Bivalent Chromatin Research Articles

Chromatin-based mechanisms contribute to the exquisite regulation of gene expression during animal development. But how those mechanisms evolved remains elusive. Here we investigate chromatin regulatory features in the closest relatives of animals, choanoflagellates. In a model choanoflagellate Salpingoeca rosetta, we compare chromatin accessibility and histone modifications to gene expression. Accessible genomic regions in S. rosetta primarily correspond to gene promoters, and we find no evidence of distal gene regulatory elements that resemble enhancers deployed to regulate developmental genes in animals. Remarkably, the histone modification H3K27me3 decorates genes with cell type-specific expression, revealing a functional similarity in S. rosetta and animals. Additionally, H3K27me3 marks LTR retrotransposons, retaining a potential ancestral role in regulating these elements. We further uncover a putative bivalent chromatin state at cell type-specific genes that consists of H3K27me3 and H3K4me1. Together, these data support the emergence of gene-associated histone modification states that underpin development before the evolution of animal multicellularity.

BackgroundAlthough liquid-liquid phase separation (LLPS) proteins are known to participate in genome organization and transcriptional regulation through the formation of biomolecular condensates, their functional interplay with other regulatory proteins and histone modifications in chromatin loop formation remains poorly characterized. By combining Hi-C chromatin interaction data with ChIP-seq profiles of 12, 27, and 24 LLPS proteins in GM12878, K562, and HepG2 cell lines, respectively, we identified chromatin loops associated with LLPS proteins and systematically analysed patterns of cooperative protein binding and histone modification enrichment within these loop-associated peaks.ResultsWe identified 162, 313, and 431 chromatin loops associated with LLPS proteins in GM12878, K562, and HepG2 cell lines, respectively. These loops were relatively small in size and predominantly anchored at enhancer regions. Examination of cooperative binding of proteins within loop-associated peaks revealed that transcriptional repressor IKZF1, HDAC1, and SAP130 most frequently co-localized with LLPS proteins in GM12878, K562, and HepG2 cells, respectively. Further analysis of histone modification enrichment patterns revealed that active histone modifications, such as H3K4me2, H3K4me3, H3K9ac, and H3K27ac, co-localized at loop-associated peaks, with H3K4me1 exhibiting additional specific co-localization with these four histone modifications at enhancer-localized loop-associated peaks. Notably, bivalent chromatin domains where H3K27me3 co-localized with active histone modifications were identified at promoter-localized loop-associated peaks in HepG2 cells, and elevated H3K27me3 occupancy at these peaks was associated with transcriptional repression of target genes. Moreover, quantitative RNA-seq analysis revealed that the expression of target genes associated with enhancer-promoter loops was correlated with both the binding of LLPS proteins and the enrichment patterns of histone modifications within their ChIP-seq peaks at loop anchors.ConclusionsOur study suggests that LLPS proteins may cooperate with transcriptional repressors to facilitate chromatin looping. Furthermore, local enrichment of histone modifications at loop-associated peaks provides additional regulatory control over chromatin architecture and gene transcription.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13072-025-00632-3.

Glucosinolates (GSLs) are secondary metabolites central to plant defence in the Brassicaceae family. While the role of histone modifications in developmental gene regulation is well studied, their function in stress-induced secondary metabolism remains unclear. Here, we show that GSL biosynthetic genes in Arabidopsis thaliana are regulated by bivalent chromatin bearing both active (histone acetylation) and repressive (H3K27me3) histone marks. Components of the Polycomb Repressive Complex 2 (PRC2), including CLF, SWN and LHP1, suppress GSL gene expression, and their loss enhances GSL accumulation. Genome-wide analyses revealed that indolic and aliphatic GSL genes are enriched with H3K27me3, with indolic genes also marked by active histone acetylation. Time-course transcriptome and metabolite analyses using HPLC following wounding revealed distinct temporal activation patterns, with indolic GSL genes induced during the early phases and aliphatic GSL genes activated at later stages. These findings suggest that bivalent histone modifications orchestrate temporal gene expression of GSL pathways under stress, revealing a previously unrecognised epigenetic mechanism underlying plant metabolic responses to environmental stimuli.

