H3K4 Methylation Research Articles

ABSTRACT Histone H4K20 methylation is critical in regulating the cell cycle, DNA damage response, and gene repression in proliferating cells. However, its role in the heart remains poorly understood. Our previous work revealed that histone H4K20 tri-methylation is elevated in acute cell models of cardiomyocyte hypertrophy but is reduced in mouse models of cardiac hypertrophy and ischemia. Although these findings highlight the dynamic nature of this modification and its significance in regulating gene expression, the data on enzymes regulating H4K20 methylation is sparse. To build upon this work and investigate H4K20 di-methylation and the enzymes modulating this site in cardiac pathology, we quantified histone H4K20 di-methylation and 12 methyltransferases and demethylases across one cell model, two mouse models of cardiac dysfunction, and cardiac tissue from heart failure patients. While we observed no global changes in H4K20 di-methylation, we detected alterations in methyltransferases KMT5C and SMYD5 and demethylases RAD23A and KDM7C in humans and mice. These findings suggest changes in H4K20 di-methylation may occur on an individual gene basis but do not lead to global alterations in H4K20 di-methylation. Additionally, this work identified four enzymes differentially modulated in cardiac dysfunction to advance our understanding of epigenetic mechanisms involved in heart disease.

De novo DNA methylation is mediated by DNA methyltransferases DNMT3A and DNMT3B, in cooperation with the catalytically inactive paralogs DNMT3L and DNMT3B3. DNMT3L is predominantly expressed in embryonic stem cells to establish methylation patterns and is silenced upon differentiation, with DNMT3B3 substituting in somatic cells. Here we present high-resolution cryo-electron microscopy structures of nucleosome-bound, full-length DNMT3A2-3L and its oligomeric assemblies in the nucleosome-free state. We identified the critical role of DNMT3L as a histone modification sensor, guiding chromatin engagement through a mechanism distinct from DNMT3B3. The structures show a 180° rotated 'switching helix' in DNMT3L that prevents direct interaction with the nucleosome acidic patch. Instead, nucleosome binding is mediated by the DNMT3L ADD domain, while the DNMT3A PWWP domain exhibits reduced engagement in the absence of H3K36 methylation. The oligomeric arrangement of DNMT3A2-3L in nucleosome-free states highlights its dynamic assembly and potential allosteric regulation. We further capture dynamic structural movements of DNMT3A2-3L on nucleosomes. These findings uncover a previously unknown mechanism by which DNMT3A-3L mediates de novo DNA methylation on chromatin through complex assembly, histone tail sensing, dynamic DNA search and regulated nucleosome engagement, providing insights into epigenetic regulation.

Epstein-Barr virus (EBV) infects >95% of adults and contributes to several human cancers. EBV can remain latent where viral lytic genes are silenced, precluding the use of antiviral agents such as ganciclovir. Little is known about the host factors involved in EBV latency. Here we performed a human genome-wide CRISPR-Cas9 screen in Burkitt lymphoma B cells, which identified lysine-specific histone demethylase 1 (LSD1) and its corepressors REST corepressor 1 (CoREST) and zinc finger protein 217 (ZNF217) as critical for EBV latency. Gene knockout or LSD1 inhibition triggered EBV reactivation, and the latter sensitized cells to ganciclovir cytotoxicity, including in murine tumour xenografts. Mechanistically, ZNF217 recruits LSD1 and CoREST to form a complex that binds a specific DNA motif associated with regions implicated in EBV reactivation. It removes histone 3 lysine 4 (H3K4) methylation marks and restricts host DNA looping. Alternatively, the H3K4 lysine methyltransferase 2D supports EBV lytic reactivation. Our results highlight H3K4 methylation as a major EBV lytic switch regulator and therapeutic target.

Microglial hyperactivation and NLRP3 methylation mediated by SETD3 after anesthesia/surgery: Unraveling new mechanisms of perioperative neurocognitive disorder.

