects are associated with reduced efficiency of crossing over between the X and Y chromosomes. Oocytes from Ubc9K14R/K14R females show related but distinct defects in recombination and synapsis. Moreover, maintenance of the primordial follicle reserve is defective in Ubc9K14R/K14R females, and their fecundity is reduced. Finally, we identify the meiosis-specific homolog-axis protein SYCP3 as a direct target of UBC9 in vitro and show that SYCP3 modification is strongly stimulated by K14 auto-sumoylation. We infer that auto-sumoylation enables UBC9 to modify a subset of targets in vivo, helping to coordinate key events of meiotic prophase and enhance the survival of primordial follicles to maximize fecundity.

During the Drosophila maternal-to-zygotic transition, maternally deposited RNAs are cleared in a temporally controlled manner. The RNA-binding protein Smaug is a major regulator of maternal mRNA decay and its expression at both the RNA and protein level is limited to a narrow temporal window during the MZT. Here we show that Pumilio RNA-binding protein promotes degradation of smaug mRNA at the end of the MZT through Pumilio-binding cis-elements in the smaug 3 untranslated region, and that disruption of this regulation leads to ectopic Smaug protein expression beyond its normal developmental window. We further find that another RNA-binding protein, Brain tumor, also contributes to repression of ectopic Smaug expression. Transcriptome-wide analyses of embryos lacking Pumilio reveal that it directly regulates hundreds of maternal mRNAs after zygotic genome activation, with many of these Pumilio targets also regulated by Brain tumor. The ectopic Smaug protein that results from loss of either Pumilio or Brain tumor causes widespread downregulation of mRNAs that contain Smaug binding sites. Together, these findings define a post-transcriptional regulatory pathway in which Pumilio and Brain tumor ensure orderly progression of the Drosophila maternal-to-zygotic by clearing smaug mRNA and preventing ectopic Smaug activity.

The chromatin remodeler ATRX and its orthologs maintain genome function by regulating repetitive DNA and dynamic chromatin, and their activities have been canonically associated with replication-independent deposition of the histone H3.3 variant. This model is difficult to reconcile with fungi, which encode ATRX orthologs but lack H3 variants that may separately support replication-coupled and replication-independent deposition. Here we show that the fungal ATRX ortholog SAD-6 instead relies on a highly divergent histone H4 variant (H4v) to mediate broad genome surveillance and defense. Deposition of H4v is strictly SAD-6-dependent and thus provides a sensitive genome-wide readout of SAD-6 activity, revealing its functions at telomeres, tRNA and rDNA loci, AT-rich DNA, artificial transgenes, decaying mobile elements, and many genic regions. We further show that SAD-6 is required for a pathway of repeat-induced point mutation (RIP) that also requires DIM-5, a conserved SUV39 methyltransferase that mediates trimethylation of histone H3 lysine-9 in heterochromatin. Together, these findings establish ATRX-like remodelers as broad regulators of genome surveillance and defense in fungi that act through a highly divergent histone H4 variant rather than H3.3. Given that RIP is proposed to recognize repetitive DNA via recombination-independent homologous pairing, the requirement for SAD-6 in RIP suggests that ATRX-like remodelers may couple DNA pairing to heterochromatin nucleation on repeats.

X-chromosome inactivation involves chromosome-wide gene silencing accompanied by extensive chromatin changes, as well the loss of topologically associating domains. Yet discrete regions of the inactive X chromosome retain activity within localised 3D domains, which contain active genes that variably escape from X inactivation. The transcription factor and architectural protein CTCF has been proposed to be implicated in escape by insulating escape domains or sustaining their topology via cohesin-mediated loop extrusion. Here, we test the role of CTCF and cohesin in escape using acute degron-mediated depletion of CTCF and RAD21 in neural progenitor cells with established escape profiles. Although CTCF occupancy correlates with escape status on the inactive X chromosome, its removal - together with loss of loop extrusion - does not disrupt escapee gene expression, or domain organization, nor does it result in spreading of silencing or activation of genes in cis. Rather, we show that facultative escape regions are self-sustaining compartments of active chromatin enriched in H3K27 acetylation and depleted in H3K27 methylation, with the magnitude of compartment strength scaling up with the degree of transcriptional activity on the inactive X chromosome. These active escapee compartments are propagated independently of CTCF and RAD21-dependent 3D architecture. Our findings identify chromatin compartmentalization as the primary feature of facultative escapee domains.