Bivalency regulates developmental genes during lineage commitment. However, mechanisms governing bivalent domain establishment, maintenance and resolution in early embryogenesis remain unclear. Here we comprehensively trace bivalent chromatin remodelling throughout mouse peri-implantation development, revealing bifurcated establishment modes that partition epiblast and primitive endoderm regulatory programmes. We identify transiently maintained bivalent domains (TB domains) enriched in the epiblast, where gradual resolution fine-tunes pluripotency progression. Through targeted screening in embryos, we uncover 22 TB domain regulators, including the essential factor ZBTB17. Genetic ablation or degradation of ZBTB17 causes peri-implantation arrest. Mechanistically, ZBTB17 collaborates with KDM6A/B to resolve bivalency by removing H3K27me3 and priming the activation of key pluripotency genes. Remarkably, TB domain dynamics are evolutionarily shared in human pluripotent transitions, with ZBTB17 involvement despite species differences. Our work establishes a framework for bivalent chromatin regulation in early mammalian development and elucidates how its resolution precisely controls lineage commitment.

The genome is compacted in the nucleus through a hierarchical chromatin organization, ranging from chromosome territories to compartments, topologically associating domains (TADs), and individual nucleosomes. Nucleosome remodeling complexes hydrolyze ATP to translocate DNA and thereby mobilize histone proteins. While nucleosome remodeling complexes have been extensively studied for their roles in regulating nucleosome positioning and accessibility, their contributions to higher-order chromatin architecture remain less well understood. Here, we investigate the roles of two key nucleosome remodelers, esBAF and INO80C, in shaping 3D genome organization in mouse embryonic stem cells. Using Hi-C, we find that loss of either remodeler has minimal effects on global compartment or TAD structures. In contrast, subcompartment organization is notably altered, suggesting that esBAF and INO80C contribute to finer-scale chromatin topology. To overcome the limited resolution of Hi-C for detecting regulatory loops, we employed promoter capture Micro-C (PCMC), which revealed that the loss of esBAF or INO80C alters a subset of promoter anchored looping interactions. Although these changes occur at distinct genomic loci for each remodeler, the affected sites are commonly enriched for bivalent chromatin regions bound by OCT4, SOX2, and NANOG (OSN), as well as BRG1 and INO80 themselves. Together, our findings reveal that esBAF and INO80C selectively influence subcompartment identity and enhancer–promoter communication at key regulatory loci, highlighting a previously underappreciated role for nucleosome remodelers in higher-order chromatin organization.

The dentate gyrus (DG), a crucial region of the hippocampus responsible for learning, spatial encoding, and memory formation, undergoes its main development and maturation after birth. Despite its importance, the regulatory mechanisms underlying postnatal DG development remain poorly understood. This study is aimed to investigate the role of H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 27 trimethylation (H3K27me3) in the development and function of the postnatal DG. We show robust enrichment of H3K4me3 in the subgranular zone (SGZ), a primary neurogenic region, while high levels of H3K27me3 were mainly presented in granule cell layer. Enhanced H3K4me3 level facilitated proliferation and development of neonatal mouse neural stem cells (NSCs), promoted differentiation towards GABA neurons, as well as improved mouse spatial learning and memory. Enhancing H3K27me3 level exerts the opposite function, additionally promoting NSCs entry into a quiescent-like state. During the neuronal differentiation of NSCs, the integration of RNA-Seq and ChIP-Seq datasets reveals that H3K4me3 and H3K27me3 co-regulate the expression of genes essential for neural development, such as Gli1, through the formation of bivalent domains. Manipulation activation of the Shh/Gli1 pathway abolishes the effect of alterations in the levels of H3K4me3 and H3K27me3 in NSCs. Based on these findings, we propose that H3K4me3 and H3K27me3 serve as molecular "switches" to dynamically regulate NSCs proliferation and differentiation and in turn, influence the postnatal developmental progression of DG, additionally to provide potential therapeutic targets for treating diseases associated with abnormal hippocampal development. During dentate gyrus development in neonatal mice, the active transcription mark H3K4me3 and the repressive mark H3K27me3 are co-localized at the promoter regions of essential neurodevelopmental genes, and thus forming bivalent chromatin domains in neural stem cells. These domains serve as a "molecular switch" that regulates the dynamic processes of cell proliferation and differentiation. The enhanced ratio of H3K4me3 to H3K27me3 markedly upregulates the expression related genes, thereby promoting cell proliferation and neuronal differentiation, ultimately leading to improved spatial learning and memory. Conversely, decreasing this ratio has the opposite effect.

