Methylation Of Histone H3 Lysine Research Articles

The RPD3L deacetylation complex is required for facultative heterochromatin repression in Neurospora crassa

Repression of facultative heterochromatin is essential for developmental processes in numerous organisms. Methylation of histone H3 lysine 27 (H3K27) by Polycomb repressive complex 2 is a prominent feature of facultative heterochromatin in both fungi and higher eukaryotes. Although this methylation is frequently associated with silencing, the detailed mechanism of repression remains incompletely understood. We utilized a forward genetics approach to identify genes required to maintain silencing at facultative heterochromatin genes in Neurospora crassa and identified three previously uncharacterized genes that are important for silencing: sds3 (NCU01599), rlp1 (RPD3L protein 1; NCU09007), and rlp2 (RPD3L protein 2; NCU02898). We found that SDS3, RLP1, and RLP2 associate with N. crassa homologs of the Saccharomyces cerevisiae Rpd3L complex and are required for repression of a subset of H3K27-methylated genes. Deletion of these genes does not lead to loss of H3K27 methylation but increases acetylation of histone H3 lysine 14 at up-regulated genes, suggesting that RPD3L-driven deacetylation is a factor required for silencing of facultative heterochromatin in N. crassa, and perhaps in other organisms.

H3K9 methylation regulates heterochromatin silencing through incoherent feedforward loops.

Histone H3 lysine-9 methylation (H3K9me) is a hallmark of the condensed and transcriptionally silent heterochromatin. It remains unclear how H3K9me controls transcription silencing and how cells delimit H3K9me domains to avoid silencing essential genes. Here, using Arabidopsis genetic systems that induce H3K9me2 in genes and transposons de novo, we show that H3K9me2 accumulation paradoxically also causes the deposition of the euchromatic mark H3K36me3 by a SET domain methyltransferase, ASHH3. ASHH3-induced H3K36me3 confers anti-silencing by preventing the demethylation of H3K4me1 by LDL2, which mediates transcriptional silencing downstream of H3K9me2. These results demonstrate that H3K9me2 not only facilitates but orchestrates silencing by actuating antagonistic silencing and anti-silencing pathways, providing insights into the molecular basis underlying proper partitioning of chromatin domains and the creation of metastable epigenetic variation.

Phase 1b study of EZH1/2 inhibitor valemetostat in combination with trastuzumab deruxtecan in patients with HER2 low/ultra-low/null metastatic breast cancer.

TPS1120 Background: In the DAISY study (NCT04132960), meaningful clinical response was observed with Trastuzumab deruxtecan (T-DXd) in HER2 IHC 0 MBC. Valemetostat is an oral, selective dual inhibitor of enhancer of zeste homolog 1 and 2 (EZH1/2) methyltransferases that specifically methylate histone H3 lysine 27. EZH2-mediated PP2A inactivation confers resistance to HER2-targeted therapy. Additionally, valemetostat upregulates Schlafen11 (SLFN11), a putative DNA/RNA helicase, that regulates the sensitivity to DNA damaging agents such as topoisomerase I inhibitors. Based on these preclinical studies, this study examines the safety and anti-tumor activity of valemetostat in combination with T-DXd in subjects with HER2 low/ultra-low/null MBC. Methods: This is a single-arm, phase-1b study to evaluate the safety and clinical activity of T-DXd in combination with valemetostat in patients with HER2 low/ultra-low/null MBC (NCT05633979). The dosing for T-DXd is 5.4 mg/kg Q3W administered intravenously as indicated for current clinical use by FDA approval. Valemetostat will be evaluated at three dose levels (100 mg, 150 mg, and 200 mg orally), with a starting dose (level 1) at 100 mg QD. The dose-limiting toxicity (DLT) evaluation period will be the first 2 treatment cycles (42 days). Major eligibility criteria: Pathologically confirmed HER2 low/ultra-low/null breast cancer; ECOG performance status ≤1; Measurable disease; Received at least one line of chemo in the metastatic setting; Progressed and would no longer benefit from endocrine therapy; Normal organ and marrow function. Exclusion criteria: symptomatic brain metastases, interstitial lung disease, cord compression, prior treatment with any anti-HER2 therapy. Objectives: To evaluate the safety and determine the maximum tolerated dose (MTD)/recommended dose for expansion (RDE); To evaluate the objective response rate (ORR); To determine the duration of response (DoR), clinical benefit rate (CBR), progression-free survival (PFS), and overall survival (OS); To evaluate the pharmacokinetics and pharmacodynamic markers. Statistical methods: Approximately 12 evaluable patients will be enrolled for the dose-escalation portion based on the Bayesian optimal interval design with a target DLT rate of 25%. Patients will be enrolled in cohorts of 3. The expansion will be performed at the RDE using the 2-stage Bayesian optimal dose-expansion design. In the first stage, 13 evaluable patients will be enrolled. If <5 patients respond in the first stage, the study will be stopped for futility. If ≥5 responses are observed, 13 additional evaluable patients will be enrolled. If 11 or more responses are observed among 26 patients, the treatment will be regarded as promising. This two-stage design yields 78% power under the alternative hypothesis of ORR = 50% (null ORR = 30%) while controlling the one-sided type I error at 10%. Clinical trial information: NCT05633979 .

