Epigenetics & Chromatin

Human cell line models, including the neuronal precursor line LUHMES, are important for investigating developmental transcriptional dynamics within imprinted regions, particularly the 15q11-q13 Angelman (AS) and Prader-Willi (PWS) syndrome locus. AS results from loss of maternal UBE3A in neurons, where the paternal allele is silenced by a convergent antisense transcript UBE3A-ATS, a lncRNA that normally terminates at PWAR1 in non-neurons. qRT-PCR analysis confirmed the exclusive and progressive increase in UBE3A-ATS in differentiating LUHMES neurons, validating their use for studying UBE3A silencing. Genome-wide transcriptome analyses revealed changes to 11,834 genes during neuronal differentiation, including the upregulation of most genes within the 15q11-q13 locus. To identify dynamic changes in chromatin loops linked to transcriptional activity, we performed a HiChIP validated by 4C, which identified two neuron-specific CTCF loops between MAGEL2-SNRPN and PWAR1-UBE3A. To determine if allele-specific differentially methylated regions (DMR) may be associated with CTCF loop anchors, whole genome long-read nanopore sequencing was performed. We identified a paternally hypomethylated DMR near the SNRPN upstream loop anchor exclusive to neurons and a paternally hypermethylated DMR near the PWAR1 CTCF anchor exclusive to undifferentiated cells, consistent with increases in neuronal transcription. Additionally, DMRs near CTCF loop anchors were observed in both cell types, indicative of allele-specific differences in chromatin loops regulating imprinted transcription. These results provide an integrated view of the 15q11-q13 epigenetic landscape during LUHMES neuronal differentiation, underscoring the complex interplay of transcription, chromatin looping, and DNA methylation. They also provide insights for future therapeutic approaches for AS and PWS.

Chromatin-based mechanisms regulating developmental transitions during meiosis are fundamental but understudied aspects of male gametogenesis. Indeed, chromatin undergoes extensive remodeling during meiosis, leading to specific patterns of gene expression and chromosome organization, which ultimately controls fundamental meiotic processes such as recombination and homologous chromo-some associations. Recent game-changing advances have been made by analysis of chromatin binding sites of meiotic specific proteins genome-wide in mouse spermatocytes. However, further progress is still highly dependent on the reliable isolation of sufficient quantities of spermatocytes at specific stages of prophase I. Here, we describe a combination of methodologies adapted for rapid and reliable isolation of synchronized fixed mouse spermatocytes. We show that chromatin isolated from these cells can be used to study chromatin binding sites by ChIP-seq. High quality data we obtained from INO80 ChIP-seq in zygotene cells was used for functional analysis of chromatin binding sites.

Long non-coding RNAs (lncRNAs) represent a diversity of transcripts that can regulate gene expression and chromatin remodelling. DNAAF3-AS1 is an lncRNA with a strong genome-wide correlation between DNAAF3-AS1 expression and the histone mark H3K36me3, according to the HiMoRNA database. To validate this association, we performed DNAAF3-AS1 knockdown in human dermal fibroblasts using antisense oligonucleotides following H3K36me3 ChIP-seq. Our results demonstrate that DNAAF3-AS1 depletion leads to a significant redistribution of H3K36me3, with increased signal in intergenic regions and the first exon, and reduced enrichment across gene bodies. Additionally, differential expression analysis revealed that DNAAF3-AS1 knockdown induces promoter switching, with downregulation of gene-body promoters downstream of TSS. These findings establish DNAAF3-AS1 as a potential regulator of H3K36me3 deposition and transcriptional architecture, providing mechanistic insight into lncRNA-mediated epigenetic control.