In the plant innate immune system, pattern recognition receptor (PRR) and nucleotide-binding domain leucine-rich repeat (NLR) proteins recognize pathogens and activate defenses. To prevent excessive immune responses that could affect growth, plants regulate PRRs and NLRs at the transcriptional and post-transcriptional levels. Poised or bivalent chromatin states, marked by the simultaneous presence of active and repressive epigenetic modifications, maintain genes in a transcriptionally primed state, keeping their expression low while enabling their rapid activation in response to stress. Here, we investigated how poised chromatin states regulate PRR and NLR genes in soybean (Glycine max). Our integrative epigenomic and transcriptomic analysis revealed that although NLR and PRR genes both harbor abundant active and repressive histone modifications and exhibit high chromatin accessibility, their basal expression levels remain relatively low. Moreover, clustered NLR and PRR genes residing within the same topologically associating domains shared similar chromatin states and expression dynamics, suggesting coordinated control. These gene families had distinct epigenetic features: NLR genes displayed narrow H3K27me3 peaks together with strong pausing of RNA Polymerase II at their 5′ ends, whereas PRR genes were characterized by broader H3K27me3 peaks. Together, our results shed light on the role of poised chromatin states in coordinating growth and defense responses in soybean.Supplementary InformationThe online version contains supplementary material available at 10.1007/s42994-025-00233-4.

Histone H3 lysine 4 methylation (H3K4me) is generally associated with active transcription and bivalent chromatin, but can also contribute to repression. In metazoans, H3K4 methylation is catalysed by KMT2 methyltransferases assembled with the core scaffolding proteins WDR5, ASH2L, and RBBP5. RBBP5 mediates complex assembly and nucleosome binding, whilst WDR5 stabilises interactions to promote tri-methylation. However, WDR5 also exhibits additional 'moonlighting' functions, leaving its specific roles in H3K4 methylation and transcription regulation unclear. Using C. elegans embryos, spike-in ChIP-seq, and null alleles of wdr-5(-) and rbbp-5(-), we dissected the contributions of these scaffolds towards H3K4 mono-, di-, and tri-methylation as well as gene expression during C. elegans embryogenesis. We show that C. elegans RBBP-5 is essential for both mono- and multi-methylated H3K4 deposition. On the other hand, WDR-5 is primarily required for H3K4me3, but can influence H3K4me2 and H3K4me1 deposition either positively or negatively depending on the genomic feature involved. We additionally performed RNA-seq on these mutants and found that rbbp-5 deletion was largely tolerated with mis-regulation of ~ 700 genes, whereas the wdr-5 deletion led to widespread transcriptomic disruption (~ 3000 genes). We initially hypothesised that these broad changes were driven by the altered H3K4me1 and H3K4me2 landscapes in the wdr-5(-) mutant. However, transcriptomic profiling of the wdr-5(-); rbbp-5(-) double mutant, which lacks H3K4 methylation, revealed a high degree of similarity to the wdr-5(-) single mutant. This refuted our initial hypothesis and indicates that the changes in H3K4 methylation are unlikely to underlie the transcriptional effects of the wdr-5 deletion. Our findings strongly indicate that WDR-5 profoundly shapes gene expression through mechanisms beyond H3K4 methylation. Distinguishing between H3K4me-dependent and independent functions of WDR-5 will further understanding of its roles in development and disease.