H3K36me3 is a hallmark of actively and recently transcribed genes and contributes to cellular memory and identity. The deposition of H3K36me3 occurs co-transcriptionally when the methyltransferase SETD2 associates with RNA polymerase II. Here we present three cryo-EM structures of SETD2 bound to RNA polymerase II elongation complexes at different states of nucleosome passage. Together with functional probing, our results suggest a 3-step mechanism of transcription-coupled H3K36me3 deposition. First, binding to the elongation factor SPT6 tethers the catalytic SET domain in proximity to the upstream DNA. Second, RNA polymerase II nucleosome passage leads to the transfer of a hexasome from downstream to upstream, poised for methylation. Finally, continued transcription leads to upstream nucleosome reassembly, partial dissociation of the histone chaperone FACT and sequential methylation of both H3 tails, completing H3K36me3 deposition of an upstream nucleosome after RNA polymerase II passage.

Glioblastoma (GBM) is characterized by the highly infiltrative growth of cancer cells into the surrounding brain parenchyma. DnaJ Heat Shock Protein Family (Hsp40) Member C10 (DNAJC10, also known as ERDJ5and PDIA19), involved in endoplasmic reticulum-associated degradation (ERAD), has been identified as a tumor suppressor in several cancers. However, its precise role and underlying mechanism in GBM remain unclear. We found that DNAJC10 expression is downregulated in GBM patients and correlated with poor survival outcomes. Overexpression of DNAJC10 reduced GBM cell migration and invasion in vitro, while its knockdown promotes these processes. Moreover, DNAJC10 overexpression inhibits infiltrative growth of GBM cells, suppresses tumor propagation and prolongs survival in xenografted mice. Mechanistically, DNAJC10 regulates multiple molecules and pathways involved in cell motility, including the epidermal growth factor receptor (EGFR) pathway. Importantly, DNAJC10 overexpression decreases EGFR transcription by inhibiting spliced X-box binding protein 1 (XBP-1s). DNAJC10 regulates XBP-1s splicing through the inositol-requiring enzyme 1α (IRE1α) branch of the unfolded protein response (UPR). XBP-1s binds the EGFR promoter and enhances recruitment of SET7/9 methyltransferase, H3K4me3, and H3K4me1. Pharmacological inhibition of histone methylation attenuates XBP-1s-induced EGFR transcription, indicating XBP-1s promotes EGFR expression via recruiting SET7/9 for H3K4 methylation. XBP-1s overexpression reverses DNAJC10-mediated EGFR downregulation. Collectively, DNAJC10 suppresses EGFR transcription by inhibiting the UPR IRE1α-XBP-1s axis, reducing SET7/9 recruitment and H3K4 methylation at the EGFR promoter. Targeting DNAJC10 or XBP-1s could be a potential approach for inhibiting GBM infiltration and may represent a novel avenue for GBM treatment.

Mechanisms of X chromosome dosage compensation have been studied in model organisms with distinct sex chromosome ancestry. However, the diversity of mechanisms as a function of sex chromosome evolution is largely unknown. Here, we anchor ourselves to the nematode Caenorhabditis elegans, where dosage compensation is accomplished by an X chromosome specific condensin that belongs to the family of structural maintenance of chromosomes (SMC) complexes. By combining a phylogenetic analyses of the C. elegans dosage compensation complex with a comparative analysis of its epigenetic signatures, such as X-specific topologically associating domains (TADs) and enrichment of H4K20me1, we show that the condensin-mediated mechanism evolved recently in the lineage leading to Caenorhabditis following an SMC-4 duplication. Unexpectedly, we found an independent duplication of SMC-4 in Pristionchus pacificus along with X-specific TADs and H4K20me1 enrichment, which suggests that condensin-mediated dosage compensation evolved more than once in nematodes. Differential expression analysis between sexes in several nematode species indicates that dosage compensation itself precedes the evolution of X-specific condensins. In Rhabditina, X-specific condensins may have evolved in the presence of an existing mechanism linked to H4K20 methylation as Oscheius tipulae X chromosomes are enriched for H4K20me1 without SMC-4 duplication or TADs. In contrast, Steinernema hermaphroditum lacks H4K20me1 enrichment, SMC-4 duplication, and TADs. Together, our results indicate that dosage compensation mechanisms continue to evolve in species with shared X chromosome ancestry, and SMC complexes may have been co-opted at least twice in nematodes, suggesting that the process of evolving chromosome wide gene regulatory mechanisms are constrained.