Regulation of cytokine and chemokine expression by histone lysine methyltransferase MLL1 in rheumatoid arthritis synovial fibroblasts

Histone lysine methylation is thought to play a role in the pathogenesis of rheumatoid arthritis (RA). We previously reported aberrant expression of the gene encoding mixed-lineage leukemia 1 (MLL1), which catalyzes methylation of histone H3 lysine 4 (H3K4), in RA synovial fibroblasts (SFs). The aim of this study was to elucidate the involvement of MLL1 in the activated phenotype of RASFs. SFs were isolated from synovial tissues obtained from patients with RA or osteoarthritis (OA) during total knee joint replacement. MLL1 mRNA and protein levels were determined after stimulation with tumor necrosis factor α (TNFα). We also examined changes in trimethylation of H3K4 (H3K4me3) levels in the promoters of RA-associated genes (matrix-degrading enzymes, cytokines, and chemokines) and the mRNA levels upon small interfering RNA-mediated depletion of MLL1 in RASFs. We then determined the levels of H3K4me3 and mRNAs following treatment with the WD repeat domain 5 (WDR5)/MLL1 inhibitor MM-102. H3K4me3 levels in the gene promoters were also compared between RASFs and OASFs. After TNFα stimulation, MLL1 mRNA and protein levels were higher in RASFs than OASFs. Silencing of MLL1 significantly reduced H3K4me3 levels in the promoters of several cytokine (interleukin-6 [IL-6], IL-15) and chemokine (C–C motif chemokine ligand 2 [CCL2], CCL5, C-X-C motif chemokine ligand 9 [CXCL9], CXCL10, CXCL11, and C-X3-C motif chemokine ligand 1 [CX3CL1]) genes in RASFs. Correspondingly, the mRNA levels of these genes were significantly decreased. MM-102 significantly reduced the promoter H3K4me3 and mRNA levels of the CCL5, CXCL9, CXCL10, and CXCL11 genes in RASFs. In addition, H3K4me3 levels in the promoters of the IL-6, IL-15, CCL2, CCL5, CXCL9, CXCL10, CXCL11, and CX3CL1 genes were significantly higher in RASFs than OASFs. Our findings suggest that MLL1 regulates the expression of particular cytokines and chemokines in RASFs and is associated with the pathogenesis of RA. These results could lead to new therapies for RA.

Abstract PO3-19-10: Phase 1b study of EZH1/2 inhibitor valemetostat in combination with trastuzumab deruxtecan in subjects with HER2 low/ultra-low/null metastatic breast cancer