Silencing evolutionary young retrotransposons by cytosine DNA methylation is essential for spermatogenesis, as failure to methylate their promoters leads to reactivation, meiotic failure, and infertility. How retrotransposons reactivate in the absence of DNA methylation is poorly understood. We show that upon defective DNA methylation, distinct retrotransposon families display unique expression patterns and chromatin landscapes during mouse spermatogenesis. We find that their reactivation in meiotic spermatocytes correlates with the loss of bivalent H3K4me3-H3K27me3 chromatin marks. Through proteomics and chromatin profiling, we identify NRF1 as a DNA methylation-sensitive transcription factor that transactivates unmethylated retrotransposons. Conditional germline knockout of Nrf1 in the absence of DNA methylation rescues the silencing of the most mutagenic retrotransposon in mice, namely Intracisternal A-particle or IAP. Our findings reveal that chromatin modifications together with a DNA methylation-sensitive transcription factor regulate retrotransposon expression in the absence of DNA methylation in spermatogenesis, revealing a mechanism by which retrotransposons proliferate in the germline after evading DNA methylation-based silencing.

Bivalent chromatin instructs lineage specification during hematopoiesis.

The Microrchidia (MORC) proteins are conserved GHKL-type ATPases required for chromatin condensation and gene silencing in animals and plants. Here we show that MORC proteins function with Polycomb-Repressive Complex 2 (PRC2) to control chromatin structure, gene expression and stress responses in rice. Rice MORC6b interacts with and stabilizes PRC2 for trimethylated histone H3 lysine 27 (H3K27me3) deposition preferentially at bivalent domains marked by both H3K4me3 and H3K27me3 to repress genes enriched for stress responses. The MORC-binding sites perfectly overlap with a set of PRC2 targets and colocalize with chromatin loop boundaries. High-throughput chromatin conformation capture combined with chromatin immunoprecipitation (Hi-ChIP) analysis revealed that the morc mutation reduces the number of H3K27me3-marked chromatin loops mainly at bivalent domains compressing many defence-related genes and affects rice plant tolerance to biotic and abiotic stresses. MORC function in H3 K27 trimethylation and gene expression is partly inhibited by ALKBH1, a DNA 6mA demethylase that impairs PRC2 binding and H3K27me3 deposition at bivalent chromatin domains and has an opposite function to MORC in stress responses. These findings identify MORCs and ALKBH1 as an antagonistic couple controlling PRC2 function in regulating chromatin structure and gene expression preferentially at bivalent chromatin domains for stress responses, which is instructive for understanding the regulation of chromatin dynamics in other eukaryotic organisms.

Bivalent chromatin domains regulate hematopoietic stem and progenitor cell differentiation.

DNA methylation clocks have been widely used for accurate age prediction, but most studies have been carried out on mammals. Here we present an epigenetic clock for the aquatic frog Xenopus tropicalis, a widely used model organism in developmental biology and genomics. To construct the clock, we collected DNA methylation data from 192 frogs using targeted bisulfite sequencing at genomic regions containing CpG sites previously shown to have age-associated methylation in Xenopus. We found highly positively and negatively age-correlated CpGs are enriched in heterochromatic regions marked with H4K20me3 and H3K9me3. Positively age-correlated CpGs are enriched in bivalent chromatin and gene bodies with H3K36me3, and tend to be proximal to lowly expressed genes. These epigenetic features of aging are similar to those found in mammals, suggesting evolutionary conservation of epigenetic aging mechanisms. Our clock enables future aging biology experiments that leverage the unique properties of amphibians.

Abstract Chromatin bivalency plays an important role in cell lineage specification during development. Cancer therapy resistance involves an extensive reprogramming of gene expression resulting in tumor lineage plasticity (LP). However, the underlying mechanisms are poorly understood. Rev-erb-alpha, a member of the nuclear receptor transcription factor family, plays a key role in regulation of circadian rhythm and metabolism primarily through repression of its target genes. We performed sequential ChIP-seq epigenome profiling of tumor tissues from the clinic and PDX models of prostate cancer and integrated analysis of tumor epigenome and transcriptome. We also treated the PDX models with small-molecule inhibitors of the receptor. We found that most of the tumor LP genes, including those in programs of neurodevelopment, neural signaling, stemness and EMT, are controlled by bivalent promoters with concurrent marking of H3K27me3 and H3K4me3. Our further studies revealed that the bivalent chromatin state resolves during the development of cancer therapy resistance. Interestingly, Rev-erb-alpha plays a critical role in promoting the bivalency resolution and activation of the LP programs. Its pharmacological targeting effectively reverses the bivalency resolution and diminishes the LP in anti-AR therapy-resistant tumors. Therefore, our study establishes that resolution of chromatin bivalency is a major epigenetic mechanism of tumor LP and demonstrates that therapeutic targeting of Rev-erb-alpha represents a novel treatment of advanced prostate cancer. This work was supported by NIH and UC Davis comprehensive cancer center and U.S. Department of Defense. Citation Format: Yatian Yang, Xiong zhang, hongye zou, Eva Corey, Ronald M. Evans, Amina Zoubeidi, Hongwu Chen. A circadian regulator drives tumor lineage plasticity through chromatin bivalency resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2726.