Paternally-inherited epigenetic information can influence phenotype in offspring (1). Here, we identify a critical mechanistic contribution of KDM6A (UTX), an X-linked histone modifier and tumor suppressor, in regulating transmissible epigenetic information in mammalian sperm. Paternal loss of KDM6A increases cancer risk in genetically wild type offspring, but how Kdm6a knockout sperm transmit this effect at the molecular level is unknown (2). We find that KDM6A functions in spermatogenesis to promote methylation of histone H3 lysine 4 (H3K4) via selective interaction with the COMPASS complex methyltransferase KMT2C (MLL3). KMT2C and KDM6A are coordinately recruited to promoters of active genes in spermatogenic cells, contrasting with recruitment to intergenic enhancers in other cell types (3, 4). Loss of KDM6A disrupts H3K4 methylation at promoters of tumor suppressor genes in spermatogonia, and some of these defects persist in epididymal sperm and correspond to impaired expression in preimplantation embryos. These genes are also misregulated in normal and malignant hematopoietic tissue of genetically wild type offspring, indicating that impaired H3K4 methylation in KDM6A-deficient male germ cells may preferentially alter regulation of tumor suppressor gene networks in development across generations.

Accurate chromosome segregation requires the spatiotemporally regulated removal of sister chromatid cohesion. Cohesin cleavage by the endopeptidase separase depends on destruction of its inhibitor securin by the ubiquitin-proteasome system (UPS) and on phosphorylation-mediated priming of the cohesin kleisin subunit. Whether the UPS also contributes to cohesin priming has remained unknown. Here, we show that the 26S proteasome mediates cohesin removal during meiosis II through branches of two parallel pathways. Using separation-of-function mutants targeting either the proteasome's core or regulatory particles, we identify a proteasome function that is required specifically for centromeric cohesin removal during meiosis II but dispensable for separase activation. Defects in this proteasome function causes centromeric accumulation of phosphatase anchor shugoshin (Sgo1), impaired cleavage of meiotic kleisin Rec8, and frequent failure of sister chromatid segregation. Bypassing the requirement for Rec8 priming, either through a phosphomimetic rec8 allele or by separase-independent Rec8 cleavage, restores chromosome segregation, demonstrating that the proteasome mediates cohesin removal independently of its established role in activating separase. Consistent with a direct role, proteasomes localize prominently to kinetochores during meiosis II. Together, these findings identify the proteasome as a dual-function regulator that mediates both separase activation and cohesin priming, revealing how a single proteolytic machine coordinates the two molecular pathways underlying stepwise chromosome segregation during meiosis.

The Mediator complex, a vital transcriptional coregulator in eukaryotes, partners with transcription factors to orchestrate gene transcription and in turn many developmental and physiological processes. Mediator subunit MED15 is required for the pre-natal development and post-natal maturation of pancreatic {beta}-cells in mice. However, whether MED15 plays a role in {beta}-cell function after initial development and throughout adulthood is unknown. To investigate the role of MED15 in {beta}-cells post-maturation, we induced a {beta}-cell specific Med15 knockout at six weeks age in male and female mice. This post-developmental Med15 ablation led to glucose intolerance and impaired insulin secretion. RNA-sequencing revealed downregulation of {beta}-cell maturation markers, indicating that MED15 is continuously required to maintain {beta}-cell functionality. Further, we implanted insulin pellets into Med15 knockout mice to lower blood glucose and used RNA-seq to validate that the transcriptional changes we observed are a direct consequence of Med15 loss and not an indirect effect of hyperglycemia arising in the knockout mice. In sum, our study shows that MED15 is continuously required after weaning to maintain functional {beta}-cell maturity.