Abstract Background Low HER2-expressing breast cancers have traditionally been classified as HER2-negative and treated as TNBC or hormone receptor (HR)-positive. Trastuzumab deruxtecan (T-DXd), an antibody-drug conjugate, composed of an anti-HER2 antibody conjugated to topoisomerase I payload, is approved for clinical use in HER2-low (IHC 1+ or IHC 2+/ISH negative) metastatic breast cancer (MBC). In the DAISY study (NCT04132960), meaningful clinical response was observed with T-DXd in HER2 IHC 0 MBC. Valemetostat is an oral, selective dual inhibitor of enhancer of zeste homolog 1 and 2 (EZH1/2, methyltransferases that specifically methylate histone H3 lysine 27. It is currently approved for patients with relapsed or refractory adult T-cell leukemia/lymphoma in Japan. EZH2-mediated PP2A inactivation has been shown to confer resistance to HER2-targeted therapy. Additionally, valemetostat has been shown to upregulate Schlafen11 (SLFN11), a putative DNA/RNA helicase, that regulates the sensitivity to DNA damaging agents such as topoisomerase I inhibitors. Consequently, this study examines the safety and anti-tumor activity of valemetostat in combination with T-DXd in subjects with HER2 low/ultra-low/null MBC. Trial Design This is a single-arm, phase-1b study to evaluate the safety and clinical activity of T-DXd in combination with valemetostat in patients with HER2 low/ultra-low/null MBC. The dosing for T-DXd is 5.4 mg/kg Q3W administered intravenously as indicated for current clinical use. Valemetostat will be evaluated at three dose levels (100 mg, 150 mg, and 200 mg orally), with starting dose (level 1) at 100 mg QD. The dose-limiting toxicity (DLT) evaluation period will be the first 2 treatment cycles (42 days). Eligibility criteria Specific aims Statistical methods Approximately 12 evaluable patients will be enrolled for the dose-escalation portion based on the Bayesian optimal interval design with a target DLT rate of 25%. Patients enrolled in cohorts of 3. The expansion will be performed at the RDE using the 2-stage Bayesian optimal dose-expansion design. In the first stage, 13 evaluable patients (including those treated at the RDE in the dose-escalation part) will be enrolled. If < 5 patients respond in the first stage, the study will be stopped for futility. If ≥ 5 responses are observed, 13 additional evaluable patients will be enrolled. If 11 or more responses are observed among the total of 26 patients the treatment will be regarded as promising. This two-stage design yields 78% power under the alternative hypothesis of ORR=50% (null ORR = 30%) while controlling the one-sided type I error at 10%. Contact information for people with a specific interest in the trial sdamodaran@mdanderson.org (NCT05633979) Citation Format: Senthil Damodaran, Toshiaki Iwase, Angela Marx, Jie Willey, Funda Meric-Bernstam, Debu Tripathy, Carlos Barcenas, Jangsoon lee, Naoto Ueno. Phase 1b study of EZH1/2 inhibitor valemetostat in combination with trastuzumab deruxtecan in subjects with HER2 low/ultra-low/null metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-19-10.

A zinc-finger protein Moc3 functions as a transcription activator to promote RNAi-dependent constitutive heterochromatin establishment in fission yeast.

In fission yeast, Schizosaccharomyces pombe, constitutive heterochromatin defined by methylation of histone H3 lysine 9 (H3K9me) and its binding protein Swi6/HP1 localizes at the telomere, centromere, and mating-type loci. These loci contain DNA sequences called dg and dh, and the RNA interference (RNAi)-dependent system establishes and maintains heterochromatin at dg/dh. Bi-directional transcription at dg/dh induced by RNA polymerase II is critical in RNAi-dependent heterochromatin formation because the transcribed RNAs provide substrates for siRNA synthesis and a platform for assembling RNAi factors. However, a regulator of dg/dh transcription during the establishment of heterochromatin is not known. Here, we found that a zinc-finger protein Moc3 localizes dh and activates dh-forward transcription in its zinc-finger-dependent manner when heterochromatin structure or heterochromatin-dependent silencing is compromised. However, Moc3 does not localize at normal heterochromatin and does not activate the dh-forward transcription. Notably, the loss of Moc3 caused a retarded heterochromatin establishment, showing that Moc3-dependent dh-forward transcription is critical for RNAi-dependent heterochromatin establishment. Therefore, Moc3 is a transcriptional activator that induces RNAi to establish heterochromatin.

Histone Lysine Demethylase KDM5 Inhibitor CPI-455 Induces Astrocytogenesis in Neural Stem Cells.