The Human gamma-herpesviruses Kaposi's sarcoma herpesvirus (KSHV) and Epstein-Barr virus (EBV) are causally associated to a wide range of cancers. While the default infection program for these viruses is latent, sporadic lytic reactivation supports virus dissemination and oncogenesis. Despite its relevance, the repertoire of host factors governing the transition from latent to lytic phase is not yet complete, leaving much of this complex process unresolved. Here we show that heat shock factor 2 (HSF2), a transcription factor involved in regulation of stress responses and specific cell differentiation processes, promotes gamma-herpesvirus lytic gene expression. In lymphatic endothelial cells infected with KSHV and in gastric cancer cells positive for EBV, ectopic HSF2 enhances the expression of lytic genes; While knocking down HSF2 significantly decreases their expression. HSF2 overexpression is accompanied by decreased levels of repressive histone marks at the promoters of the lytic regulators KSHV ORF50 and EBV BZLF1, both characterized by poised chromatin features. Our results demonstrate that endogenous HSF2 binds to the promoters of KSHV ORF50 and EBV BZLF1 genes and shifts the bivalent chromatin state towards a more transcriptionally permissive state. We detected HSF2 binding to the ORF50 promoter in latent cells, in contrast, in lytic cells, HSF2 occupancy at the ORF50 promoter is lost in conjunction with its proteasomal degradation. These findings identify HSF2 as a regulator of gamma-herpesvirus lytic gene expression in latency and offer new insights on the function of this transcription factors at poised gene promoters, improving our understanding of its role in differentiation and development.

Epigenetics plays a key role in cellular differentiation and maintaining cell identity, enabling cells to regulate their genetic activity without altering the DNA sequence. Epigenetic regulation occurs within the context of hierarchically folded chromatin, yet the interplay between the dynamics of epigenetic modifications and chromatin architecture remains poorly understood. In addition, it remains unclear what mechanisms drive the formation of rugged epigenetic patterns, characterised by alternating genomic regions enriched in activating and repressive marks. In this study, we focus on post-translational modifications of histone H3 tails, particularly H3K27me3, H3K4me3, and H3K27ac. We introduce a mesoscopic stochastic model that incorporates chromatin architecture and competition of histone-modifying enzymes into the dynamics of epigenetic modifications in small genomic loci comprising several nucleosomes. Our approach enables us to investigate the mechanisms by which epigenetic patterns form on larger scales of chromatin organisation, such as loops and domains. Through bifurcation analysis and stochastic simulations, we demonstrate that the model can reproduce uniform chromatin states (open, closed, and bivalent) and generate previously unexplored rugged profiles. Our results suggest that enzyme competition and chromatin conformations with high-frequency interactions between distant genomic loci can drive the emergence of rugged epigenetic landscapes. Additionally, we hypothesise that bivalent chromatin can act as an intermediate state, facilitating transitions between uniform and rugged landscapes. This work offers a powerful mathematical framework for understanding the dynamic interactions between chromatin architecture and epigenetic regulation, providing new insights into the formation of complex epigenetic patterns.