Dysregulated hormone secretion and erosion of endocrine cell identity are features of type 1 and type 2 diabetes, but the transcriptional programs maintaining adult human islet identity and function remain poorly defined. The large MAF transcription factor MAFB is expressed in human - and {beta}-cells, marks their most functionally mature subpopulations, and is downregulated in diabetes, but its role in adult human islets has not been tested directly. Using shRNA-mediated MAFB knockdown (KD) in whole and CD26+ -cell-enriched human pseudoislets, we found that whole pseudoislet MAFB KD impaired glucagon synthesis and secretion while only modestly reducing insulin content and cAMP-potentiated insulin release. Single-cell profiling detected no {beta}-cell transcriptional response beyond MAFB KD itself, consistent with buffering by the related {beta}-cell-enriched MAFA transcription factor. In contrast, -cell-restricted MAFB KD unmasked a cell-autonomous requirement for MAFB in stimulus-secretion coupling. MAFB deficiency also destabilized -cell identity, downregulating canonical -cell and neuroendocrine secretory genes while ectopically inducing mesenchymal and extracellular matrix remodeling programs. In addition, MAFB-dependent downregulation of electron transport chain genes was confined to a large -cell subcluster, manifesting as impaired islet-wide mitochondrial respiration within the broader -cell population. Together, these findings identify MAFB as an essential adult human -cell maintenance factor that links diabetes-associated downregulation to impaired glucagon secretion, -cell identity erosion, and mitochondrial dysfunction. RESEARCH IN CONTEXTO_LIWhat is already known about this subject? O_LIMAFB is expressed in adult human - and {beta}-cells, marks their most functionally mature subpopulations, and is downregulated in type 1 and type 2 diabetes C_LIO_LIIn human stem cell models, MAFB is essential for generating insulin-producing {beta}-like cells, whereas glucagon-producing -like cells are reduced but still formed C_LIO_LINeither model addresses adult human islets: rodent MafB becomes -cell restricted after birth, and stem cell models capture differentiation, not maintenance C_LI C_LIO_LIWhat is the key question? O_LIIs MAFB required to maintain identity and secretory function in adult human islet cells? C_LI C_LIO_LIWhat are the new findings? O_LIMAFB knockdown in primary human pseudoislets impaired glucagon synthesis and secretion but minimally affected {beta}-cells, consistent with buffering by MAFA C_LIO_LIKnockdown in CD26+ -cell-enriched pseudoislets revealed a cell-autonomous requirement for MAFB in stimulus-secretion coupling, and destabilized -cell identity by inducing mesenchymal and extracellular matrix programs C_LIO_LIMAFB loss downregulated electron transport chain genes in the largest -cell subcluster and reduced mitochondrial respiration C_LI C_LIO_LIHow might this impact on clinical practice in the foreseeable future? O_LIPreserving MAFB activity in adult human -cells may represent a strategy to limit -cell dysfunction in diabetes C_LI C_LI

MYC family members have been extensively studied as undruggable transcription factors regulating oncogenic signaling in highly aggressive tumors such as small cell lung cancer (SCLC), via promoter binding. Here, leveraging the previously described Myc-driven SCLC mouse model (RPM), we generated RPM-Miz1{Delta}POZ (RPMM) mice to uncover a Myc-dependent regulation of neuroendocrine (NE) differentiation, via enhancers. Our functional and genomic analyses reveal that Miz1 facilitates Myc binding to low-affinity E-boxes at distal chromosomal regions, thereby enabling Myc occupancy at sites with otherwise limited intrinsic affinity. We further show that SCLC patients and cellular models share an enrichment of low-affinity E-Box Myc binding motifs at enhancer regions that loop to genes of classic neuroendocrine differentiation. Integrated epigenetic and genomic analyses with AI-modeling implicate Myc/Miz1 binding at enhancers as the determinant for the expression of bona-fide neuroendocrine genes. In RPMM tumors, the suppression of neuroendocrine identity is paralleled by a redistribution of Myc protein towards promoter-proximal regions, hyper-activation of Myc transcriptional programs, apoptotic priming and enhanced sensitivity to etoposide. Together, these findings uncover Miz1/Myc-engaged enhancers as a central hub for neuroendocrine lineage programs and provide a mechanistic basis for a targeted inhibition of Miz1 to boost chemosensitivity in SCLC.