Lysine-specific histone demethylase 5A (KDM5A) is known to facilitate proliferation in cancer cells and maintain stemness to repress the astrocytic differentiation of neural stem cells (NSCs). In the study presented here, we investigated the effect of a KDM5 inhibitor, CPI-455, on NSC fate control. CPI-455 induced astrocytogenesis in NSCs during differentiation. Kdm5a, but not Kdm5c, knockdown induced glial fibrillary acidic protein (Gfap) transcription. CPI-455 induced signal transducer and activator of transcription 3, increased bone morphogenetic protein 2 expression, and enhanced mothers against decapentaplegic homolog 1/5/9 phosphorylation. The treatment of CPI-455 enhanced the methylation of histone H3 lysine 4 in the Gfap promoter when compared to that of the dimethyl sulfoxide control. In addition, CPI-455 treatment significantly reduced the recruitment of KDM5A to the Gfap promoter. Our data suggest that the KDM5 inhibitor CPI-455 effectively controls NSC cell fate via KDM5A inhibition and induces astrocytogenesis.

The Association between Long-Term DDT or DDE Exposures and an Altered Sperm Epigenome-a Cross-Sectional Study of Greenlandic Inuit and South African VhaVenda Men.

The organochlorine dichlorodiphenyltrichloroethane (DDT) is banned worldwide owing to its negative health effects. It is exceptionally used as an insecticide for malaria control. Exposure occurs in regions where DDT is applied, as well as in the Arctic, where its endocrine disrupting metabolite, -dichlorodiphenyldichloroethylene (-DDE) accumulates in marine mammals and fish. DDT and -DDE exposures are linked to birth defects, infertility, cancer, and neurodevelopmental delays. Of particular concern is the potential of DDT use to impact the health of generations to come via the heritable sperm epigenome. The objective of this study was to assess the sperm epigenome in relation to -DDE serum levels between geographically diverse populations. In the Limpopo Province of South Africa, we recruited 247 VhaVenda South African men and selected 50 paired blood serum and semen samples, and 47 Greenlandic Inuit blood and semen paired samples were selected from a total of 193 samples from the biobank of the INUENDO cohort, an EU Fifth Framework Programme Research and Development project. Sample selection was based on obtaining a range of -DDE serum levels (). We assessed the sperm epigenome in relation to serum -DDE levels using MethylC-Capture-sequencing (MCC-seq) and chromatin immunoprecipitation followed by sequencing (ChIP-seq). We identified genomic regions with altered DNA methylation (DNAme) and differential enrichment of histone H3 lysine 4 trimethylation (H3K4me3) in sperm. Differences in DNAme and H3K4me3 enrichment were identified at transposable elements and regulatory regions involved in fertility, disease, development, and neurofunction. A subset of regions with sperm DNAme and H3K4me3 that differed between exposure groups was predicted to persist in the preimplantation embryo and to be associated with embryonic gene expression. These findings suggest that DDT and -DDE exposure impacts the sperm epigenome in a dose-response-like manner and may negatively impact the health of future generations through epigenetic mechanisms. Confounding factors, such as other environmental exposures, genetic diversity, and selection bias, cannot be ruled out. https://doi.org/10.1289/EHP12013.

Sequential deregulation of histone marks, chromatin accessibility and gene expression in response to PROTAC-induced degradation of ASH2L

The trithorax protein ASH2L is essential for organismal and tissue development. As a subunit of COMPASS/KMT2 complexes, ASH2L is necessary for methylation of histone H3 lysine 4 (H3K4). Mono- and tri-methylation at this site mark active enhancers and promoters, respectively, although the functional relevance of H3K4 methylation is only partially understood. ASH2L has a long half-life, which results in a slow decrease upon knockout. This has made it difficult to define direct consequences. To overcome this limitation, we employed a PROTAC system to rapidly degrade ASH2L and address direct effects. ASH2L loss resulted in inhibition of proliferation of mouse embryo fibroblasts. Shortly after ASH2L degradation H3K4me3 decreased with its half-life varying between promoters. Subsequently, H3K4me1 increased at promoters and decreased at some enhancers. H3K27ac and H3K27me3, histone marks closely linked to H3K4 methylation, were affected with considerable delay. In parallel, chromatin compaction increased at promoters. Of note, nascent gene transcription was not affected early but overall RNA expression was deregulated late after ASH2L loss. Together, these findings suggest that downstream effects are ordered but relatively slow, despite the rapid loss of ASH2L and inactivation of KMT2 complexes. It appears that the systems that control gene transcription are well buffered and strong effects are only beginning to unfold after considerable delay.

