Histone Tails Research Articles

H3.3K122A results in a neomorphic phenotype in mouse embryonic stem cells

Canonical histone H3 and histone variant H3.3 are posttranslationally modified with the genomic distribution of these marks denoting different features and these modifications may influence transcription. While the majority of posttranslational modifications occur on histone tails, there are defined modifications within the globular domain, such as acetylation of H3K122/H3.3K122. To understand the function of the amino acid H3.3K122 in transcriptional regulation, we attempted to generate H3.3K122A mouse embryonic stem (mES) cells but were unsuccessful. Through multi-omic profiling of mutant cell lines harboring two or three of four H3.3 targeted alleles, we have uncovered that H3.3K122A is neomorphic and results in lethality. This is surprising as prior studies demonstrate H3.3-null mES cells are viable and pluripotent but exhibit a reduced differentiation capacity. Together, these studies have uncovered a novel dependence of a globular domain residue within H3.3 for viability and broadened our understanding of how histone variants contribute to transcription regulation and pluripotency in mES cells.

The BET protein inhibitor apabetalone (RVX-208) prevents doxorubicin-induced endothelial senescence

Abstract Background Accumulation of senescent endothelial cells (ECs) plays a pivotal role in cardiometabolic disorders and lifespan reduction(1). Yet, only a few senolytics are known to date, partly due to the poor grasp of the molecular mechanisms that control the senescence survival programme. Bromo-and extraterminal domain (BET) proteins, known epigenetic reader proteins, recognize acetylated histone tails and regulate gene transcription. RVX-208, an FDA-approved BET inhibitor, has shown to modulate transcriptional programs involved in inflammation, cancer, and renal disease(2). Yet, its potential role in EC senescence and cardio-oncology remains elusive. Purpose Our study aims to investigate whether pharmacological modulation of BET proteins by RVX-208 can mitigate doxorubicin-induced endothelial senescence. Methods Human aortic endothelial cells (HAECs) were exposed to different concentrations of Doxo (50nM-500nM) for 48 hours. Cell morphology changes, cell cycle arrest, senescence associated secreted phenotype (SASP) release, upregulation of SA-b gal activity, DNA damage response (gH2AX) and expression of p21, p16 and p53 were used as molecular markers of cellular senescence. Conditioned media was collected from Doxo-treated ECs and processed for molecular analyses or used for treatment of healthy, non-senescent ECs. To test whether RVX-208 exerts any senolytic or senomorphic effect, HAECs were treated with Doxo (100 nM) in presence or absence of RVX-208 (5mM-20mM) for 48 hrs. RNA-seq and proteomic profiling were performed in vehicle and Doxo-treated ECs. Results Doxo-treated ECs showed a dose-dependent increase in senescence markers (p16, p21 and p53) and DNA-damage response (gH2AX). FACS analysis revealed an accumulation of cells in G2/M phase in Doxo-treated ECs (100 nM). Of interest, treatment with RVX-208 (15mM) prevented the upregulation of senescent markers (p16, p21 and p53) while rescuing alterations in cell morphology and reducing the number of SA-b gal positive cells. RVX-208 prevented Doxo-induced upregulation of genes involved in inflammation, oxidative stress and mitochondrial dysfunction as shown by RNA-seq and proteomic analysis. Conditioned media from Doxo-treated ECs showed an enrichment of SASP-associated markers (IL6, CCL2, IL8, TIMP2 and PDGF-b) as shown by dot-blot arrays. Exposure of healthy ECs to conditioned media from senescent ECs recapitulated aging features. Notably, RVX-208 blunted the release of SASP markers (IL6, CCL2 and IL8) from Doxo-treated ECs. Mechanistically, we found enhanced BET protein occupancy (BRD4) with concomitant enrichment of activating chromatin marks (H3K27Ac and H3K4me) on the promoter of senescence genes COL1A2, CDKN1C and SESN3. Conclusion Targeting BET proteins with RVX-208 may offer a promising therapeutic strategy to prevent endothelial aging in cancer patients undergoing cardiotoxic therapies.