Abiotic stresses, including drought, salinity, temperature fluctuations, and nutrient deficiencies, challenge plant growth and productivity, requiring adaptive mechanisms for survival. Histone modifications, especially histone methylation, participate in gene expression regulation in response to these stresses. Notably, bivalent H3K4me3-H3K27me3 modifications play a central role in fine-tuning stress-responsive genes, allowing plants to adapt to environmental changes. Recent studies have highlighted the dynamic switching of these bivalent chromatin marks at specific loci during stress, facilitating plant acclimatization to adverse environments. This review focuses on the four major histone H3 methylation modifications-H3K4, H3K9, H3K27, and H3K36-examining the roles of the associated methyltransferases and demethylases in mediating histone methylation dynamics. We synthesize recent findings on how these modifications regulate plant responses to various abiotic stresses, such as drought, salinity, heat, light stress, heavy metal exposure, and nutrient stress. By exploring these molecular mechanisms, we aim to deepen our understanding of how histone methylation shapes plant stress responses at both transcriptional and epigenetic levels. Furthermore, we also discuss the functional interaction of histone methylation with histone acetylation. These insights are critical for advancing breeding strategies aimed at improving plant tolerance to environmental stressors, ensuring food security, and supporting sustainable agricultural practices amid climate change.

Stem cells are undifferentiated cells that exhibit a bivalent chromatin state that determines their fate. These cells have potential applications in human and animal health and livestock production. Somatic cell nuclear transfer or cloning is currently being used to produce genetically edited animals. A highly differentiated genome is the main obstacle to correcting epigenetic reprogramming by enucleated oocytes during cloning. Activation of pluripotency genes in the somatic genome is a promising strategy to contribute to more efficient epigenetic reprogramming, improving this technique. Recently, epigenome editing has emerged as a new generation of clustered regularly interspaced short palindromic repeats-clustered regularly interspaced short palindromic repeats-associated protein 9 technology with the aim of modifying the cellular epigenome to turn genes on or off without modifying DNA. Here, we characterize the DNA methylation profile of the CpG island spanning the 5' untranslated region to intron 1 of the bovine octamer-binding transcription factor (Oct4) gene in gametes, embryos, and fibroblasts. DNA methylation patterns were categorized into three levels: low (0%-20%), moderate (21%-50%), and high (51%-100%). Sperm and embryos showed a hypomethylation pattern, whereas oocytes exhibited a hypo- to moderate methylation pattern. Fetal and adult skin fibroblasts were hypomethylated and moderately methylated, respectively. These results are essential for future studies aimed at manipulating the expression of Oct4. Thus, epigenome editing can be used to turn on the Oct4 in somatic cells to generate induced pluripotent stem cells. This strategy could potentially convert a fully differentiated cell into a cell with certain degree of pluripotency, facilitating nuclear reprogramming by the enucleated oocyte and improving cloning success rates.

The mechanistic link between the complex mutational landscape of de novo methyltransferase DNMT3A and the pathology of acute myeloid leukemia (AML) has not been clearly elucidated so far. Motivated by a recent discovery of the significance of DNMT3A-destabilizing mutations (DNMT3AINS) in AML, we here investigate the common characteristics of DNMT3AINS AML methylomes through computational analyses. We present that methylomes of DNMT3AINS AMLs are considerably different from those of DNMT3AR882 AMLs in that they exhibit increased intratumor DNA methylation heterogeneity in bivalent chromatin domains. This epigenetic heterogeneity was associated with the transcriptional variability of developmental and membrane-associated factors shaping stem cell niche, and also was a predictor of the response of AML cells to hypomethylating agents, implying that the survival of AML cells depends on stochastic DNA methylations at bivalent domains. Altogether, our work provides a novel mechanistic model suggesting the genomic origin of the aberrant epigenomic heterogeneity in disease conditions.

Embryonic development follows a conserved sequence of events across species, yet the pace of development is highly variable and particularly slow in humans. Species-specific developmental timing is largely recapitulated in stem cell models, suggesting a cell-intrinsic clock. Here we use directed differentiation of human embryonic stem cells into neuroectoderm to perform a whole-genome CRISPR-Cas9 knockout screen and show that the epigenetic factors Menin and SUZ12 modulate the speed of PAX6 expression during neural differentiation. Genetic and pharmacological loss-of-function of Menin or SUZ12 accelerate cell fate acquisition by shifting the balance of H3K4me3 and H3K27me3 at bivalent promoters, thereby priming key developmental genes for faster activation upon differentiation. We further reveal a synergistic interaction of Menin and SUZ12 in modulating differentiation speed. The acceleration effects were observed in definitive endoderm, cardiomyocyte and neuronal differentiation paradigms, pointing to chromatin bivalency as a general driver of timing across germ layers and developmental stages.