Conformationally constrained potent inhibitors for enhancer of zeste homolog 2 (EZH2)

The enhancer of zeste homolog 2 (EZH2) plays the role of the main catalytic subunit of polycomb repressive complex 2 (PRC2) that catalyzes the methylation of histone H3 lysine 27 (H3K27). Overexpression or mutation of EZH2 has been observed in many types of hematologic malignancies and solid tumors, such as myeloma, lymphoma, prostate, breast, kidney, and lung cancers. EZH2 has been demonstrated as a promising therapeutic target for the treatment of tumors. Based on the structure of 1 (EPZ-6438), a series of novel conformationally constrained derivatives were designed and synthesized aiming to improve the EZH2 inhibition activity, especially for mutated EZH2. Structure and activity relationship (SAR) exploration and optimization at both enzymatic and cellular levels led to the discovery of 28. In vitro, 28 displayed potent EZH2 inhibition activity with an IC50 value of 0.95 nM, which is comparable to EPZ-6438 (1). 28 exhibited high anti-proliferation activity against different lymphoma cell lines including WSU-DLCL2, Pfeiffer and Karpas-422 (IC50 = 2.36, 1.73, and 1.82 nM, respectively). In vivo, 28 showed acceptable pharmacokinetic characteristics (oral bioavailability F = 36.9%) and better efficacy than 1 in both Pfeiffer and Karpas-422 xenograft mouse models, suggesting that it can be further developed as a potential therapeutic candidate for EZH2-associated cancers.

Cotranscriptional demethylation induces global loss of H3K4me2 from active genes in Arabidopsis.

Based on studies of animals and yeasts, methylation of histone H3 lysine 4 (H3K4me1/2/3, for mono-, di-, and tri-methylation, respectively) is regarded as the key epigenetic modification of transcriptionally active genes. In plants, however, H3K4me2 correlates negatively with transcription, and the regulatory mechanisms of this counterintuitive H3K4me2 distribution in plants remain largely unexplored. A previous genetic screen for factors regulating plant regeneration identified Arabidopsis LYSINE-SPECIFIC DEMETHYLASE 1-LIKE 3 (LDL3), which is a major H3K4me2 demethylase. Here, weshow that LDL3-mediated H3K4me2 demethylation depends onthe transcription elongation factor Paf1C and phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII). In addition, LDL3 binds to phosphorylated RNAPII. These results suggest that LDL3 is recruited to transcribed genes by binding to elongating RNAPII and demethylates H3K4me2 cotranscriptionally. Importantly, the negative correlation between H3K4me2 and transcription is significantly attenuated in the ldl3 mutant, demonstrating the genome-wide impacts of the transcription-driven LDL3 pathway to control H3K4me2 in plants. Our findingsimplicate H3K4me2 demethylation in plants as chromatin records of transcriptional activity, which ensures robust gene control.

ASH2L, a COMPASS core subunit, is involved in the cell invasion and migration of triple-negative breast cancer cells through the epigenetic control of histone H3 lysine 4 methylation

ASH2L (Absent-Small-Homeotic-2-Like protein) is a core subunit of the COMPASS (COMplex of Proteins ASsociated with Set1) complex, the most notable writer of the methylation of histone H3 lysine 4 (H3K4). The COMPASS complex regulates active promoters or enhancers for gene expression, and its dysfunction is associated with aberrant development and disease. Here, we demonstrated that ASH2L mediated the cell invasion and migration activity of triple-negative breast cancer cells through the interaction with the COMPASS components and the target genomic regions. Transcriptome analysis indicated a potential correlation between ASH2L and the genes involved in inflammatory/immune responses. Among them, we found that the intrinsic expression of IL1B (interleukin 1 beta), an essential proinflammatory gene, was directly regulated by ASH2L. These results revealed a novel role of ASH2L on the maintenance of breast cancer malignancy possibly through H3K4 methylation of the target inflammatory/immune responsive genes.

DOT1L promotes spermatid differentiation by regulating expression of genes required for histone-to-protamine replacement.

