Genes & Development

The PR-DUB complex is responsible for erasing the repressive histone modification, H2AK119ub1. ASXL1-3 proteins are mutually exclusive catalytic partners of BAP1 in the PR-DUB complex. Somatic heterozygous ASXL1-3 variants are associated with cancer, including myeloid malignancies, while de novo germline variants cause neurodevelopmental disorders such as Bohring-Opitz syndrome. These pathogenic variants are almost exclusively nonsense and frameshift and have been proposed to act as gain-of-function. However, the precise catalytic impact and mechanism of variant ASXL1-3 remains elusive. Using an isogenic embryonic stem cell model we have discovered that ASXL1 BOS variants drive reductions - but not global ablations - in H2AK119ub1, consistent with gain-of-function. This catalytic change occurs through the production of a truncated ASXL1 protein with enhanced stability. Hyper-stabilised ASXL1 drives a stoichiometric shift in PR-DUB assembly away from ASXL2 complexes. The drop in H2AK119ub1 levels ultimately reduces PRC2 binding and H3K27me3 deposition. Surprisingly, this phenotype is shared across PR-DUB loss-of-function models and indeed is emerging as a common phenotype across genetically and mechanistically distinct Polycomb-related chromatinopathies.

The rhombic lip (RL) gives rise to all cerebellar glutamatergic cell types, including unipolar brush cells (UBCs). Disruptions to UBC development can lead to the neurodevelopmental disorder Dandy-Walker Syndrome and the pediatric brain tumor medulloblastoma, but these diseases have not been adequately modeled in mice. To evaluate conservation of UBC development in mouse and human, we examined UBC localization, lineage decisions, and the underlying molecular mechanisms of UBC differentiation using multiplex immunofluorescence and single-cell RNA-seq of wild-type and conditional knockout animals of the primary UBC transcription factor Eomes. Similar to the human RL, the murine RL is molecularly compartmentalized, cycling EOMES+ UBC progenitors are highly abundant, and persist after birth. Eomes regulates the transcriptional networks important for UBC differentiation and migration, but not UBC fate. Overall, our findings suggest that murine UBC development recapitulates many features of human UBC development, with EOMES playing a central role in UBC maturation.

In female mammals, Xist, the master regulator of X-chromosome inactivation (XCI), is expressed monoallelically. This pattern is established during early embryonic development, when the active Xist allele is chosen at random in each cell. How this choice is made remains incompletely understood. Combining knock-down and overexpression strategies in differentiating mouse embryonic stem cells, which recapitulate the onset of random XCI, we identify a role for the repressive chromatin mark H3K9me3 in the XCI initiation. We show that H3K9me3 accumulates at the promoter-proximal region of the silent Xist allele in female cells as monoallelic expression is established. Unexpectedly, this accumulation requires prior transcription of Xist itself, likely during the initial phase of upregulation, when Xist is frequently transcribed in male cells and from both X chromosomes in females. A repressive function of Xist-dependent H3K9me3 accumulation is supported by our finding that premature, transient Xist overexpression primes an allele for future silencing and skews the choice of the inactive X. Xist-dependent H3K9me3 recruitment does not require its antisense transcript Tsix, which can nonetheless enhance subsequent maintenance of the mark. In addition, the X-linked Xist activator RNF12 counteracts H3K9me3 formation, independently of its known target REX1. Our results thus point to facultative heterochromatin formation as a key contributor to choice at the onset of XCI, where activating and repressing mechanisms are intertwined to establish monoallelic Xist expression.

The olfactory epithelium of adult mammals contains two populations of stem cells that support its remarkable ability to regenerate neuronal and non-neuronal cell types throughout life. How olfactory epithelial cell types are established during development, however, is not well understood. Here, we use genetic lineage tracing and single-cell RNA sequencing of the perinatal mouse olfactory epithelium to construct a developmental trajectory consisting of multiple lineages. We identify transitional states and lineage relationships between individual cells and establish Ascl1+ cells as the primary multipotent progenitors in the perinatal olfactory epithelium. Further, Ascl1+ cells become progressively restricted in their cell fate capacity over developmental time and appear to be transcriptionally primed toward specific lineages. We also predict signaling pathways that may contribute to lineage plasticity and niche permissiveness. Together, these results contribute to our understanding of how cell-intrinsic and-extrinsic signals contribute to the establishment of a stem cell niche.

