Polycomb repressive complex 2 (PRC2)-mediated histone H3 K27 trimethylation (H3K27me3) recruits canonical PRC1 (cPRC1) to maintain heterochromatin. In early development, Polycomb-regulated genes can display long-range three-dimensional interactions, many of which resolve during lineage differentiation. Here we report that Polycomb-anchored looping is controlled by H3K27me3 spreading and regulates target gene silencing to influence cell fate specification. Using glioma-derived H3 Lys27-to-Met (H3K27M) mutations as tools to restrict H3K27me3 spreading, we show that H3K27me3 confinement concentrates the chromatin pool of cPRC1, resulting in heightened three-dimensional interactions that mirror the chromatin architecture of pluripotency. Conversely, H3K27me3 spread in pluripotent stem cells dilutes local cPRC1 chromatin concentration, weakening Polycomb loop contact frequencies. Disruption of cPRC1 binding or aggregation compromises stringent repression of Polycomb genes and induces differentiation and tumor regression of H3K27M-mutant glioma. These results identify the regulatory principles and disease implications of Polycomb looping and show that histone-modification-guided distribution of reader complexes is an important mechanism for nuclear compartment organization.

To elucidate possible genetic etiology in two patients with recurrent hydatidiform moles (RHM). Whole exome sequencing was performed on two women presented with RHM. Exome data were filtered, and selected candidate variants were validated by Sanger sequencing. Additionally, we reviewed the literature for GTN rates in patients with RHM and compared them between different genetic etiologies. Exome sequencing identified a heterozygous deleterious variant in FOXL2 p.(Gly187Asp), reported in patients with premature ovarian insufficiency (POI), and a very rare homozygous variant in KASH5 p.(Arg196His). The patient harboring the KASH5 variant experienced recurrent GTNs, and her sister who also had recurrent moles died of metastatic GTN. This study describes the identification of variants in FOXL2 and KASH5 in two patients with RHM. Our findings further support the association of variants in these genes with RHM while highlighting the potential for severe, life-threatening progression to metastatic GTN. Reviewing GTN incidence per patient summarizes current data and their limitations that need to be taken into consideration when interpreting overall risk.

Cerebro–Costo–Mandibular Syndrome (CCMS) is a rare congenital disorder due to pathogenic variants in the core spliceosomal gene SNRPB. Affected individuals present with axial skeletal abnormalities, including cleft palate, micrognathia, posterior rib gaps, and a bell–shaped thorax. The molecular basis of these axial defects remains poorly understood, limiting the development of targeted interventions. To study the temporal and molecular requirements of SNRPB during axial development, we generated a mouse model of CCMS using an inducible Cre–lox system to conditionally delete Snrpb. Mosaic deletion of exons 2–3 of Snrpb after gastrulation resulted in the full spectrum of CCMS-like abnormalities, including micrognathia, posterior rib gaps and a reduced thoracic cavity. Despite normal somite morphology and patterning, transcriptomic analysis of E9.5 mutant somites revealed upregulation of p53 pathway genes and mis-expression of retinoic acid (RA) signaling components, consistent with reduced RA signaling. Alternatively spliced genes in Snrpb mutant somites were associated with post-transcriptional regulation, including chromatin modifiers. To test if RA pathway supplementation can rescue axial defects, we performed dietary RA supplementation; however, this failed to rescue mutant phenotypes, suggesting that p53 activation and chromatin dysregulation additionally contribute to disease pathogenesis. Together, these findings establish the first in vivo model of CCMS–associated axial skeletal abnormalities and indicate that Snrpb dysfunction disrupts the early specification of axial skeletal identity without overtly altering somite patterning.

Female infertility is a prevalent reproductive disorder with high genetic heterogeneity. Previous reports have demonstrated the causal role of biallelic pathogenic variants in the Subcortical Maternal Complex (SCMC) genes in female reproductive failure with some leading to infertility, early embryonic loss, and molar pregnancies, while others are compatible with live birth with and without multilocus imprinting disorders (MLID). Here, we report two deleterious protein-truncating variants, c.1326delG, p.Leu443Phefs*78 and c.2802_2803del, p.Arg935Metfs*15, in heterozygous state in the NLRP2 gene of a patient with primary infertility, four early miscarriages, and one failed attempt of intracytoplasmic sperm injection. We show that the two variants mediate mRNA decay in EBV-transformed lymphoblastoid cells from the patient, lead to decreased NLRP2 protein levels, and alter NLRP2 interactions with other members of the SCMC in vitro. This study emphasizes the importance of performing clinical exomes for patients with recurrent reproductive failure and reporting their variants and reproductive histories to improve patient counseling.