Unique chromatin remodeling factors orchestrate dramatic changes in nuclear morphology during differentiation of the mature sperm head. A crucial step in this process is histone-to-protamine exchange, which must be executed correctly to avoid sperm DNA damage, embryonic lethality and male sterility. Here, we define an essential role for the histone methyltransferase DOT1L in the histone-to-protamine transition. We show that DOT1L is abundantly expressed in mouse meiotic and postmeiotic germ cells, and that methylation of histone H3 lysine 79 (H3K79), the modification catalyzed by DOT1L, is enriched in developing spermatids in the initial stages of histone replacement. Elongating spermatids lacking DOT1L fail to fully replace histones and exhibit aberrant protamine recruitment, resulting in deformed sperm heads and male sterility. Loss of DOT1L results in transcriptional dysregulation coinciding with the onset of histone replacement and affecting genes required for histone-to-protamine exchange. DOT1L also deposits H3K79me2 and promotes accumulation of elongating RNA Polymerase II at the testis-specific bromodomain gene Brdt. Together, our results indicate that DOT1L is an important mediator of transcription during spermatid differentiation and an indispensable regulator of male fertility.

Abstract 3567: Cancer-epigenetic mouse models for medulloblastoma

Abstract Medulloblastoma (MB) is the most common childhood brain tumors (up to 20% of all childhood brain tumors) and occurs mainly in children (<15 years old. MB is located in the cerebellum in the brain and can spread to the spine or to other parts of brain. Because MB growth is very fast, MB is categorized as a type of high-grade malignant primary tumors. MB development is often associated with the dysregulation of signaling pathways, such as the wingless (WNT) and sonic hedgehog (SHH). Methylated histone H3 lysine 4 (H3K4) is a major methylation mark. Trimethyl H3K4 (H3K4me3) occupies as much as 75% of all human gene promoters. We previously reported that the H3K4 methyltransferase KMT2D (also called MLL4, MLL2, and ALR; a transcriptional coactivator, is required for neuronal differentiation of human neuron-lineage NT2/D1 stem cells. Interestingly, our analysis of several databases has shown that Kmt2d is the most frequently mutated epigenetic modifier (8%‒10%) in medulloblastoma (MB). Recently, we showed that brain-specific knockout of Kmt2d alone induces spontaneous MB in mice. Kmt2d loss highly upregulated several oncogenic signaling programs and downregulated tumor suppressive genes. In this study, we sought to determine whether Kmt2d loss cooperates with another oncogenic event in MB genesis. Particularly, PTCH (also known as PTCH1) is one of the most frequently mutated genes in MB, and KMT2D is known to be a shared driver gene mutation in MB. For these reasons, we examined the effect of Kmt2d loss on of Ptch+/−-driven MB genesis. A heterozygous loss of Kmt2d increased the incidence and progression of Ptch+/−-driven MB genesis. We have shown that Kmt2d loss reduces epigenomic signals for super-enhancers/enhancers and broad H3K4me3 in 4-month-old cerebella. In addition, we determine the effect of heterozygous Kmt2d loss on cell proliferation by comparing the Nes-Cre Kmt2dfl/+ Ptch+/- (KP) cerebella to Ptch+/- cerebella at 2- & 4-month-old time points using IHC staining of Ki-67 (a proliferation marker). MB’s from KP mice showed two different mechanisms by activating oncogenic cellular signaling kinases (e.g., phospho [p]-AKT and p-p38) levels as well as MYCN signaling pathway. Our transcriptomic analysis revealed several tumor suppressive and transcriptional corepressor genes to be downregulated such as Ncor2 and Dnmt3a. We found Ncor2 depletion in Ptch-/+ neurospheres shows upregulation of similar oncogenic genes as in KP mice tumors. Kmt2d loss reduced cerebellar granule cell differentiation. Our analysis for oncogenic kinases may provide a basis for a novel MB treatment plan involving the use of cellular kinase inhibitors. As KMT2D is among the most often mutated genes in human MBs, this mouse model may be useful for future preclinical therapeutic experiments for the treatment of MB patients. KMT2D in medulloblastoma genesis using mouse models will provide epigenetic and mechanistic insights into how heterozygous KMT2D loss promotes medulloblastoma. Citation Format: Shilpa S. Dhar, Ali S. Rizvi, Lauren Reed, Calena Brown, Min Gyu Lee. Cancer-epigenetic mouse models for medulloblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3567.

Regulation of the SUV39H Family Methyltransferases: Insights from Fission Yeast.