Pre-mRNA splicing is essential for gene expression, yet how disruption of core spliceosomal factors produces tissue- and developmental stage-specific phenotypes remains poorly understood. Here, we investigated the in vivo function of the conserved spliceosomal kinase PRPF-4 in C. elegans using endogenous reporter analysis, conditional protein depletion, and transcriptome-wide analysis of alternative splicing and gene expression. We found that PRPF-4 is broadly expressed throughout development and is continuously required for postembryonic development, with distinct requirements in the pharynx, nervous system, and germline. Acute PRPF-4 depletion rapidly disrupts alternative splicing across thousands of transcripts, with exon skipping representing the predominant class of affected events. In addition, PRPF-4 depletion results in a robust transcriptome shift with induction of components of the spliceosome and repression of ciliary and ion transport-related transcripts. These findings establish PRPF-4 as a central regulator of RNA metabolism and demonstrate the far-reaching effects on gene expression caused by loss of core spliceosomal components.

The piRNA pathway maintains genome integrity by silencing transposons cotranscriptionally in the nucleus through recognition of nascent transposon RNAs and recruitment of endogenous transcriptional and chromatin-level repressive mechanisms to transposon loci. However, the molecular link between the SFiNX complex, which recognizes nascent transposon RNA, and downstream effector complexes has remained elusive. Here, we demonstrate that the Small ovary (Sov) protein mediates this connection. By mapping the functional activities of its structural elements, we reveal that Sov contributes to transposon silencing through two distinct molecular mechanisms. First, Sov specifically directs nascent transposon transcripts toward nuclear RNA exosome-mediated degradation by physically interacting with the RNA decay factor TEsup1. Second, Sov directly binds the heterochromatin protein HP1a via multiple conserved motifs and undergoes phase separation, facilitating heterochromatin formation and genome-wide gene repression. Genetic analyzes of sov mutants reveal that these functions are separable: RNA-mediated transcriptional silencing is essential for piRNA pathway activity, while phase separation-dependent heterochromatin regulation is critical for stable transposon repression. We propose that Sov acts as a molecular scaffold in piRNA-guided transposon silencing, integrating transposon recognition with cotranscriptional RNA decay and chromatin-based regulatory pathways.