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.

Polycomb Repressive Complexes PRC1 and PRC2 establish and maintain chromatin architectures critical for cell-type specific gene expression. In the developing spinal cord, PRC1 is essential for implementing Hox-dependent transcriptional programs that diversify motor neuron identities. Although PRC1 can assemble into subnuclear condensates, whether these structures contribute to the specification and maintenance of neuronal subtype fates remains unclear. Using chick and mouse models, we show that canonical PRC1 components - BMI1, PHC2, CBX2, RING1B - coalesce into subnuclear assemblies as neural progenitors differentiate into motor neurons. Disrupting PHC2 polymerization in either neural progenitors or postmitotic neurons dismantles BMI1-containing PRC1 assemblies, triggering derepression of caudal Hox genes and a cell autonomous loss of segment-specific motor neuron fates. Mouse mutants lacking the core PRC1 subunit Ring1 fail to form BMI1-containing assemblies, despite intact PHC2 and CBX2 polymers. In contrast, motor neuron-restricted deletion of core PRC2 subunits (Ezh1/2) depletes H3K27me3 histone marks but retains PRC1 assemblies and Hox boundaries. Together, these findings indicate that PHC2-dependent PRC1 assemblies stabilize Hox transcriptional boundaries and motor neuron segmental identities.

Eukaryotic RNA polymerase II transcripts carry a signature m7G cap (Cap0) structure that is co-transcriptionally added to the 5 end, and is essential for translation and RNA stability. Higher eukaryotes carry additional essential ribose methylations on the first and second cap-proximal nucleotides, termed as Cap1 and Cap2, respectively. The ubiquitous Cap1 modification protects cellular RNAs from being recognized by the innate immune sensors. Cap2 is also implicated in such an innate immune role, but here we use our genetic analyses of two human cell lines and three mouse tissues to reveal that loss of CMTR2 does not result in activation of the innate immune response. Germline deletion of mouse CMTR2 shows that it is required for male and female fertility. While mutant germ cells proceed into the meiotic pachytene spermatocyte stage, their transcriptome fails to keep pace and transition from the preceding leptotene/zygotene stages. Such a meiotic role is not conserved in other vertebrates like zebrafish, as cmtr2 mutants are fertile, instead it has a role in defining sex, as all mutants are exclusively males. Taken together, our study reveals that CMTR2 does not influence innate immune response in human cells and mouse tissues, but shapes gene expression during developmental transitions.

Structural remodeling of neuronal projections in response to developmental cues, injury, or disease is essential for adaptive circuit rewiring. This dynamic process, characterized by pruning and regrowth phases, requires the coordinated execution of neurite regrowth and branching to establish functional neuronal circuits. Yet, how these processes are regulated in space and time at the post-transcriptional level remains poorly understood. Here, we identify the conserved RNA-binding protein Imp (IGF2BP) as a central regulator of developmental axonal remodeling in Drosophila CCAP/Bursicon neurons. We show that Imp acts within a restricted time window during late metamorphosis to control both late regrowth and branching of adult CCAP/Bursicon axons. Combining functional approaches, high-resolution imaging and single-molecule mRNA detection, we further show that Imp controls these temporally distinct programs through genetically separable regulatory mechanisms. While axonal elongation is mediated by Imp-dependent stabilization of profilin mRNA, axonal branching is mediated by an independent mechanism that may involve local regulation in axons. Together, our findings demonstrate that axonal regrowth and branching, two morphogenetic events essential for neuronal circuit maturation in vivo, are controlled independently, yet coordinated through a common and conserved post-transcriptional framework. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/740905v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1ca291corg.highwire.dtl.DTLVardef@15108daorg.highwire.dtl.DTLVardef@11c5741org.highwire.dtl.DTLVardef@1a4773a_HPS_FORMAT_FIGEXP M_FIG C_FIG