Germ cells are unique in that they tailor chromatin toward generating totipotency. Accordingly, mammalian spermatogonia, including spermatogonial stem cells that constitute the source for male gametes, acquire distinctive chromatin organization with weak insulation, but the underlying mechanism remains unknown. Here we show that STAG3, so far known to exclusively form meiotic cohesins, generates a mitotic cohesin for male germline nucleome programming in mice. Owing to its shorter chromatin residence, STAG3-cohesin attenuates topologically associating domains, rewires enhancer-promoter and Polycomb-mediated repressive interactions, and facilitates finer and more strengthened compartments, establishing a distinctive spermatogonial nucleome. Moreover, in the absence of STAG3-cohesin, spermatogonial stem cells show an impaired differentiation priming for spermatogenesis. Mitotic STAG3-cohesin is also expressed in human B cells and their malignant variations, promoting their propagation. Our findings on mitotic STAG3-cohesin elucidate a principle of male germline nucleome programming, demonstrate an unexpected mitotic role for STAG3 and might potentially improve understanding of human malignancies.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with limited therapeutic options. Loss of the Polycomb repressive complex 2 (PRC2), via inactivating mutations in SUZ12 or EED, occurs frequently in MPNSTs and is associated with poor prognosis. However, the downstream chromatin and signaling consequences of these mutations remain incompletely understood. Here, we show that PRC2 deficiency in MPNST cells induces coordinated chromatin remodeling, characterized by loss of repressive H3K27me3 and gain of activating marks, including H3K27ac and H3K36me2. Integrative epigenomic, transcriptomic, and proteomic profiling revealed that this chromatin reprogramming activates a fetal-like growth signature centered on insulin-like growth factor 2 (IGF2) and its post-transcriptional regulators, Insulin-like Growth Factor 2 mRNA-Binding Protein (IGF2BP1-3). Functional studies demonstrate that PRC2-deficient cells are selectively dependent on IGF2 for proliferation, and that restoration of SUZ12 suppresses IGF2 expression and reduces growth. Analysis of human MPNST tumors confirms upregulation of the IGF2-IGF2BP axis in PRC2-deficient tumors, highlighting its clinical relevance. Together, these findings link PRC2 loss to activation of fetal growth factor-driven oncogenic signaling and identify IGF2 and its regulatory network as potential vulnerabilities in this aggressive tumor subtype. ### Competing Interest Statement The authors have declared no competing interest. Sigrid Jusélius Foundation Emil Aaltosen Säätiö, https://ror.org/005rt3g54 Otto A. Malm Foundation, https://ror.org/05srazs86

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration and neuropsychiatric abnormalities. Loss of full-length dystrophins is both necessary and sufficient to initiate DMD. These isoforms are expressed in the hippocampus, cerebral cortex (Dp427c), and cerebellar Purkinje cells (Dp427p). However, our understanding of the consequences of their absence, which is crucial for developing targeted interventions, remains inadequate. We combined RNA sequencing with genome-scale metabolic modelling (GSMM), immunodetection, and mitochondrial assays to investigate dystrophic alterations in the brains of the mdx mouse model of DMD. The cerebra and cerebella were analysed separately to discern the roles of Dp427c and Dp427p, respectively. Investigating these regions at 10 days (10d) and 10 weeks (10w) followed the evolution of abnormalities from development to early adulthood. These time points also encompass periods before onset and during muscle inflammation, enabling assessment of the potential damage caused by inflammatory mediators crossing the dystrophic blood–brain barrier. For the first time, we demonstrated that transcriptomic and functional dystrophic alterations are unique to the cerebra and cerebella and vary substantially between 10d and 10w. The common anomalies involved altered numbers of retained introns and spliced exons across mdx transcripts, corresponding with alterations in the mRNA processing pathways. Abnormalities in the cerebra were significantly more pronounced in younger mice. The top enriched pathways included those related to metabolism, mRNA processing, and neuronal development. GSMM indicated dysregulation of glucose metabolism, which corresponded with GLUT1 protein downregulation. The cerebellar dystrophic transcriptome, while significantly altered, showed an opposite trajectory to that of the cerebra, with few changes identified at 10 days. These late defects are specific and indicate an impact on the functional maturation of the cerebella that occurs postnatally. Although no classical neuroinflammation markers or microglial activation were detected at 10 weeks, specific differences indicate that inflammation impacts DMD brains. Importantly, some dystrophic alterations occur late and may therefore be amenable to therapeutic intervention, offering potential avenues for mitigating DMD-related neuropsychiatric defects.