Characterization of Medusavirus encoded histones reveals nucleosome-like structures and a unique linker histone

The organization of DNA into nucleosomes is a ubiquitous and ancestral feature that was once thought to be exclusive to the eukaryotic domain of life. Intriguingly, several representatives of the Nucleocytoplasmic Large DNA Viruses (NCLDV) encode histone-like proteins that in Melbournevirus were shown to form nucleosome-like particles. Medusavirus medusae (MM), a distantly related giant virus, encodes all four core histone proteins and, unique amongst most giant viruses, a putative acidic protein with two domains resembling eukaryotic linker histone H1. Here, we report the structure of nucleosomes assembled with MM histones and highlight similarities and differences with eukaryotic and Melbournevirus nucleosomes. Our structure provides insight into how variations in histone tail and loop lengths are accommodated within the context of the nucleosome. We show that MM-histones assemble into tri-nucleosome arrays, and that the putative linker histone H1 does not function in chromatin compaction. These findings expand our limited understanding of chromatin organization by virus-encoded histones.

A "Goldilocks Zone" for Recruiting BET Proteins with Bromodomain-1-Selective Ligands.

Synthetic genome readers/regulators (SynGRs) are bifunctional molecules that are rationally designed to bind specific genomic sequences and engage cellular machinery that regulates the expression of targeted genes. The prototypical SynGR1 targets GAA trinucleotide repeats and recruits the BET family of transcriptional regulatory proteins via a flexibly tethered ligand, JQ1. This pan-BET ligand binds both tandem bromodomains of BET proteins (BD1 and BD2). Second-generation SynGRs, which substituted JQ1 with bromodomain-selective ligands, unexpectedly revealed that BD1-selective ligands failed to functionally engage BET proteins in living cells despite displaying the ability to bind BD1 in vitro. Mechanistically, recruiting a BET protein via BD1- or BD2-selective SynGRs should have resulted in indistinguishable functional outcomes. Here we report the conversion of inactive BD1-targeting SynGRs into functional gene regulators by a structure-guided redesign of the chemical linker that bridges the DNA-binding molecule to the highly selective BD1 ligand GSK778. The results point to an optimal zone for positioning the BD1-selective ligand for functional engagement of BET proteins on chromatin, consistent with the preferred binding of BD1 domains to distal acetyllysine residues on histone tails. The results not only resolve the mechanistic conundrum but also provide insight into domain-selective targeting and nuanced design of chemo probes and therapeutics.

Histone Tail Cleavage as a Mechanism for Epigenetic Regulation.

Histones are essential for DNA packaging and undergo post-translational modifications that significantly influence gene regulation. Among these modifications, histone tail cleavage has recently garnered attention despite being less explored. Cleavage by various proteases impacts processes such as stem cell differentiation, aging, infection, and inflammation, though the mechanisms remain unclear. This review delves into recent insights on histone proteolytic cleavage and its epigenetic significance, highlighting how chromatin, which serves as a dynamic scaffold, responds to signals through histone modification, replacement, and ATP-dependent remodeling. Specifically, histone tail cleavage is linked to critical cellular processes such as granulocyte differentiation, viral infection, aging, yeast sporulation, and cancer development. Although the exact mechanisms connecting histone cleavage to gene expression are still emerging, it is clear that this process represents a novel epigenetic transcriptional mechanism intertwined with chromatin dynamics. This review explores known histone tail cleavage events, the proteolytic enzymes involved, their impact on gene expression, and future research directions in this evolving field.

Molecular basis of global promoter sensing and nucleosome capture by the SWR1 chromatin remodeler.

The SWR1 chromatin remodeling complex is recruited to+1 nucleosomes downstream of transcription start sites of eukaryotic promoters, where it exchanges histone H2A for the specialized variant H2A.Z. Here, we use cryoelectron microscopy (cryo-EM) to resolve the structural basis of the SWR1 interaction with free DNA, revealing a distinct open conformation of the Swr1 ATPase that enables sliding from accessible DNA to nucleosomes. A complete structural model of the SWR1-nucleosome complex illustrates critical roles for Swc2 and Swc3 subunits in oriented nucleosome engagement by SWR1. Moreover, an extended DNA-binding α helix within the Swc3 subunit enables sensing of nucleosome linker length and is essential for SWR1-promoter-specific recruitment and activity. The previously unresolved N-SWR1 subcomplex forms a flexible extended structure, enabling multivalent recognition of acetylated histone tails by reader domains to further direct SWR1 toward the+1 nucleosome. Altogether, our findings provide a generalizable mechanism for promoter-specific targeting of chromatin and transcription complexes.