Remodelling of chromatin architecture is known to regulate gene expression and has been well characterized in cell lineage development but less so in response to cell perturbation. Activation of T cells, which triggers extensive changes in transcriptional programs, serves as an instructive model to elucidate how changes in genome organization orchestrate gene expression in response to cell perturbation. To characterize coordinate changes at different levels of chromatin architecture, we analysed chromatin accessibility, chromosome conformation and gene expression after activation of human T cells. T cell activation led to widespread changes in chromatin interactions and accessibility that were mostly shared between CD4+ and CD8+ T cells. Differential chromatin interactions were associated with upregulation or downregulation of linked target genes. Moreover, activation was associated with the formation of shorter chromatin interactions, partitioning of topologically associating domains (TADs) and acquisition of new TAD boundaries characterized by higher nucleosome occupancy, and lower chromatin accessibility and gene expression. These findings render an integrated and multiscale characterization of activation-induced re-organization of chromatin architecture underlying gene transcription in human T cells.

Canonical Wnt signaling is crucial for intestinal homeostasis as the major Wnt signaling effector in the intestines, TCF4, is required for stem cell maintenance. The capability of TCF4 to maintain the stem cell phenotype is contingent upon {beta}-catenin, a potent transcriptional activator which interacts with histone acetyltransferases and chromatin remodeling complexes. In colorectal cancer, mutations result in high levels of nuclear {beta}-catenin causing aberrant cell growth. Here, we used RNAi to explore the influence of TCF4 on chromatin structure (Hi-C) and gene expression (RNA sequencing) across a 72-hour time series in colorectal cancer. We found that TCF4 reduction results in a disproportionate upregulation of gene expression genome-wide, including a powerful induction of SOX2. Hi-C analysis revealed a general increase in chromatin compaction across the entire time series, though this did not influence gene expression. Analysis of local chromosome organization demonstrated a TAD boundary loss which influenced the expression of a cluster of CEACAM genes on chromosome 19. Four-dimensional nucleome (4DN) analysis, which integrates structural (Hi-C) and functional (RNA sequencing) data, identified EMT and E2F as the two most deregulated pathways and LUM, TMPO, and AURKA as highly influential genes in these networks. Results from gene expression, chromatin structure, and centrality analyses were then integrated to generate a list of candidate transcription factors for reprogramming of colorectal cancer cells to a vulnerable state. The top ranked transcription factor in our analysis was c-JUN, an oncoprotein known to interact with TCF4 and {beta}-catenin.

Sex differences shape human physiology and disease risk, yet their autosomal epigenetic basis remains incompletely understood. Using whole genome bisulfite sequencing data from iMETHYL database ([~]100 adults) across three purified blood cell types (CD4 T cells, monocytes, neutrophils), we performed comprehensive mapping of cell-type specific sex-associated DNA methylation. Using genome-wide Z-test and confidence-interval method, we identified thousands of autosomal differentially methylated sites (DMSs), the majority of which were cell type-specific. We found that neutrophils exhibited pronounced female-biased hypermethylation, whereas CD4 T cells and monocytes showed more balanced patterns. DMSs were enriched within gene bodies and annotated to neuronal and adhesion functions, with T-cell activation uniquely associated in CD4 T cells. Transcription factor binding site enrichment indicated hematopoietic regulators, suggesting that sex-associated methylation is tightly linked to early developmental processes within blood lineages. Differentially methylated regions overlapped genome-wide association study loci for lifetime smoking, multiple sclerosis, and psoriasis, indicating convergence between sex-associated epigenetic states and genetic susceptibility. Evolutionary analysis revealed limited conservation but identified a conserved intronic CpG within FIGN. This study provides the first genome-wide, cell type-resolved map of autosomal sex-associated DNA methylation in human blood and establishes a foundation for mechanistic and translational studies in sex-informed biology and medicine. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/682299v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@18524c0org.highwire.dtl.DTLVardef@2fd8cdorg.highwire.dtl.DTLVardef@142b445org.highwire.dtl.DTLVardef@4949be_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundDNA methylation is an important factor in the regulation of gene expression and genome stability. High DNA methylation levels are associated with transcriptional repression. In mammalian systems, unmethylated, low methylated and fully methylated regions (UMRs, LMRs, and FMRs, respectively) can be distinguished. UMRs are associated with proximal regulatory regions, while LMRs are associated with distal regulatory regions. Although DNA methylation is mainly limited to the CG context in mammals, while it occurs in CG, CHG and CHH contexts in plants, UMRs and LMRs were expected to occupy similar genomic sequences in both mammals and plants. ResultsThis study investigated major model and crop plants such as Arabidopsis thaliana, tomato (Solanum lycopersicum), rice (Oryza sativa) and maize (Zea mays), and shows that plant genomes can also be subdivided in UMRs, LMRs and FMRs, but that LMRs are mainly present in the CHG context rather than the CG context. Strikingly, the identified CHG LMRs were enriched in transposable elements rather than regulatory regions. Maize candidate regulatory regions overlapped with UMRs. LMRs were enriched for heterochromatic histone modifications and depleted for DNase accessibility and H3K9 acetylation. CHG LMRs form a distinct, abundant cluster of loci, indicating they have a different role than FMRs. ConclusionsBoth mammalian and plant genomes can be segmented in three distinct classes of loci, UMRs, LMRs and FMRs, indicating similar underlying mechanisms. Unlike in mammals, distal regulatory sequences in plants appear to overlap with UMRs instead of LMRs. Our data indicate that LMRs in plants have a different function than those in mammals.