Therapeutic targeting of WDR5-MLL1 by EMBOW-derived peptides suppresses leukemia progression.

The transition from vegetative growth to reproduction in flowering plants is often timed by seasonal changes in day length (photoperiod). In the long-day (LD) plant Arabidopsis thaliana, the photoperiod pathway induces a daily rhythmic activation of the florigen gene FLOWERING LOCUS T (FT) to promote the floral transition. Under inductive LDs, FT expression is activated around dusk, but to be repressed overnight and into the early afternoon the next day. Here, we report that AtING1 and AtING2, Arabidopsis homologs of the mammalian Inhibitor of Growth (ING) proteins, read di- and tri-methylated histone-3 lysine 4 (H3K4me2/me3) on FT chromatin and further recruit Polycomb-repressive complex 2 (PRC2) to repress FT expression at night and into the early afternoon the next day, following FT activation at dusk. This prevents precocious flowering under inductive LDs. Our study reveals that the H3K4me2/me3-ING1/2-PRC2 module timely represses FT expression following the daily rhythmic FT activation, to prevent excessive FT expression and thus precisely control flowering time, in response to inductive photoperiodic signals.

The KMT2 class of histone methyltransferases regulates methylation of histone 3 lysine 4 (H3K4), a conserved post-translational modification associated with active transcription. However, previous studies have highlighted catalytic-independent functions of KMT2 members and questioned the influence of H3K4 methylation on gene expression. Here, we address this by generating catalytically inactive mutants of SET-2 and SET-16, the two KMT2 members in Caenorhabditis elegans. Through chromatin analysis, we determined the effect of SET-2 and SET-16 catalytic activities on H3K4me3 deposition and identified shared and distinct targets. Gene expression profiling showed that simultaneous inactivation of SET-2 and SET-16 catalytic activities results in gene deregulation independent of H3K4me3 status at transcription start sites. Finally, we examined the relevance of SET-2 and SET-16 catalytic activity on phenotypes identified in null mutants and found that SET-2 catalytic activity is essential for proper somatic development, whereas SET-16 enzymatic activity has cell type-specific roles. Interestingly, animals lacking SET-2 and SET-16 catalytic activity are viable and fertile under normal growth conditions. Our results reveal catalytic-dependent and -independent roles of KMT2 members, and that combined loss of SET-16 and SET-2 is compatible with life in C. elegans.

Histone methylation, catalyzed by SET domain-containing lysine methyltransferases, is a conserved epigenetic mechanism regulating gene expression in eukaryotes. However, the evolutionary dynamics of SET domain proteins and their functional interplay in fungi remain poorly understood. Here, we analyzed 18,718 SET domain proteins from 1038 fungal genomes and identified three major clusters, with Cluster 1 enriched for canonical histone methyltransferases. The evolution of the SET domain protein family coordinates with genome expansion in fungi. Functional characterization of seven Cluster 1 proteins in Fusarium graminearum, a globally significant fungal pathogen, reveals diverse roles in growth, development, and virulence. In-depth analyses of two H3K36-specific methyltransferases, Set2 and Ash1, uncover their distinct regulatory functions. Set2-mediated H3K36me3 is enriched in gene bodies of euchromatic regions and facilitates transcription elongation. In contrast, Ash1-mediated H3K36me3 localizes to promoters within facultative heterochromatin and represses transcription. Notably, Ash1-mediated H3K36me3 cooperates with Polycomb repressive complex 2 (PRC2)-dependent H3K27me3 to silence secondary metabolite (SM) gene clusters. Deletion of ASH1 reduces H3K27me3 levels and derepresses SM gene expression. Conversely, Set2-mediated H3K36me3, facilitated by Ctk1-dependent RNA polymerase II phosphorylation, promotes transcriptional elongation of SM genes. Together, these findings reveal evolutionary features of fungal SET domain proteins and uncover a synergistic interplay between H3K36me3 and H3K27me3 in regulating fungal secondary metabolism and virulence. This study advances our understanding of epigenetic regulation in fungi and provides potential targets for controlling fungal pathogens.