Chronic thermal stress on Octopus maya embryos down-regulates epigenome-related genes and those involved in the nervous system development and morphogenesis

Red Octopus maya is strongly influenced by temperature. Recent studies have reported negative reproduction effects on males and females when exposed to temperatures higher than 27 °C. Embryos under thermal stress show morphological and physiological alterations; similar phenotypes have been reported in embryos from stressed females, evidencing transgenerational consequences. Transcriptomic profiles were characterized along embryo development during normal-under thermal stress and epigenetic alterations through DNA methylation and damage quantification. Total RNA in organogenesis, activation, and growth stages in control and thermal stress were sequenced with Illumina RNA-Seq. Similarly, total DNA was used for DNA methylation and damage quantification between temperatures and embryo stages. Differential gene expression analyses showed that embryos express genes associated with oxygen transport, morphogenesis, nervous system, neuroendocrine cell differentiation, spermatogenesis, and male sex differentiation. Conversely, embryos turn off genes involved mainly in nervous system development, morphogenesis, and gene expression regulation when exposed to thermal stress – consistent with O. maya embryo phenotypes showing abnormal arms, eyes, and body development. No significant differences were observed in quantifying DNA methylation between temperatures but they were for DNA damage quantification. Epigenetic alterations are hypothesized to occur since several genes found downregulated belong to the epigenetic machinery but at histone tail level.

H3.3K122A results in a neomorphic phenotype in mouse embryonic stem cells.

The histone variant H3.3 acts in coordination with histone posttranslational modifications and other chromatin features to facilitate appropriate transcription. Canonical histone H3 and histone variant H3.3 are post-translationally modified with the genomic distribution of these marks denoting different features and with more recent evidence suggesting that these modifications may influence transcription. While the majority of posttranslational modifications occur on histone tails, there are defined modifications within the globular domain, such as acetylation of H3K122/H3.3K122. To understand the function of the residue H3.3K122 in transcriptional regulation, we attempted to generate H3.3K122A mouse embryonic stem (mES) cells but were unsuccessful. Through multi-omic profiling of mutant cell lines harboring two or three of four H3.3 targeted alleles, we have uncovered that H3.3K122A is neomorphic and results in lethality. This is surprising as prior studies demonstrate H3.3-null mES cells are viable and pluripotent, albeit with reduced differentiation capacity. Together, these studies have uncovered a novel dependence of a globular domain residue of H3.3 for viability and broadened our understanding of how histone variants contribute to transcription regulation and pluripotency in mES cells.

Single-nucleosome imaging unveils that condensins and nucleosome–nucleosome interactions differentially constrain chromatin to organize mitotic chromosomes

For accurate mitotic cell division, replicated chromatin must be assembled into chromosomes and faithfully segregated into daughter cells. While protein factors like condensin play key roles in this process, it is unclear how chromosome assembly proceeds as molecular events of nucleosomes in living cells and how condensins act on nucleosomes to organize chromosomes. To approach these questions, we investigate nucleosome behavior during mitosis of living human cells using single-nucleosome tracking, combined with rapid-protein depletion technology and computational modeling. Our results show that local nucleosome motion becomes increasingly constrained during mitotic chromosome assembly, which is functionally distinct from condensed apoptotic chromatin. Condensins act as molecular crosslinkers, locally constraining nucleosomes to organize chromosomes. Additionally, nucleosome-nucleosome interactions via histone tails constrain and compact whole chromosomes. Our findings elucidate the physical nature of the chromosome assembly process during mitosis.

Deciphering the dynamics: Exploring the impact of mechanical forces on histone acetylation.

Living cells navigate a complex landscape of mechanical cues that influence their behavior and fate, originating from both internal and external sources. At the molecular level, the translation of these physical stimuli into cellular responses relies on the intricate coordination of mechanosensors and transducers, ultimately impacting chromatin compaction and gene expression. Notably, epigenetic modifications on histone tails govern the accessibility of gene-regulatory sites, thereby regulating gene expression. Among these modifications, histone acetylation emerges as particularly responsive to the mechanical microenvironment, exerting significant control over cellular activities. However, the precise role of histone acetylation in mechanosensing and transduction remains elusive due to the complexity of the acetylation network. To address this gap, our aim is to systematically explore the key regulators of histone acetylation and their multifaceted roles in response to biomechanical stimuli. In this review, we initially introduce the ubiquitous force experienced by cells and then explore the dynamic alterations in histone acetylation and its associated co-factors, including HDACs, HATs, and acetyl-CoA, in response to these biomechanical cues. Furthermore, we delve into the intricate interactions between histone acetylation and mechanosensors/mechanotransducers, offering a comprehensive analysis. Ultimately, this review aims to provide a holistic understanding of the nuanced interplay between histone acetylation and mechanical forces within an academic framework.