DNA methylation is a regulator of bacterial gene expression and adaptation, influencing traits such as virulence and antimicrobial resistance. The dynamic nature of DNA methylation enables rapid responses to changing environments and is a source of heterogeneity in bacterial populations. However, condition-dependent DNA methylation and consequences for transcriptional output remain poorly understood. We applied Oxford Nanopore sequencing to profile DNA methylation during exponential growth and late stationary phase of Salmonella enterica serovar Typhimurium and integrated these data with transcriptomic analyses. We found that each DNA methyltransferase (MTases) exhibits a distinct activity pattern across growth stages, which could not be explained by transcriptional levels of the corresponding enzymes. As predicted, DNA methylation patterns determined by regulatory MTases were dynamic across growth conditions whereas methylation patterns of MTases belonging to R-M systems were comparatively stable. We identified growth stage-specific methylation patterns for all studied MTases and correlations between methylation states and gene expression patterns. Together, these findings chart DNA methylation networks in the epigenetic regulation of bacterial physiology. Author summaryDNA methylation in bacteria is best known for its role protecting DNA from endonucleases, such as restriction-modification, and coordinating chromosome replication and mutation repair, yet DNA methylation also regulates gene expression and cell physiology. Previous studies primarily examined bacterial DNA methylation at single time points or in limited genomic regions, providing only a partial view of its biological significance. In this study, we used Oxford Nanopore sequencing to compare DNA methylation patterns in Salmonella enterica during exponential growth and late stationary phase then integrated these data with corresponding gene expression profiles. We identified numerous methylation target motifs, all of which demonstrated constitutively methylated or unmethylated regions. This systems-level analysis clarifies the role of DNA methylation in bacterial adaptation across growth stages and demonstrates the utility of Oxford Nanopore sequencing for genome-wide methylation profiling.