NSD2 catalyses the epigenetic modification H3K36me2 (refs. 1,2) and is a candidate convergent downstream effector of oncogenic signalling in diverse malignancies3-5. However, it remains unclear whether the enzymatic activity of NSD2 is therapeutically targetable. Here we characterize a series of clinical-grade small-molecule catalytic NSD2 inhibitors (NSD2i) and show that the pharmacological targeting of NSD2 constitutes an epigenetic dependency with broad therapeutic efficacy in KRAS-driven preclinical cancer models. NSD2i inhibits NSD2 with single-digit nanomolar half-maximal inhibitory concentration potency and high selectivity over related methyltransferases. Structural analyses reveal that the specificity of NSD2i for NSD2 is due to competitive binding with S-adenosylmethionine and catalytic disruption through a binary-channel obstruction mechanism. Proteo-epigenomic and single-cell strategies in pancreatic and lung cancer models support a mechanism in which sustained NSD2i exposure reverses pathological H3K36me2-driven chromatin plasticity, re-establishing silencing at H3K27me3-legacy loci to curtail oncogenic gene expression programs. Accordingly, NSD2i impairs the viability of pancreatic and lung cancer cells and the growth of patient-derived xenograft tumours. Furthermore, NSD2i, which is well-tolerated in vivo, prolongs survival in advanced-stage autochthonous KRASG12C-driven pancreatic and lung tumours in mouse models to a comparable level as KRAS inhibition with sotorasib6. In these models, treatment with both a NSD2 inhibitor and sotorasib synergize to confer sustained survival with extensive tumour regression and elimination. Together, our work uncovers targeting of the NSD2-H3K36me2 axis as an actionable vulnerability in difficult to treat cancers and provides support for the evaluation of NSD2 and KRAS inhibitor combination therapies in a clinical setting.

This study explores the cell fate reprogrammability of H3K27M-mutant pediatric high-grade gliomas (pHGG) using neuronal transdifferentiation as a potential targeted therapy. We treated the BT245 patient-derived glioma cell line with pharmacological combinations targeting neuronal differentiation pathways and performed bulk RNA sequencing to characterize gene expression patterns driving cell fate transitions. Our findings reveal that the drug combinations induce transcriptomic changes consistent with differentiation towards neuronal phenotypes, including the upregulation of synaptic and dendritic signaling genes and the downregulation of malignant signatures. In comparison, astrocytic differentiation media (DM) and H3K27M knockout (KO) promote residual astrocytic phenotypes, suggesting neuronal transdifferentiation as a more effective strategy for mitigating tumor aggressiveness and progression. Differentially expressed genes such as GRIK1, GRIN1, NRXN3, NRXN1, CALB2, SCGN, SLC32A1, SLC1A2, KCNC3, and neurodevelopmental regulators including WNT7A, DLX6, ERBB4, ARX, BCL11B, SEMA3C, and FGFBP3 were identified as key markers regulating the neuron-like lineage transition. This study demonstrates that pHGGs can be phenotypically redirected toward neuronal-like identities through modulating cell fate differentiation programs. These findings advance the concept of ‘differentiation therapy’ as a promising intervention to reduce phenotypic plasticity and malignancy in pHGG ecosystems. While these are early in vitro findings, the potential ability to steer and control glioma cells toward stable, less malignant fates offers promising translational potential for patient-centered targeted therapies.