A DNA condensation code for linker histones

Linker histones play an essential role in chromatin packaging by facilitating compaction of the 11-nm fiber of nucleosomal "beads on a string." The result is a heterogeneous condensed state with local properties that range from dynamic, irregular, and liquid-like to stable and regular structures (the 30-nm fiber), which in turn impact chromatin-dependent activities at a fundamental level. The properties of the condensed state depend on the type of linker histone, particularly on the highly disordered C-terminal tail, which is the most variable region of the protein, both between species, and within the various subtypes and cell-type specific variants of a given organism. We have developed an in vitro model system comprising linker histone tail and linker DNA, which although very minimal, displays surprisingly complex behavior, and is sufficient to model the known states of linker histone-condensed chromatin: disordered "fuzzy" complexes ("open" chromatin), dense liquid-like assemblies (dynamic condensates), and higher-order structures (organized 30-nm fibers). A crucial advantage of such a simple model is that it allows the study of the various condensed states by NMR, circular dichroism, and scattering methods. Moreover, it allows capture of the thermodynamics underpinning the transitions between states through calorimetry. We have leveraged this to rationalize the distinct condensing properties of linker histone subtypes and variants across species that are encoded by the amino acid content of their C-terminal tails. Three properties emerge as key to defining the condensed state: charge density, lysine/arginine ratio, and proline-free regions, and we evaluate each separately using a strategic mutagenesis approach.

Structural basis for linker histone H5–nucleosome binding and chromatin fiber compaction

The hierarchical packaging of chromatin fibers plays a critical role in gene regulation. The 30-nm chromatin fibers, a central-level structure bridging nucleosomal arrays to higher-order organizations, function as the first level of transcriptional dormant chromatin. The dynamics of 30-nm chromatin fiber play a crucial role in biological processes related to DNA. Here, we report a 3.6-angstrom resolution cryogenic electron microscopy structure of H5-bound dodecanucleosome, i.e., the chromatin fiber reconstituted in the presence of linker histone H5, which shows a two-start left-handed double helical structure twisted by tetranucleosomal units. An atomic structural model of the H5-bound chromatin fiber, including an intact chromatosome, is built, which provides structural details of the full-length linker histone H5, including its N-terminal domain and an HMG-motif-like C-terminal domain. The chromatosome structure shows that H5 binds the nucleosome off-dyad through a three-contact mode in the chromatin fiber. More importantly, the H5-chromatin structure provides a fine molecular basis for the intra-tetranucleosomal and inter-tetranucleosomal interactions. In addition, we systematically validated the physiological functions and structural characteristics of the tetranucleosomal unit through a series of genetic and genomic studies in Saccharomyces cerevisiae and in vitro biophysical experiments. Furthermore, our structure reveals that multiple structural asymmetries of histone tails confer a polarity to the chromatin fiber. These findings provide structural and mechanistic insights into how a nucleosomal array folds into a higher-order chromatin fiber with a polarity in vitro and in vivo.

Haspin mediates H3.3S31 phosphorylation downstream of Aurora B in mouse embryonic stem cells.

Histone phosphorylation is instrumental in regulating diverse cellular processes across eukaryotes. Unraveling the kinases that target specific histone sites is key to deciphering the underlying mechanisms. Among the various sites on histone tails that can undergo phosphorylation, the kinase responsible for H3.3S31 phosphorylation remained elusive. Since both H3.3S31ph and H3T3ph occur specifically during mitosis, and Haspin is the known kinase for H3T3 phosphorylation, we investigated its potential role in H3.3S31 phosphorylation. We employed CRISPR/Cas9, RNA interference, and specific small molecule inhibitors to eliminate Haspin function in various cell types. Our data consistently revealed a link between Haspin and H3.3S31ph. Furthermore, in vitro kinase assays provided evidence supporting Haspin's contribution to H3.3S31ph. Loss- and gain-of-function experiments targeting Haspin and Aurora B further suggested a hierarchical relationship. Haspin acts as a downstream kinase of Aurora B, specifically orchestrating H3.3S31 phosphorylation in mESCs. This study unveils a novel role for Haspin as a kinase in regulating H3.3S31 phosphorylation during mitosis. This discovery holds promise for expanding our understanding of the functional significance of Haspin and H3.3S31ph in mammals.