During spermatogenesis, germ cells undergo extensive chromatin remodeling to promote highly diverse and stage-specific transcriptional programs that drive differentiation, meiosis, and spermiogenesis. However, conventional loss-of-function approaches do not allow systematic interrogation of gene function across spermatogenesis in vivo. Here, we introduce a protein-degron strategy coupled with intra-seminiferous tubule injection (dTAG-IS) to enable rapid, simultaneous, and stage-resolved functional investigation of NFYA during spermatogenesis. Acute NFYA-depletion induces apoptosis in differentiating spermatogonia (diff-Spg), pachytene spermatocytes (pSpc), and round spermatids (rStd), revealing an essential requirement across spermatogenesis. Mechanistically, NFYA exhibits stage-specific chromatin-binding patterns and directly regulates distinct transcriptional programs, including cell cycle progression and histone assembly in diff-Spg; chromosome organization and DNA damage response in pSpc; and antioxidative stress programs in rStd. Together, our study underscores dTAG-IS as a versatile strategy for in vivo loss-of-function studies during spermatogenesis and establishes NFYA as a multifaceted regulator that drives stage-specific transcriptional programs to ensure spermatogenesis progression.

During meiosis, accurate chromosome segregation requires significant condensation and compaction. These processes are mediated by condensins, cohesins, and histone tail modifications. We identified that MET-2, a histone methyltransferase that catalyzes the dimethylation of histone H3 lysine 9 (H3K9me2), differentially impacts chromosome size in the male vs. female C. elegans germline. In met-2 null worms, autosomes during spermatogenesis are significantly larger than wild type, while chromosome size during oogenesis is unaffected. X-univalent size in males is also unaffected by loss of MET-2, indicating MET-2 differentially regulates autosomal and X-chromosome compaction in male spermatogenesis. Autosome size is not changed when males harbor a catalytically deficient MET-2 (met-2CD) or have mutations preventing germline histone H3K9 methylation (H3K9R). In addition, met-2 males, in contrast to met-2CD or H3K9R males, have more active RNA pol II in later stages of meiosis. These data suggest MET-2 plays a noncatalytic role in mediating chromosome structure and transcription. In met-2 male germ lines, genes on the X chromosome, which is typically enriched in H3K9me2, are significantly more likely to be upregulated than genes on autosomes, even though X-univalent size is unchanged. These results suggest that MET-2 plays a sex-specific role that is not limited to its enzymatic activity.

Centromeres are essential for chromosome segregation and are epigenetically defined by CENH3/CENP-A nucleosomes. Centromere position along chromosomes varies within and between species, ranging from telocentric to metacentric architectures. Yet, how centromere position influences chromosome inheritance and karyotype evolution remains poorly understood. To reposition the centromere, we used CRISPR-Cas9 to break the centromeric satellite array of Arabidopsis thaliana chromosome 3. Fission chromosomes rapidly acquired telomeres, converting a metacentric into two stable telocentric neo-chromosomes. Neo-centromere formation involved genetic restructuring and de novo satellite higher-order repeat formation, together with epigenetic remodeling of CENH3 and DNA methylation. Crossing the six-chromosome fission line to five-chromosome wild type produced a meiosis-specific trivalent that mis-segregated and generated aneuploidy. Trivalent recombination doubled through two obligate crossovers, which were shifted towards the telomeres. Inheritance was strongly distorted in favor of the wild type centromere, as telocentrics segregated into inviable monosomic gametes. The reciprocal trisomic gametes were associated with centromere-proximal recombination, demonstrating that crossover position governs trivalent segregation. Distortion further increased when CENH3 was over-expressed, implicating centromere strength in the outcomes of trivalent meiosis. Our results reveal rapid centromere remodeling following karyotype change, and how trivalent centromere architecture distorts inheritance, with implications for hybrid incompatibility and synthetic chromosome design.