BACKGROUND:The decreasing costs of sequencing, along with the growing understanding of epigenetic mechanisms driving diseases, have led to the increased application of chromatin immunoprecipitation (ChIP), Cleavage Under Targets & Release Using Nuclease (CUT&RUN) and Cleavage Under Targets and Tagmentation (CUT&TAG) sequencing-which are designed to map DNA or chromatin-binding proteins to their genome targets-in biomedical research. Existing software tools, namely peak-callers, are available for analyzing data from these technologies, although they often struggle with diffuse and broad signals, such as those associated with broad histone post-translational modifications (PTMs). RESULTS:To address this limitation, we present ChIPbinner, an open-source R package tailored for reference-agnostic analysis of broad PTMs. Instead of relying on pre-identified enriched regions from peak-callers, ChIPbinner divides (bins) the genome into uniform windows. Thus, users are provided with an unbiased method to explore genome-wide differences between two samples using scatterplots, principal component analysis (PCA), and correlation plots. It also facilitates the identification and characterization of differential clusters of bins, allowing users to focus on specific genomic regions significantly affected by treatments or mutations. We demonstrated the effectiveness of this tool through simulated datasets and a case study assessing H3K36me2 depletion following NSD1 knockout in head and neck squamous cell carcinoma, highlighting the advantages of ChIPbinner in detecting broad histone mark changes over existing software. CONCLUSIONS:Binned analysis provides a more holistic view of the genomic landscape, allowing researchers to uncover broader patterns and correlations that may be missed when solely focusing on individual peaks. ChIPbinner offers researchers a convenient tool to perform binned analysis. It improves on previously published software by providing a clustering approach that is independent of each bin's differential enrichment status and more precisely identifies differentially bound regions for broad histone marks, while also offering additional features for downstream analysis of these differentially enriched bins.

Research question: How can the effectiveness of exome sequencing be improved for diagnosing infertility, and what are the key challenges and lessons learned from analysing nine unrelated cases? Design: Nine unrelated infertility cases referred between 2019 and 2024 were analysed in this study. Exome sequencing was conducted on probands and other family members when needed. Sanger sequencing was used for segregation analysis, and consequences on splicing were investigated on mRNA from patient cells and minigene assay. Results: Nine causative variants were identified, including six novel, in nine genes, TUBB8, PATL2, CCDC39, STAG3, KIAA0319, FBXO43, AGBL5/BBS7, PLCZ1 and HS6ST1, across diverse reproductive phenotypes, including oocyte maturation arrest, early embryonic arrest, spermatogenic failure and syndromic infertility. KIAA0319 was also identified as a novel candidate gene for male infertility. Conclusions: Variant segregation in family members and the possibility of re-contacting the patients for further evaluations and questions were crucial to maximize diagnostic yield and reach robust conclusions. This study underscores the need for multidisciplinary collaboration between reproductive medicine specialists and geneticists to facilitate the complexity of infertility, improve its diagnostic yield and counsel patients.

H3K36 methylation is a key epigenetic mark with critical roles in development and disease. Here, we systematically dissect its functions using CRISPR-engineered mouse mesenchymal stem cells lacking combinations of the five H3K36 methyltransferases, culminating in quintuple knockout cells devoid of H3K36me2/3. We show that H3K36me2 influences enhancer activity, supports the expression of their target genes, and safeguards active genes from encroachment of the repressive marks, H3K27me2/3. In addition, we find that the loss of H3K36me triggers redistribution of large heterochromatic H3K9me3 domains into euchromatin, in part mediated by SUV39H1, leading to global epigenomic remodelling, constitutive heterochromatin erosion, and a collapse of 3D genome organization. Parallel analyses in human HNSCC cells overexpressing the H3K36M oncohistone reveal conserved disruptions to the epigenome and chromatin architecture. Together, these results establish H3K36 methylation as a pivotal regulator of chromatin state and genomic structure.