Abstract BACKGROUND ATRX mutations define a clinically significant subset of gliomas with limited targeted treatment options. We hypothesised that genes inducing synthetic sterility with xnp-1 (ATRX orthologue) in C. elegans could uncover actionable vulnerabilities in ATRX-deficient human cancers. To explore this, we performed a cross-species screen linking developmental genetics to cancer cell viability. MATERIAL AND METHODS Synthetic sterility was quantified in xnp-1 mutant worms following RNAi knockdown. Fertility defects were assessed, and candidate genes were mapped to human orthologues. Corresponding small-molecule inhibitors—TAK-981 (SUMO pathway), UNC-0642 (H3K9 methylation), and G007-LK (tankyrase inhibition)—were evaluated in ATRX-deficient HeLa-LT and SF188 cell lines. Cell viability was measured under single-agent and combination treatments with DNA-damaging agents (doxorubicin, bleomycin). RESULTS RNAi of top hits, including gei-17 and set-25, induced significant fertility defects in xnp-1 mutants. However, pharmacological inhibition of the corresponding human pathways failed to produce selective lethality in ATRX-deficient cells, even when combined with DNA damage. Pathway modulation was confirmed (e.g., SUMO suppression by TAK-981), yet no differential sensitivity emerged. These findings suggest that synthetic sterility in C. elegans may reflect germline-specific stress responses not conserved in cancer biology. CONCLUSION Although the candidate compounds identified through C. elegans synthetic sterility screens did not produce selective cytotoxicity in ATRX-deficient human cancer cells, this outcome provides valuable insight into the translational landscape of cross-species screening. The fertility-based phenotype in xnp-1 mutants likely captures gene interactions relevant to germline development and stress responses, which may not be conserved in cancer biology. By reporting these results, we aim to contribute to a more informed and efficient use of model organisms in neuro-oncology drug discovery.

The histone crosstalk code in plants: Deciphering epigenetic complexity.

Commonly used in cancer therapy, topoisomerase II (TOP2) poisons are designed to stabilize the normally transient DNA TOP2 cleavage complexes in chromatin, leading to deleterious DNA double-strand breaks. TOP2 poisons are often associated with significant side effects, highlighting the need to identify strategies aimed at improving the efficacy of TOP2 poisons in order to lower the required dosage. Here, we demonstrated that inhibiting histone H4-lysine 20 (H4K20) methyltransferases SUV4-20H1 and SUV4-20H2 induced synthetic lethality in combination with the TOP2 poison etoposide in prostate cancer. Remarkably, the loss of the SUV4-20H enzymes, which prevents the conversion of H4K20 mono-methylation to higher methylation states, increased replication fork velocity without impacting prostate cancer cell behavior. However, these apparently innocuous epigenetic changes significantly enhanced the trapping of TOP2 complexes in chromatin and increased DNA damage in response to etoposide. Furthermore, SUV4-20H depletion and the subsequent changes in H4K20 methylation impaired the repair of TOP2-induced DNA breaks by disrupting BRCA1-mediated homologous recombination processes, ultimately leading to extensive cancer cell death and significant inhibition of prostate tumor growth in vivo. Overall, these findings demonstrate that targeting the epigenetic activity of SUV4-20H is a powerful strategy to enhance the efficacy of TOP2 poisons and may represent a therapeutic alternative in prostate cancer, where SUV4-20H2 expression emerges as a potential marker of aggressive disease and high metastatic risk.