Long, dosage-sensitive mRNAs encoding neurodevelopmental regulators are selectively dependent on fragile X messenger ribonucleoprotein (FMRP) for efficient translation across animal systems, but the mechanism underlying this length-dependent requirement remains unclear. Here, we show that FMRP maintains translation of long target mRNAs by preventing their inappropriate sequestration into a Me31B/DDX6-dependent P-body repression pathway. In Drosophila oocytes, loss of FMRP caused target mRNAs, but not bulk polyadenylated RNA, to accumulate in Me31B-marked P-bodies. Improved individual-nucleotide resolution crosslinking and immunoprecipitation (iiCLIP) revealed that FMRP and Me31B co-occupy many long coding sequences, suggesting that FMRP targets are intrinsically vulnerable to repressive Me31B-associated machinery. A screen targeting [~]120 candidate genes, followed by proteomic analysis, identified an FMRP-associated ribonucleoprotein (RNP) assembly containing the stress granule-linked proteins Rin/G3BP, Lig/UBAP2L, and Capr/Caprin1. These factors supported translation or localization of distinct FMRP target subsets. Finally, inhibition of Me31B-dependent repressive complex assembly restored translation of the majority of FMRP targets in FMRP-deficient oocytes, supporting a model in which translational failure results from excessive repression. Together, these findings reveal a cytoplasmic RNP assembly that safeguards mRNAs by antagonizing promiscuous P-body repression and provide a mechanistic explanation for the selective vulnerability of long neurodevelopmental transcripts to FMRP loss.

The STAG1 and STAG2 paralogs are mutually exclusive subunits of cohesin, a large protein complex that controls different aspects of genome organization. Loss-of-function mutations in STAG2 are common in a variety of cancers, and have been associated with increased sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPi). This provides a possible entry point for therapeutic exploitation. However, insights in the underlying mechanism and additional determinants of this sensitivity are currently lacking. Here we show that STAG2-deficient cell lines exhibit variable PARPi sensitivity. While wildtype TP53 enhances PARPi sensitivity in RPE1 cells, STAG2 depletion does not trigger p53 or affect PARPi sensitivity in RPE1-TP53wt cells. PARPi sensitivity is determined by the capacity of STAG1 to compensate for the lack of STAG2. It appears that STAG2 is not uniquely required for the PARPi response, and its loss predominantly sensitizes cells through a general defect in cohesin function. PARP trapping underlies this sensitivity and is strongly influenced by basal PARP1 expression levels and further enhanced by oncogenic signaling. Perturbations in sister chromatid cohesion also enhances PARPi sensitivity, correlating with aggravated cohesion defects and increased chromosome breakage. Mechanistically, cohesin impairment does not appear to compromise repair of nickase-induced DNA lesions, suggesting that cohesin-defective cells are particularly vulnerable to a distinct class of lesions associated with trapped PARP. Together, our findings identify residual cohesin function and PARP1 expression as key determinants of PARPi response and suggest that successful clinical application of PARP inhibitors in STAG2-mutant cancers will require biomarker-guided patient stratification rather than STAG2 mutation status alone.

DNA lesions continually challenge genome replication and threaten genome integrity. DNA damage tolerance pathways, including translesion DNA synthesis (TLS), allow cells to bypass lesions and prevent stalled forks from collapsing into double-strand breaks. Because TLS polymerases are intrinsically error-prone, however, this pathway must be tightly restrained; persistent or deregulated TLS can increase mutagenesis, create therapeutic vulnerabilities, and promote aggressive cancer phenotypes. Through integrated transcriptional profiling, genome-wide CRISPR/Cas9 screening for replication-stress sensitivity, and complementary proteomic analyses, we identify F-box protein 44 (FBXO44) as a late p53-responsive regulator of the TLS mediator RAD18. FBXO44 promotes RAD18 ubiquitination during recovery from replication stress and facilitates shutdown of RAD18-dependent PCNA monoubiquitination. Consistently, FBXO44 loss delays resolution of replication stress and TLS signaling, increases mutation frequency, and is associated with elevated mutational burden and therapy resistance in experimental models and patient datasets. These findings define a p53-FBXO44-RAD18 regulatory axis that limits mutagenic TLS and helps safeguard genome integrity after replication stress.

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.