Zellweger spectrum disorder (ZSD) results from biallelic variants in any one of 13 PEX genes involved in peroxisome biogenesis and function. The majority of ZSD cases result from pathogenic variants in PEX1. Here, we present 3 patients with suspected PEX1-related ZSD and non-diagnostic whole exome sequencing and describe the use of multiple modalities to ascertain their diagnosis. We confirmed peroxisomal dysfunction in the patients by demonstrating abnormal peroxisome metabolite levels in blood and peroxisome import dysfunction in patient fibroblasts. RNA studies including RNA-seq and RT-PCR, followed by Sanger sequencing showed leaky splice variants including an intron 13 variant causing exon 14 skipping (Patient 1), an intron 22 variant causing intron 22 retention (Patient 2), and a synonymous splice-site variant causing exon 16 skipping (Patient 3). All three patients had very low amounts of canonical PEX1 transcripts on RNA-seq, as well as residual but reduced PEX1 protein levels on immunoblotting, which likely explains their non-severe ZSD phenotype. This study suggests that a multi-modality approach combining biochemical testing, functional assays in fibroblasts and molecular investigations including sequencing of non-coding regions and RNA analysis may aid in diagnosis of patients with suspected PBD-ZSD and inconclusive WES.

Background:Methylation of histone 3 lysine 36 (H3K36me) has emerged as an essential epigenetic component for the faithful regulation of gene expression. Despite its importance in development, disease, and cancer, how the molecular agents collectively shape the H3K36me landscape is unclear.Results:We use a mouse mesenchymal stem cell model to perturb the H3K36me deposition machinery and infer the activities of the five most prominent players: SETD2, NSD1, NSD2, NSD3, and ASH1L. We find that H3K36me2 is the most abundant of the three methylation states and is predominantly deposited at intergenic regions by NSD1, and partly by NSD2. In contrast, H3K36me1/3 are most abundant within exons and are positively correlated with gene expression. We demonstrate that while SETD2 deposits most H3K36me3, it also deposits H3K36me2 within transcribed genes. Additionally, loss of SETD2 results in an increase of exonic H3K36me1, suggesting other H3K36 methyltransferases (K36MTs) prime gene bodies with lower methylation states ahead of transcription. Through a reductive approach, we uncover the distribution patterns of NSD3- and ASH1L-catalyzed H3K36me2. While NSD1/2 establish broad intergenic H3K36me2 domains, NSD3 deposits H3K36me2 peaks on active promoters and enhancers. Meanwhile, the activity of ASH1L is restricted to the regulatory elements of developmentally relevant genes, and our analyses implicate PBX2 as a potential recruitment factor.Conclusions:Within genes, SETD2 deposits both H3K36me2/3, while the other K36MTs are capable of depositing H3K36me1/2 independently of SETD2 activity. For the deposition of H3K36me1/2, we find a hierarchy of K36MT activities where NSD1>NSD2>NSD3>ASH1L. While NSD1 and NSD2 are responsible for most genome-wide propagation of H3K36me2, the activities of NSD3 and ASH1L are confined to active regulatory elements.

Over the past two decades, scientists have increasingly realized the importance of the three-dimensional (3D) genome organization in regulating cellular activity. Hi-C and related experiments yield 2D contact matrices that can be used to infer 3D models of chromosome structure. Visualizing and analyzing genomes in 3D space remains challenging. Here, we present ARGV, an augmented reality 3D Genome Viewer. ARGV contains more than 350 pre-computed and annotated genome structures inferred from Hi-C and imaging data. It offers interactive and collaborative visualization of genomes in 3D space, using standard mobile phones or tablets. A user study comparing ARGV to existing tools demonstrates its benefits.

Posterior fossa group A (PFA) ependymoma is a lethal brain cancer diagnosed in infants and young children. The lack of driver events in the PFA linear genome led us to search its 3D genome for characteristic features. Here, we reconstructed 3D genomes from diverse childhood tumor types and uncovered a global topology in PFA that is highly reminiscent of stem and progenitor cells in a variety of human tissues. A remarkable feature exclusively present in PFA are type B ultra long-range interactions in PFAs (TULIPs), regions separated by great distances along the linear genome that interact with each other in the 3D nuclear space with surprising strength. TULIPs occur in all PFA samples and recur at predictable genomic coordinates, and their formation is induced by expression of EZHIP. The universality of TULIPs across PFA samples suggests a conservation of molecular principles that could be exploited therapeutically.