Urothelial cancer (UC) cells of the luminal subtype exhibit partial, incomplete differentiation towards umbrella cells that line bladder lumen, seen by morphology and gene expression. Differentiation is stalled even though the cells express master transcription factors (MTFs) that drive luminal urothelial differentiation, e.g., FOXA1 and CEBPB, at levels seen in normal differentiated urothelium. We therefore analyzed the FOXA1/CEBPB MTF hub by mass spectrometry. SWI/SNF coactivator complex (CoA) components, e.g., SMARCA4, ARID1A, that read the epigenetic activation mark histone 3 lysine 27 acetylation (H3K27ac) and use ATP-hydrolysis to open chromatin, were the most abundant proteins pulled-down with FOXA1/CEBPB. However, genes for these and other CoA, e.g., CREBBP, EP300 that write H3K27ac, were mutated/deleted in >95% of UCs in clinical series. Also contained in the hub were corepressors (CoR) that erase H3K27ac and close chromatin, e.g., HDAC1, CHD4 - genes for these CoR were recurrently gained in UCs. Chromatin analyses showed H3K27ac-centered remodeling was needed to activate umbrella but not constitutively accessible cell growth/division/housekeeping genes. Restoring ARID1A into ARID1A-mutated UC cells using lentiviral transduction, or inhibiting CoR with siRNA or small molecules, activated umbrella genes and terminated replications. In summary, UC-genesis selects for loss- and gain-of-function of CoA and CoR respectively in the urothelial-lineage MTF hub; small molecule CoR-inhibitors are candidate remedies to renew maturation towards terminal differentiated-fates. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/744501v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@19e853org.highwire.dtl.DTLVardef@e3c934org.highwire.dtl.DTLVardef@ae7874org.highwire.dtl.DTLVardef@66331b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Insulin production is a cardinal feature of pancreatic {beta} cells. Studies in rodents show that {beta} cells can switch between low and high insulin gene activity states and that elevated insulin production makes {beta} cells more vulnerable to stresses associated with diabetes. In people, genetically elevated insulin production increases the risk of type 1 diabetes. Via effects on obesity, hyperinsulinemia contributes to the pathogenesis of type 2 diabetes. Here, we characterize {beta} cells in low and high INS gene activity states sorted from primary human islets transduced with INS-GFP adenovirus and differentiated INS-EGFP knock-in embryonic stem cells (SC{beta} cells). We profile {beta} cell function, protein synthesis, resilience to diabetes associated stress, single {beta} cell transcriptomes and their co-activity networks, and purified {beta} cell proteomes. We show that human {beta} cells transition between distinct states. High INS cells have elevated maturity marker mRNAs and proteins, increased protein translation, are larger, but also more susceptible to cell death when exposed to diabetes-relevant stresses. We also catalogue thousands of differences in proteins in high INS stem cell-derived {beta} cells compared directly with high INS primary {beta} cells. Our study improves our understanding of the delicate balance between insulin production and {beta} cell resilience and guides the engineering of better {beta} cells. Blurbtranscriptional, proteomic, and functional analyses of insulin gene expression states in human {beta} cells from donor islets and stem cells Key findingsO_LIWe identify high and low INS gene activity states in human insulin-producing cells from donor islets and embryonic stem cell differentiations. C_LIO_LIWe characterize the relationship between insulin production and fragility, demonstrating that increased insulin production comes at a cost of reduced resilience to multiple stresses. C_LIO_LIFunctional, transcriptomic, and proteomic analyses identify similarities and differences between how primary and stem cell-derived {beta} cells manage stress and insulin production. C_LIO_LIWe report a comprehensive side-by-side proteomic analysis of purified primary and stem cell- derived {beta} cells in the high INS state and identify differences in protein production and secretion machinery, providing a roadmap for making better {beta} cells. C_LI

Mammalian genomes encode multiple transposable element (TE) silencing pathways that distinguish their targets through different molecular features, with KRAB zinc finger proteins recognizing DNA sequence, the HUSH complex sensing intronless transcripts, and the PIWI-piRNA pathway using small RNA guides. How chromatin state itself contributes to TE recognition remains less defined. Here we identify Spindlin1 (SPIN1), a three-Tudor-domain histone reader implicated in germline piRNA-directed DNA methylation, as a transcriptional repressor of evolutionarily young TEs in mouse embryonic stem cells. SPIN1 selectively binds LINE and ERV loci carrying H3K4me3 and H3K9me3, a chromatin signature enriched at young, transcription-permissive elements, and recognition of these marks by Tudor domains 1 and 2 is required for TE targeting. SPIN1 engages SPINDOC as a Tudor 1 and 3-dependent cofactor whose loss phenocopies SPIN1 depletion, and associates with the H3K9 methyltransferases SETDB1 and G9a. SPIN1 loss reduces H3K9me3 and is accompanied by increased chromatin accessibility, without altering DNA methylation. Thus, SPIN1 uses a histone-state-based mechanism to identify and repress young TEs in pluripotent cells, mechanistically distinct from its germline mode in which SPIN1 cooperates with the PIWI-piRNA pathway to promote DNA methylation, illustrating how a single histone reader engages distinct silencing machineries across cellular contexts.