Three-dimensional organization of the eukaryotic genome is directly affected by the nuclear {beta}-actin pool that regulates enhancer function by affecting H3K27 acetylation levels. This actin-based mechanism, in turn, influences enhancer-dependent transcriptional regulation and plays a crucial role in driving gene expression changes observed upon compartment-switching. Using a combination of bulk RNA-seq and qPCR analyses performed on total RNA from WT mouse embryonic fibroblasts (MEFs), {beta}-actin heterozygous (HET) MEFs, and {beta}-actin KO MEFs, in this study we demonstrate that expression of several lncRNAs is directly affected by {beta}-actin depletion. Among these lncRNAs, Meg3 expression increases in a {beta}-actin dosage-dependent manner. Using ChIRP-seq, ChIRP-MS and f-RIP-qPCR, we show that {beta}-actin depletion leads to alterations in Meg3 genomic association. It also leads to Meg3 enrichment at or close to gene regulatory sites including enhancers and promoters concomitantly with increased H3K27 acetylation levels. At these sites, specific Meg3 association with H3K27 acetylation leads to loss of promoter-enhancer interactions as revealed by the Activity by Contact (ABC) model that builds on RNA-seq, H3K27acetylation ChIP-seq, ATAC-seq and HiC-seq obtained in WT and {beta}-actin KO MEFs. Results from metabolomics experiments in WT, HET and {beta}-actin KO MEFs show these mechanisms contribute to the repression of genes involved in metabolic biosynthetic pathways for chondroitin, heparan, dermatan sulfate, and phospholipases, hence impacting their synthesis. We propose that at sites of actin-dependent increase in H3K27acetylation levels Meg3 interferes with promoter-enhancer interactions, potentially impairing local genome organization (or DNA looping) and negatively regulating gene expression.

Gene expression requires the targeting of transcription factors (TFs) to regulatory sequences often occluded within nucleosomes. To comprehensively examine TF nucleosome binding, we developed Pioneer-Seq. In Pioneer-seq a library of nucleosomes containing thousands of DNA sequences with TF binding sites in all possible nucleosome orientations is bound to a TF and the protein-nucleosome complex is isolated and quantified. To demonstrate Pioneer-seq we examined nucleosome binding by OCT4, SOX2, KLF4, and c-MYC. Our results demonstrate that KLF4 and SOX2 can bind close to the nucleosome dyad with nucleosome sequence being the major factor regulating TF binding across all studied TFs.

Chromatin structure and organization has a key role in gene expression regulation. Here, we integrated ChIP-seq, RNA-seq, Hi-C, epigenetic, and cancer-related mutations data to get insight into the role of Death Inducer Obliterator gene (Dido1) in RNA pol II (RNAPII) transcription and chromatin structure regulation. Analysis of ChIP-seq data of DIDO3, the largest protein isoform of Dido1, revealed binding-sites overlap about 70% with RNAPII and H3K36me3 in the mouse genome, but also significant overlap 10-30% with Polycomb, CTCF, H3K4me3, and H3K27ac. Based on this analysis we propose that DIDO3s PHD domain interacts with H3K36me3 posttranslational modification. Integrating multi-omics data we describe how DIDO3 potentially recruit several transcription factors, including RNAPII, and also regulates genes transcribing those same transcription factors. DIDO3 regulation of the genes traduced into proteins to which it binds puts DIDO3 in the center of intricate feedback loops. We showed, by using data from a DIDO3 mutant, that DIDO3 C-terminus is responsible for most of these transcriptional regulation, and is also implicated in other very important pathways by regulating genes encoding for Polycomb-accessory proteins, subunits of the SWI/SNF chromatin remodelling, or Set1/COMPASS chromatin modifier complexes. These multi-protein complexes control gene activation or silencing and also play a role in tumour development. DIDO3 C-terminus region and splice-site for alternative DIDO2/DIDO3 protein isoforms tended to accumulate recurrent truncating mutations identified in the TCGA Pan-Cancer dataset. We hypothesize that deregulation of DIDO3, as it happens with large epigenetic complexes and long-range interactions, leads to cell differentiation deficiency and cancer development. Overall, we propose here a molecular mechanism by which DIDO3, favour RNAPII pausing and long-range chromatin interactions.