Altered histone post-translational modifications are frequently associated with cancer. Here, we apply mass-spectrometry to study the epigenetic landscapes of breast cancer subtypes, with a particular focus on triple-negative breast cancers (TNBCs), a heterogeneous group lacking well-defined molecular targets and effective therapies. The analysis of over 200 tumors reveals epigenetic signatures that discriminate TNBCs from the other BC subtypes, and that distinguish TNBC patients with different prognoses. Employing a multi-OMICs approach integrating epigenomics, transcriptomics, and proteomics data, we investigate the mechanistic role of increased H3K4 methylation in TNBCs, demonstrating that H3K4me2 sustains the expression of genes associated with the TNBC phenotype. Through CRISPR-mediated editing, we establish a causal relationship between H3K4me2 and gene expression for several targets. Furthermore, treatment with H3K4 methyltransferase inhibitors reduce TNBC cell growth in vitro and in vivo. Collectively, our results unravel a novel epigenetic pathway implicated in TNBC pathogenesis and suggest new opportunities for targeted therapy.

The clinical application of CRISPR-Cas9 remains limited by delivery challenges, particularly for oral administration. Lysine-specific demethylase 1 (Lsd1) plays a key role in colonic inflammation and tumorigenesis. Here, we developed an oral genome-editing platform (TPGS-RNP@LNP), where Lsd1-targeting ribonucleoproteins (RNPs) were encapsulated in mulberry leaf lipid nanoparticles (LNPs) and formulated with d-α-tocopherol polyethylene glycol succinate (TPGS). TPGS reinforced the lipid bilayer of LNPs, enhanced gastrointestinal stability, and facilitated colonic mucus penetration. Upon the galactose receptor–mediated endocytosis of TPGS-RNP@LNPs by macrophages, their fusion with the endosomal membrane and the presence of nuclear localization signals ensured the nuclear delivery of RNPs. TPGS-RNP@LNPs achieved 59.7% Lsd1 editing efficiency in macrophages, surpassing the commercial CRISPRMAX (43.0%). Oral TPGS-RNP@LNPs promoted H3K4 methylation to modulate epigenetic states, achieving inflammation mitigation, epithelial barrier restoration, and retardation of colitis and its associated tumorigenesis. As an LNP-based oral RNP delivery system, TPGS-RNP@LNPs provide a promising platform for precise treatment of colorectal diseases.

ABSTRACTPancreatic neuroendocrine tumors (PNETs) are a rare and understudied set of cancers, with increasing incidence. Neuroendocrine tumors are unique in the fact that they express high levels of the somatostatin receptor type 2 (SSTR2), which represents a target for both tumor imaging and therapeutics. PNET grade inversely correlates with SSTR2 tumor staining and higher tumor grade is associated with poor patient prognosis. With no known mutations, SSTR2 expression is believed to be lost through aberrant epigenetic mechanisms. Enhanced knowledge of the epigenetic biology and players controlling SSTR2 expression may allow for identification of novel PNET imaging and treatment modalities. Through in-depth studies, we found that the specificde novoDNA methyltransferase (DNMT), DNMT3B, is responsible forSSTR2gene CpG methylation and silencing. Using DNMT3B as a starting point, along with the concept of functional crosstalk between various epigenetic mechanisms, we further discovered that Polycomb Repressor Complexes 1 and 2 (PRC1 and PRC2) play important roles in silencing SSTR2. Moreover, we found several histone lysine demethylases, enzymes that remove activating histone H3K4 methylation marks, to be critical for silencing expression of SSTR2. We additionally identified several chromatin remodeling enzymes/complexes as cellular factors that negatively regulate SSTR2 expression. Finally, using the HiBiT luminescent reporter system, we exploited functional chemo-genomic screens to further expand our knowledge of SSTR2 epigenetic control. These screens both reinforced several of our initial findings and helped to identify additional silencing mechanism potentially regulating SSTR2 expression. A commonality in our findings point to the presence, or necessity, of Class I HDACs in nearly all the epigenetic silencing mechanisms characterized. Overall, our work demonstrates thatSSTR2gene expression is likely silenced through various dynamic and interconnected epigenetic events, resulting in a compacted, transcriptionally repressed chromatin environment. Our study offers novel potential therapeutic targets and combinations to best increase expression of SSTR2, which are currently being tested in pre-clinical studies from our group, with the goal of future clinical trials aimed at increasing SSTR2 expression in high-grade, SSTR2-low NET patients.