Purpose To investigate the genetic etiology of patients with female infertility. Methods Whole Exome Sequencing was performed on genomic DNA extracted from the patient’s blood. Exome data were filtered for damaging rare biallelic variants in genes with possible roles in reproduction. Sanger sequencing was used to validate the selected variants and segregate them in family members. Results A novel homozygous likely pathogenic variant, c.626G>A, p.Trp209*, was identified in the TERB1 gene of the patient. Additionally, we report a second homozygous pathogenic TERB1 variant, c.1703C>G, p.Ser568*, in an infertile woman whose azoospermic brother was previously described to be homozygous for her variant. Conclusions Here, we report for the first time two homozygous likely pathogenic and pathogenic TERB1 variants, c.626G>A, p.Trp209* and c.1703C>G, p.Ser568*, respectively, in two unrelated women with primary infertility. TERB1 is known to play an essential role in homologous chromosome movement, synapsis, and recombination during the meiotic prophase I and has an established role in male infertility in humans. Our data add TERB1 to the shortlist of Meiosis I genes associated with human infertility in both sexes.

To identify novel genes responsible for recurrent hydatidiform moles (HMs), we performed exome sequencing on 75 unrelated patients who were negative for mutations in the known genes. We identified biallelic deleterious variants in 6 genes, FOXL2, MAJIN, KASH5, SYCP2, MEIOB, and HFM1, in patients with androgenetic HMs, including a familial case of 3 affected members. Five of these genes are essential for meiosis I, and their deficiencies lead to premature ovarian insufficiency. Advanced maternal age is the strongest risk factor for sporadic androgenetic HM, which affects 1 in every 600 pregnancies. We studied Hfm1-/- female mice and found that these mice lost all their oocytes before puberty but retained some at younger ages. Oocytes from Hfm1-/- mice initiated meiotic maturation and extruded the first polar bodies in culture; however, their meiotic spindles were often positioned parallel, instead of perpendicular, to the ooplasmic membrane at telophase I, and some oocytes extruded the entire spindle with all the chromosomes into the polar bodies at metaphase II, a mechanism we previously reported in Mei1 -/- oocytes. The occurrence of a common mechanism in two mouse models argues in favor of its plausibility at the origin of androgenetic HM formation in humans.

Background Primary ciliopathies are a heterogeneous group of rare disorders predominantly caused by autosomal-recessive genetic variants that disrupt non-motile ciliary function. They often manifest as a syndromic phenotype, frequently involving the kidney. Biallelic pathogenic variants in C2CD3 disrupt ciliogenesis and Sonic Hedgehog (SHH) signaling, resulting in a severe ciliopathy (Orofaciodigital syndrome XIV, OMIM 615948). We present compound heterozygous missense variants in C2CD3 that partially disrupt ciliary function in a patient with isolated renal disease. Methods Exome sequencing identified biallelic C2CD3 missense variants (p.Pro168Leu; p.Thr2079Met). Patient-derived fibroblasts and urinary renal epithelial cells (URECs), and human RPE-1 C2CD3 knockout (KO) cell-lines were used for in vitro studies. Results Cilia length was significantly shorter in patient-derived fibroblasts compared to an unaffected sibling (2.309 vs. 2.850 mu m, P < 0.0001), while URECs showed significantly shortened cilia (2.068 vs. 2.807 mu m, P < 0.0001) and a 40.8% reduction in ciliation (P < 0.001). The latter was not observed in fibroblasts, suggesting a kidney-specific effect. SHH signaling was dysregulated in patient cells as expression of GLI3 activator protein and GLI1 mRNA was significantly reduced. C2CD3 localization to the basal body was significantly reduced in patient URECs. Finally, rescue experiments in C2CD3 KO RPE-1 cells corroborated these findings by demonstrating a reduced capacity to restore ciliogenesis for each variant. Conclusion Biallelic hypomorphic missense variants in C2CD3 may contribute to an isolated nephronophthisis phenotype with impaired ciliogenesis and SHH signaling. Our findings underscore the importance of functional testing to characterize candidate gene-disease relationships in patients with nephropathy of unknown etiology.