BackgroundReduced copy number of the D4Z4 macrosatellite at human chromosome 4q35 is associated with facioscapulohumeral muscular dystrophy (FSHD). A pervasive idea is that chromatin alterations at the 4q35 locus following D4Z4 repeat unit deletion lead to disease via inappropriate expression of nearby genes. Here, we sought to analyze transcription and chromatin characteristics across 4q35 and how these are affected by D4Z4 deletions and exogenous stresses. ResultsWe found that the 4q subtelomere is subdivided into discrete domains, each with characteristic chromatin features associated with distinct gene expression profiles. Centromere-proximal genes within 4q35 (ANT1, FAT1 and FRG1) display active histone marks at their promoters. In contrast, poised or repressed markings are present at telomere-proximal loci including FRG2, DBE-T and D4Z4. We discovered that these discrete domains undergo region-specific chromatin changes upon treatment with chromatin enzyme inhibitors or genotoxic drugs. We demonstrated that the 4q35 telomere-proximal FRG2, DBE-T and D4Z4-derived transcripts are induced upon DNA damage to levels inversely correlated with the D4Z4 repeat number, are stabilized through post-transcriptional mechanisms upon DNA damage, and are bound to chromatin. ConclusionOur study reveals unforeseen biochemical features of RNAs from clustered transcription units within the 4q35 subtelomere. Specifically, the FRG2, DBE-T and D4Z4-derived transcripts are chromatin-associated and are stabilized post-transcriptionally after induction by genotoxic stress. Remarkably, the extent of this response is modulated by the copy number of the D4Z4 repeats, raising new hypotheses about their regulation and function in human biology and disease.

The establishment of precise transcriptional programs during neuronal development depends on a complex and dynamic landscape of regulatory elements. Here, we combined histone modification ChIP-seq, chromatin accessibility profiling, and transcriptomics to map active enhancer repertoires during the differentiation of human embryonic stem cells into midbrain dopaminergic neurons. Our integrative analysis revealed thousands of stage-specific enhancers, over half of which were previously unannotated, and uncovered coordinated chromatin and transcriptional transitions linking pluripotency exit to neuronal lineage commitment. Functional enrichment of enhancer-linked target genes delineated distinct regulatory programs, pluripotency maintenance in undifferentiated cells and midbrain specification in differentiated neuron, while transcription factor motif analysis identified regulatory modules, including a dopaminergic-specific RFX4 network. We further characterized enhancer-gene relationships showing concordant changes in chromatin state and expression for neuronal genes, suggesting enhancer-driven control of dopaminergic identity. These findings provide a genome-scale framework for interpreting how dynamic enhancer landscapes encode developmental fate decisions in the human nervous system.

BackgroundRibosomal DNA (rDNA) transcription by the RNA polymerase I (Pol I) is a rate-limited step for ribosome synthesis, which is critical for cell growth, cell differentiation, and tumorigenesis. Meanwhile rDNA transcription is modulated by DNA methylation and histone epigenetic modification. Though with great progress in epigenetic research recently, it still remains much uncertain about the relationship of histone variant epigenetic modification and rDNA transcription.\n\nResultsIn this study, epigenetic profiles of silent rDNA in next-generation sequencing datasets were examined. We found that the chaperone of histone variant H3.3, the alpha-thalassemia/mental retardation X-linked syndrome protein (ATRX)/death domain-associated protein (DAXX) complex, and methyltransferase SET domain bifurcated 1 (Setdb1, also known as ESET) help maintain H3.3K9me3 modifications among the promoter and coding regions of silent rDNA. Our experiments further confirmed that DAXX depletion leads to the conversion of silent rDNA into upstream binding factor-bound active rDNA and the release of rDNA transcriptional potency. Support for this model is provided by data from a low-grade glioma in which ATRX is lost and a higher level of ribosomal biosynthesis, nucleolus activity, and proliferation are observed.\n\nConclusionsWe demonstrate a model of epigenetic regulation for rDNA with roles for the ATRX/DAXX complex and H3.3/H3K9me3 modifications identified. Thus, loss of ATRX/DAXX may represent a driving force for tumorigenesis due to its contribution to the release of rDNA transcriptional potency.