Heterochromatin formation in Schizosaccharomyces pombe requires the spreading of histone 3 (H3) Lysine 9 (K9) methylation (me) from nucleation centers by the H3K9 methylase, Suv39/Clr4, and the reader protein, HP1/Swi6. To accomplish this, Suv39/Clr4 and HP1/Swi6 have to associate with nucleosomes both nonspecifically, binding DNA and octamer surfaces and specifically, via recognition of methylated H3K9 by their respective chromodomains. However, how both proteins avoid competition for the same nucleosomes in this process is unclear. Here, we show that phosphorylation tunes oligomerization and the nucleosome affinity of HP1/Swi6 such that it preferentially partitions onto Suv39/Clr4’s trimethyl product rather than its unmethylated substrates. Preferential partitioning enables efficient conversion from di-to trimethylation on nucleosomesin vitroand H3K9me3 spreadingin vivo. Together, our data suggests that phosphorylation of HP1/Swi6 creates a regime that increases oligomerization and relieves competition with the “read-write” mechanism of Suv39/Clr4, together promoting for productive heterochromatin spreading.

Cooperative control of neuron-specific repressive chromatin states by intellectual-disability-linked KDM1A and KDM5C demethylases.

Isocitrate dehydrogenase 1 (IDH1) mutations are key drivers of glioma biology, influencing tumor aggressiveness and treatment response. To elucidate their molecular impact, we performed proteome analysis on patient-derived (PD) and U87MG glioma cell models with either mutant or wild-type IDH1. We quantified over 6000 protein groups per model, identifying 1594 differentially expressed proteins in PD-AS (IDH1MUT) vs. PD-GB (IDH1WT) and 904 in U87MUT vs. U87WT. Both IDH1MUT models exhibited enhanced MHC antigen presentation and interferon signaling, indicative of an altered immune microenvironment. However, metabolic alterations were model-dependent: PD-AS cells shifted toward glycolysis and purine salvage, while U87MUT cells retained oxidative phosphorylation, potentially due to D2-hydroxyglutarate (2OHG)-mediated HIF1A stabilization. We also observed a predominance of downregulated DNA repair proteins in IDH1MUT models, particularly those involved in homologous recombination. In contrast, RB1 and ASMTL were strongly upregulated in both IDH1MUT models, implicating them in DNA repair and cellular stress responses. We also found distinct expression patterns of proteins regulating histone methylation in IDH1MUT cells, favoring increased methylation of H3K4, H3K9, and H3K36. A key driver of this may be the upregulation of SETD2 in PD-AS, an H3K4 and H3K36 trimethyltransferase linked to the recruitment of HIF1A as well as DNA mismatch repair proteins. This study uncovers candidate biomarkers and pathways relevant to glioma progression and therapeutic targeting, but also underscores the complexity of predicting glioma pathogenesis and treatment responses based on IDH1 mutation status. While proteome profiling provides valuable insights, a comprehensive understanding of IDH1MUT gliomas will likely require integrative multi-omics approaches, including DNA/RNA methylation profiling, histone and protein post-translational modification analyses, and targeted DNA damage and repair assays.