What tunes guanine ionization potential in a nucleosome? An all-in-one systematic QM/MM assessment

Guanine radical cations are precursors to oxidatively induced DNA lesions, and the determination of oxidative DNA hot spots beyond oligonucleotides remains a current challenge. In order to rationalize the finetuned ionization properties of the ∼60 guanines in a nucleosome core particle, we report a robust molecular dynamics-then-FO-DFTB/MM (fragment-orbital tight-binding density functional theory/molecular mechanics) simulation protocol spanning 20 μs. Our work allows us to identify several factors governing guanine ionization potential and map oxidative hotspots. Our results highlight the predominant role of the proximity of positively charged histone residues in the modulation of the guanine ionization potential up to 0.6 eV. Consequently, fast, long-range hole transfer in nucleosomal DNA could be tuned by the proximity of histone tails, which differs, from a biological point of view, on the chromatin state.

P-115 GATAD2B is required for pre-implantation embryo development by delaying zygotic genome activation

Abstract Study question Major zygotic genome activation (ZGA) involving the activation of thousands of genes occurs at the late-2cell stage, but the regulatory mechanisms governing ZGA remain unclear. Summary answer Our results demonstrate that GATAD2B is essential for early embryonic development, in part through facilitating zygotic genome activation (ZGA). What is known already Acetylation modifications of histone tails promote transcriptional activation, and the maternal deletion of H4K16ac leads to failure in ZGA. GATAD2B is one of the core subunits of the Nucleosome remodeling and histone deacetylation complex(NuRD). Study design, size, duration Using mouse embryos as the research model, the study was conducted over a period of approximately two years to ensure a relatively small-scale investigation. Participants/materials, setting, methods We examined the dynamic expression of NURD complex components, including GATAD2B, during pre-implantation embryonic stages. GATAD2B expression was specifically reduced via microinjection of GATAD2B-siRNA during the zygote stage, revealing its impact on early embryonic development in mice. Zygotic genome activation (ZGA), morula compaction, and blastocyst lineage differentiation were investigated using immunofluorescence, western blot, and other techniques. Additionally, we employed RNA-seq, protein immunoprecipitation, and mass spectrometry to uncover underlying molecular mechanisms. Main results and the role of chance Our research has shown that GATAD2B exhibits specific nucleus localization and high protein expression from the late-2-cell to 8-cell. This intriguing phenomenon prompted us to investigate the relationship between GATAD2B and the ZGA. We discovered a distinctive pattern of GATAD2B, starting from the late 2-cell stage with nuclear localization. GATAD2B depletion resulted in defective embryonic development, including increased DNA damage at morula, decreased blastocyst formation rate and abnormal differentiation of ICM/TE lineages. Consistent with the delay during the cleavage stage, the transcriptome analysis of the 2-cell revealed inhibition of the cell cycle G2/M phase transition pathway. Furthermore, the GATAD2B proteomics data provided clear evidence of a certain association between GATAD2B and molecules involved in the cell cycle pathway. As hypothesized, GATAD2B-deficient 2-cell embryos exhibited abnormalities in ZGA during the maternal-to-embryonic transition, with lower expression of the major ZGA mark MERVL. Limitations, reasons for caution Given the limitations of antibody effectiveness and specificity required for the experiments, the exploration of molecular mechanisms is still ongoing. Wider implications of the findings Our findings have elucidated the impact of GATAD2B on early embryonic development through the regulation of zygotic genome activation (ZGA), providing a novel genetic explanation for clinical defects in early embryonic development. Trial registration number not applicable

Polyamines regulate cell fate by altering the activity of histone-modifying enzymes.

Polyamines are polycationic alkyl-amines abundant in proliferating stem and cancer cells. How these metabolites influence numerous cellular functions remains unclear. Here we show that polyamine levels decrease during differentiation and that inhibiting polyamine synthesis leads to a differentiated-like cell state. Polyamines concentrate in the nucleus and are further enriched in the nucleoli of cells in culture and in vivo . Loss of polyamines drives changes in chromatin accessibility that correlate with altered histone post-translational modifications. Polyamines interact electrostatically with DNA on the nucleosome core, stabilizing histone tails in conformations accessible to modifying enzymes. These data reveal a mechanism by which an abundant metabolite influences chromatin structure and function in a non-sequence specific manner, facilitating chromatin remodeling during reprogramming and limiting it during fate commitment.

Brainwide silencing of prion protein by AAV-mediated delivery of an engineered compact epigenetic editor.