Leptomeningeal metastases are the primary source of morbidity and mortality for pediatric medulloblastoma patients. Due to limited surgical sampling of metastases in patients, little is understood of the mechanisms of metastasis. Here, we identify biologically distinct quiescent small metastases (designated as micrometastases) and mitotically active larger metastases (macrometastases). Macrometastases are more metabolically active than micrometastases and contain higher levels of lipids, particularly cholesterol. Macrometastases secrete CXCL12, which attracts lipid-laden macrophages into the tumor. Lipid-laden macrophages upregulate the cholesterol transporter ABCG1, promoting the efflux of free cholesterol, which is then taken up by tumor cells via the HDL receptor SCARB1. CXCL12-driven macrophage recruitment and exogenous cholesterol are sufficient and necessary to drive progression of medulloblastoma leptomeningeal metastases in vivo. High fat diets drive metastatic progression in vivo. Dietary or pharmacological interventions targeting the CXCL12-SCARB1-cholesterol axis represent therapeutic strategies to either prevent or treat medulloblastoma leptomeningeal metastases.

The central mechanisms through which glial cells regulate whole-body metabolism remain poorly understood. Here, we identify Notch signaling in hypothalamic oligodendrocyte lineage cells as a previously unrecognized regulator of systemic energy homeostasis. Pharmacological inhibition of the Notch ligands Jagged1 (Jag1) and Jagged2 (Jag2) induces rapid and reversible weight loss across diverse physiological and metabolic contexts independently of toxicity or caloric intake. Single-nucleus transcriptomic analyses identify hypothalamic oligodendrocyte precursor cells (OPCs) as the principal Notch-responsive population following systemic Jag1/2 inhibition and reveal expansion of a metabolically specialized GPR17 intermediate state characterized by enhanced oxidative metabolism and increased predicted communication with hypothalamic neurons. This glial remodeling is accompanied by fasting-like transcriptional reprogramming of AgRP neurons, reorganization of melanocortin-autonomic circuit activity, and activation of peripheral catabolic programs. Importantly, selective deletion of Notch1/2 in hypothalamic OPCs recapitulates the major physiological and metabolic effects of systemic Jag1/2 inhibition, establishing oligodendrocyte Notch signaling as a causal regulator of whole-body metabolism. Together, our findings establish Notch-dependent oligodendrocyte state transitions as a previously unrecognized mechanism linking glial plasticity to systemic energy homeostasis.

Cancer cell plasticity underlies tumor progression, metastasis and therapeutic resistance, yet how RNA-binding proteins regulate malignant state transitions remains poorly defined. Here, we identify the splicing regulator RBFOX2 as a suppressor of oncogenic cell plasticity in Group 3 medulloblastoma (MB). OTX2 antagonizes RBFOX2 by directing an opposing alternative splicing program that mirrors developmental origins and controls cytoskeletal dynamics and cell state transitions. Mechanistically, OTX2 alters the RBFOX2 RNA-binding landscape, limiting RBFOX2 engagement at cytoskeletal loci and suppressing exon inclusion. RBFOX2 loss rewires splicing and transcriptional outputs to promote highly plastic proliferative, mesenchymal-like and undifferentiated cellular states associated with enhanced oncogenic properties. Consistent with these findings, RBFOX2low states mark highly plastic cell populations in patient tumors and xenografts, positioning RBFOX2 as a determinant of malignant cell stability. Together, these findings establish RBFOX2 as a developmental gatekeeper that restrains malignant cell plasticity through coordinated control of cytoskeletal gene programs.

Understanding the behavior of human neural progenitor cells (NPCs) requires comparative characterization of their transcriptomic landscape, particularly through comparisons with closely related primate species. The rhesus macaque represents a key non-hominoid outgroup for such analyses, and several representative transcriptomic datasets are now available. Here, we compare genes enriched in human NPCs with those enriched in rhesus macaque NPCs and found that human NPC-enriched genes are associated with gene programs supporting radial glial identity and proliferative capacity, as well as functions related to sister chromatid segregation. This analysis identified two zinc-finger transcription factors for functional investigation: the ape-specific ZNF90 and the highly conserved OVOL2. By analysing their genome-wide binding, transcriptional output, and cellular effects in cerebral organoids, we uncovered a previously uncharacterized regulatory axis with divergent but partially overlapping effects that converge on apical progenitor (AP) maintenance. Together, our findings support a model in which lineage-specific / evolutionarily young transcription factor become integrated into a conserved developmental gene regulatory network, generating novel NPC dynamics during primate corticogenesis.