The prevalence of many diseases differs by sex, potentially due to sex-specific patterns in DNA methylation. Autosomal sex-specific differences in DNA methylation have been observed in cord blood and placental tissue, but are not well studied in saliva or in diverse populations. We sought to characterize sex-specific DNA methylation on autosomal chromosomes in saliva samples from children in the Fragile Families and Child Wellbeing Study, a multi-ethnic prospective birth cohort containing an oversampling of Black, Hispanic and low-income families. DNA methylation from saliva samples were analyzed on 796 children at both ages 9 and 15 with DNA methylation measured using the Illumina HumanMethylation 450k array. An epigenome-wide association analysis of the age 9 samples identified 8,430 sex-differentiated autosomal DNA methylation sites at age 9 (P < 2.4x10-7), of which 76.2% had higher DNA methylation in female children. The strongest sex-difference was in the cg26921482 probe, in the AMDHD2 gene, with 30.6% higher DNA methylation in female compared to male children (P < 1x10-300). Treating the age 15 samples as an internal replication set, we observed highly consistent results between the age 9 and age 15 measurements, indicating stable and replicable sex-differentiation. Further, we directly compared our results to previously published DNA methylation sex differences in both cord blood and saliva and again found strong consistency. Our findings support widespread and robust sex-differential DNA methylation across age, human tissues, and populations. These findings help inform our understanding of potential biological processes contributing to sex differences in human physiology and disease.

Over the last few decades, novel methods have been developed to study how chromosome positioning within the nucleus may play a role in gene regulation. Adaptation of these methods in the human malaria parasite, Plasmodium falciparum, has recently led to the discovery that the three-dimensional structure of chromatin within the nucleus may be critical in controlling expression of virulence genes (var genes). Recent work has implicated an unusual, highly conserved var gene called var2csa in contributing to coordinated transcriptional switching, however how this gene functions in this capacity is unknown. To further understand how var2csa influences var gene switching, targeted DNA double-strand breaks (DSBs) within the sub-telomeric region of chromosome 12 were used to delete the gene and the surrounding chromosomal region. To characterize the changes in chromatin architecture stemming from this deletion and how these changes could affect var gene expression, we used a combination of RNA-seq, Chip-seq and Hi-C to pinpoint epigenetic and chromatin structural modifications in regions of differential gene expression. We observed a net gain of interactions in sub-telomeric regions and internal var gene regions following var2csa knockout, indicating an increase of tightly controlled heterochromatin structures. Our results suggest that disruption of var2csa results not only in changes in var gene transcriptional regulation but also a significant tightening of heterochromatin clusters thereby disrupting coordinated activation of var genes throughout the genome. Altogether our result confirms a strong link between the var2csa locus, chromatin structure and var gene expression. AUTHOR SUMMARYMalaria remains one of the deadliest parasite-borne diseases, causing not only over a half million deaths annually, but also infecting hundreds of millions more. Plasmodium falciparum, the protozoan parasite that is responsible for the most virulent form of human malaria, is transmitted to humans by infected female mosquitoes during a blood meal. Due to a growing resistance to all existing antimalarials, there is a need to identify novel targets to design new antimalarial strategies. Our research builds on the growing body of evidence that supports the role of genome organization or chromatin structure within the nucleus in controlling the parasite development as well as virulence factors designed to circumvent the host immune response. This study identifies genes and structural elements within the Plasmodium falciparum genome that are controlled, at least partially, by the expression of a single unique and highly conserved virulence gene.

Alternative exon usage is known to affect a large portion of genes in mammalian genomes. Importantly, different splice forms sometimes lead to distinctly different protein functions. We analyzed data from the Human Epigenome Atlas (version 9) whereby we connected the differential usage of exons in various developmental stages of human cells/tissues to differential epigenetic modifications at the exon level. In total, we analyzed 19 human tissues, adult cells, and cultured cells that mimic early developmental stages. We found that the differential occurrence of protein isoforms across developmental stages was often associated with changes in histone marks at exon boundary regions. Many of the genes that are differentially regulated at the exon level were found to be functionally associated with development and metabolism.