Prion disease is caused by misfolding of the prion protein (PrP) into pathogenic self-propagating conformations, leading to rapid-onset dementia and death. However, elimination of endogenous PrP halts prion disease progression. In this study, we describe Coupled Histone tail for Autoinhibition Release of Methyltransferase (CHARM), a compact, enzyme-free epigenetic editor capable of silencing transcription through programmable DNA methylation. Using a histone H3 tail-Dnmt3l fusion, CHARM recruits and activates endogenous DNA methyltransferases, thereby reducing transgene size and cytotoxicity. When delivered to the mouse brain by systemic injection of adeno-associated virus (AAV), Prnp-targeted CHARM ablates PrP expression across the brain. Furthermore, we have temporally limited editor expression by implementing a kinetically tuned self-silencing approach. CHARM potentially represents a broadly applicable strategy to suppress pathogenic proteins, including those implicated in other neurodegenerative diseases.

Structure- and Property-Based Optimization of Efficient Pan-Bromodomain and Extra Terminal Inhibitors to Identify Oral and Intravenous Candidate I-BET787.

The bromodomain and extra terminal (BET) family of bromodomain-containing proteins are important epigenetic regulators that elicit their effect through binding histone tail N-acetyl lysine (KAc) post-translational modifications. Recognition of such markers has been implicated in a range of oncology and immune diseases and, as such, small-molecule inhibition of the BET family bromodomain-KAc protein-protein interaction has received significant interest as a therapeutic strategy, with several potential medicines under clinical evaluation. This work describes the structure- and property-based optimization of a ligand and lipophilic efficient pan-BET bromodomain inhibitor series to deliver candidate I-BET787 (70) that demonstrates efficacy in a mouse model of inflammation and suitable properties for both oral and intravenous (IV) administration. This focused two-phase explore-exploit medicinal chemistry effort delivered the candidate molecule in 3 months with less than 100 final compounds synthesized.

Expression Proteomics and Histone Analysis Reveal Extensive Chromatin Network Changes and a Role for Histone Tail Trimming during Cellular Differentiation.

In order to understand the coordinated proteome changes associated with differentiation of a cultured cell pluripotency model, protein expression changes induced by treatment of NT2 embryonal carcinoma cells with retinoic acid were monitored by mass spectrometry. The relative levels of over 5000 proteins were mapped across distinct cell fractions. Analysis of the chromatin fraction revealed major abundance changes among chromatin proteins and epigenetic pathways between the pluripotent and differentiated states. Protein complexes associated with epigenetic regulation of gene expression, chromatin remodelling (e.g., SWI/SNF, NuRD) and histone-modifying enzymes (e.g., Polycomb, MLL) were found to be extensively regulated. We therefore investigated histone modifications before and after differentiation, observing changes in the global levels of lysine acetylation and methylation across the four canonical histone protein families, as well as among variant histones. We identified the set of proteins with affinity to peptides housing the histone marks H3K4me3 and H3K27me3, and found increased levels of chromatin-associated histone H3 tail trimming following differentiation that correlated with increased expression levels of cathepsin proteases. We further found that inhibition of cathepsins B and D reduces histone H3 clipping. Overall, the work reveals a global reorganization of the cell proteome congruent with differentiation, highlighting the key role of multiple epigenetic pathways, and demonstrating a direct link between cathepsin B and D activity and histone modification.

Cancer-associated Histone H3 N-terminal arginine mutations disrupt PRC2 activity and impair differentiation

Dysregulated epigenetic states are a hallmark of cancer and often arise from genetic alterations in epigenetic regulators. This includes missense mutations in histones, which, together with associated DNA, form nucleosome core particles. However, the oncogenic mechanisms of most histone mutations are unknown. Here, we demonstrate that cancer-associated histone mutations at arginines in the histone H3 N-terminal tail disrupt repressive chromatin domains, alter gene regulation, and dysregulate differentiation. We find that histone H3R2C and R26C mutants reduce transcriptionally repressive H3K27me3. While H3K27me3 depletion in cells expressing these mutants is exclusively observed on the minor fraction of histone tails harboring the mutations, the same mutants recurrently disrupt broad H3K27me3 domains in the chromatin context, including near developmentally regulated promoters. H3K27me3 loss leads to de-repression of differentiation pathways, with concordant effects between H3R2 and H3R26 mutants despite different proximity to the PRC2 substrate, H3K27. Functionally, H3R26C-expressing mesenchymal progenitor cells and murine embryonic stem cell-derived teratomas demonstrate impaired differentiation. Collectively, these data show that cancer-associated H3 N-terminal arginine mutations reduce PRC2 activity and disrupt chromatin-dependent developmental functions, a cancer-relevant phenotype.