RNA surveillance and decay is carried out in part by helicase containing complexes that identify, capture and sometimes modify RNA before delivering it to the RNA exosome complex for processing or degradation. The MTREC core complex includes a Mtr4-like protein (Mtl1) helicase and Red1 that works with other cofactors and the RNA exosome in Schizosaccharomyces pombe to degrade nuclear transcripts in processes that can result in formation of facultative heterochromatin. The activities of Mtl1 remain uncharacterized as do contributions of Red1 to Mtl1 within MTREC. Here, we reconstitute the MTREC core complex, resolve structures by cryo-electron microscopy, and compare MTREC activities to S. pombe Mtr4 and Mtl1. We show that Mtl1 is more active relative to MTREC and Mtr4, that MTREC binds RNA better than Mtl1, and that Red1 includes an autoinhibitory coiled-coil domain that dimerizes MTREC and contacts the Mtl1 RecA domains to disrupt its ATPase active site. Together, these data suggest that Red1 may endow MTREC to bind RNA while slowing translocation so that it remains associated with RNA long enough to chaperone it to the RNA exosome for processing or degradation.

During embryonic development, cellular competence to respond to differentiation signals changes dynamically. Although the mechanisms underlying competence acquisition have been extensively studied, those underlying competence loss remain unclear. In preimplantation mouse embryos, Hippo signaling shifts from regulating trophectoderm (TE) fate specification to promoting epiblast maturation. During the blastocyst stage, inner cell mass (ICM) cells lose TE competence in response to the Hippo signaling effector TEAD-YAP. Here, we show that the pioneer factor SOX2 terminates TE competence in the ICM. SOX2 binding to the TEAD-YAP-dependent TE enhancer (TEE) of the TE regulator Gata3 induces chromatin closure, suppressing TEE responsiveness to TEAD-YAP activity. This function of SOX2 requires its interaction with the corepressor TLE4 and histone deacetylase. Similar SOX2-dependent chromatin closure also occurs around other TE genes, including the TE enhancer of another TE regulator, Cdx2. Thus, SOX2 terminates TE competence in ICM cells by closing Hippo signaling-responsive enhancers.

The liver is organized into tightly regulated zones with distinct metabolic functions but zonation erodes with age. Cellular senescence contributes to aging and liver diseases, however, its impact on aging biology is ill-defined. As part of The Cellular Senescence Network Consortium, we used multiple spatial transcriptomics approaches (GeoMx, Visium, CosMx) with snRNA-seq to profile senescence signatures, zonation markers, and metabolic pathways in livers from wild-type (WT) mice of multiple ages. We observed a loss of canonical zone signatures in aged mouse livers characterized by "expansion" of midlobular (zone 2) marker gene expression, accompanied by diminished expression of zone 3 marker genes by middle-age (18 months), indicative of loss of cell identity. Multiple analytic approaches identified distinct age-, zone- and sex-specific senescence signatures, which were significantly associated with zonation markers changes. This was recapitulated in Ercc1 mutant models of accelerated senescence, supporting a causal role of senescent cells in liver aging. A "no-zone" hepatocyte-like cluster expanded with age and with the strongest Senescence-Associated Secretory Phenotype (SASP) profile. Gene expression profiles from senescent hepatocytes implicate decreased WNT signaling and increased BMP as contributing to age-related loss of zonation. Together, these data elucidate the role of senescent cells in driving aging biology in non-diseased liver through disruption of cell:cell signaling and the loss of metabolic and cell identity gene expression necessary for hepatocyte function.

Chromatin remodelers are generally thought to regulate gene expression through nucleosome mobilization and modulation of local chromatin accessibility. Whether these complexes can instead control transcription through higher-order chromatin organization remains poorly understood. Here we identify the BAF subunit DPF3a as an essential regulator of skeletal muscle regeneration and myogenic differentiation. DPF3a functions through association with the H3K36me2/3 reader HRP2 and preferentially localizes to a distinctive chromatin state characterized by focal depressions within broad H3K36me2 domains. Biochemical reconstitution demonstrates that H3K36 methylation directly enhances remodeling activity of the DPF3a-containing cBAF complex. Unexpectedly, despite profound transcriptional defects, loss of DPF3a produces minimal changes in local chromatin accessibility. Instead, DPF3a is required for long-range chromatin looping associated with activation of myogenic genes. Together, our findings uncover a non-canonical mechanism whereby a chromatin remodeler regulates transcription primarily through three-dimensional genome organization rather than local accessibility control, and establish histone modification-guided chromatin remodeling as a key principle in gene regulation.