Core histone proteins H2A, H2B, H3, and H4 are encoded by a large family of genes distributed across the human genome. Canonical core histones contribute the majority of proteins to bulk chromatin packaging, and are encoded in 4 clusters by 65 coding genes comprising 17 for H2A, 18 for H2B, 15 for H3, and 15 for H4, along with at least 17 total pseudogenes. The canonical core histone genes display coding variation that gives rise to 11 H2A, 15 H2B, 4 H3, and 2 H4 unique protein isoforms. Although histone proteins are highly conserved overall, these isoforms represent a surprising and seldom recognised variation with amino acid identity as low as 77% between canonical histone proteins of the same type. The gene sequence and protein isoform diversity also exceeds commonly used subtype designations such as H2A.1 and H3.1, and exists in parallel with the well-known specialisation of variant histone proteins. RNA sequencing of histone transcripts shows evidence for differential expression of histone genes but the functional significance of this variation has not yet been investigated. To assist understanding of the implications of histone gene and protein diversity we have catalogued the entire human canonical core histone gene and protein complement. In order to organise this information in a robust, accessible, and accurate form, we applied software build automation tools to dynamically generate the canonical core histone repertoire based on current genome annotations and then to organise the information into a manuscript format. Automatically generated values are shown with a light grey background. Alongside recognition of the encoded protein diversity, this has led to multiple corrections to human histone annotations, reflecting the flux of the human genome as it is updated and enriched in reference databases. This dynamic manuscript approach is inspired by the aims of reproducible research and can be readily adapted to other gene families.

Chromatin architecture governs DNA accessibility and gene expression. Thus, any perturbations to chromatin can significantly alter gene expression programs and promote disease. Prior studies demonstrate that every amino acid in a histone is functionally significant, and that even a single amino acid substitution can drive specific cancers. We previously observed that naturally occurring H2B variants are dysregulated during the epithelial to mesenchymal transition (EMT) in bronchial epithelial cells. Naturally occurring H2B variants differ from canonical H2B by only a few amino acids, yet single amino acid changes in other histone variants (e.g., H3.3) can drive cancer. We therefore hypothesized that H2B variants might function like oncohistones, and investigated how they modify chromatin architecture, dynamics, and function. We find that H2B variants are frequently dysregulated in many cancers, and correlate with patient prognosis. Despite high sequence similarity, mutations in each H2B variant tend to occur at specific "hotspots" in cancer. Some H2B variants cause tighter DNA wrapping around nucleosomes, leading to more compact chromatin structures and reduced transcription factor accessibility to nucleosomal DNA. They also altered genome-wide accessibility to oncogenic regulatory elements and genes, with concomitant changes in oncogenic gene expression programs. Although we did not observe changes in cell proliferation or migration in vitro, our Gene Ontology (GO) analyses of ATAC-seq peaks and RNA-seq data indicated significant changes in oncogenic pathways. These findings suggest that H2B variants may influence early-stage, cancer-associated regulatory mechanisms, potentially setting the stage for oncogenesis later on. Thus, H2B variant expression could serve as an early cancer biomarker, and H2B variants might be novel therapeutic targets.

Changes in the sperm epigenome induced by age and/or stressors often follow common unexplained patterns affecting genes responsible for embryonic development and neurodevelopment. The stochastic epigenetic variation (SEV) hypothesis proposes that in response to stressors naturally variable methylation regions (VMRs) associated with morphogenic genes increase in methylation variation to diversify phenotypes and improve chances of survival of the genetic lineage. Here, we test predictions from the SEV hypothesis using mouse and rat sperm DNA methylation and other -omics data. We demonstrate that the context of DNA regions determines the response of sperm methylome to various factors rather than the stressors and/or timing of these factors. We propose a model explaining age/stress-dependent shifts in methylation in VMRs by an asymmetric increase in methylation variation of these regions. Because methylation variation in VMRs increases with age, sperm methylome response to stressors may be characterized as an acceleration of epigenetic aging.