Histones, which make up nucleosomes, undergo various post-translational modifications, such as acetylation, methylation, phosphorylation, and ubiquitylation. In particular, histone methylation serves different cellular functions depending on the location of the amino acid residue undergoing modification, and is tightly regulated by the antagonistic action of histone methyltransferases and demethylases. The SUV39H family of histone methyltransferases (HMTases) are evolutionarily conserved from fission yeast to humans and play an important role in the formation of higher-order chromatin structures called heterochromatin. The SUV39H family HMTases catalyzes the methylation of histone H3 lysine 9 (H3K9), and this modification serves as a binding site for heterochromatin protein 1 (HP1) to form a higher-order chromatin structure. While the regulatory mechanism of this family of enzymes has been extensively studied in various model organisms, Clr4, a fission yeast homologue, has made an important contribution. In this review, we focus on the regulatory mechanisms of the SUV39H family of proteins, in particular, the molecular mechanisms revealed by the studies of the fission yeast Clr4, and discuss their generality in comparison to other HMTases.

Methylation of histone H3 lysine 4 is required for maintenance of beta cell function in adult mice

Aims/hypothesisBeta cells control glucose homeostasis via regulated production and secretion of insulin. This function arises from a highly specialised gene expression programme that is established during development and then sustained, with limited flexibility, in terminally differentiated cells. Dysregulation of this programme is seen in type 2 diabetes but mechanisms that preserve gene expression or underlie its dysregulation in mature cells are not well resolved. This study investigated whether methylation of histone H3 lysine 4 (H3K4), a marker of gene promoters with unresolved functional importance, is necessary for the maintenance of mature beta cell function.MethodsBeta cell function, gene expression and chromatin modifications were analysed in conditional Dpy30 knockout mice, in which H3K4 methyltransferase activity is impaired, and in a mouse model of diabetes.ResultsH3K4 methylation maintains expression of genes that are important for insulin biosynthesis and glucose responsiveness. Deficient methylation of H3K4 leads to a less active and more repressed epigenome profile that locally correlates with gene expression deficits but does not globally reduce gene expression. Instead, developmentally regulated genes and genes in weakly active or suppressed states particularly rely on H3K4 methylation. We further show that H3K4 trimethylation (H3K4me3) is reorganised in islets from the Leprdb/db mouse model of diabetes in favour of weakly active and disallowed genes at the expense of terminal beta cell markers with broad H3K4me3 peaks.Conclusions/interpretationSustained methylation of H3K4 is critical for the maintenance of beta cell function. Redistribution of H3K4me3 is linked to gene expression changes that are implicated in diabetes pathology.Graphical abstract

Histone Demethylase KDM3 (JMJD1) in Transcriptional Regulation and Cancer Progression.

Methylation of histone H3 lysine 9 (H3K9) is a repressive histone mark and associated with inhibition of gene expression. KDM3 is a subfamily of the JmjC histone demethylases. It specifically removes the mono- or di-methyl marks from H3K9 and thus contributes to activation of gene expression. KDM3 subfamily includes three members: KDM3A, KDM3B and KDM3C. As KDM3A (also known as JMJD1A or JHDM2A) is the best studied, this chapter will mainly focus on the role of KDM3A-mediated gene regulation in the biology of normal and cancer cells. Knockout mouse studies have revealed that KDM3A plays a role in the physiological processes such as spermatogenesis, metabolism and sex determination. KDM3A is upregulated in several types of cancers and has been shown to promote cancer development, progression and metastasis. KDM3A can enhance the expression or activity of transcription factors through its histone demethylase activity, thereby altering the transcriptional program and promoting cancer cell proliferation and survival. We conclude that KDM3A may serve as a promising target for anti-cancer therapies.

Polycomb Repressive Complex 2 in Oncology.

Dynamic regulation of the chromatin state by Polycomb Repressive Complex 2 (PRC2) provides an important mean for epigenetic gene control that can profoundly influence normal development and cell lineage specification. PRC2 and PRC2-induced methylation of histone H3 lysine 27 (H3K27) are critically involved in a wide range of DNA-templated processes, which at least include transcriptional repression and gene imprinting, organization of three-dimensional chromatin structure, DNA replication and DNA damage response and repair. PRC2-based genome regulation often goes wrong in diseases, notably cancer. This chapter discusses about different modes-of-action through which PRC2 and EZH2, a catalytic subunit of PRC2, mediate (epi)genomic and transcriptomic regulation. We will also discuss about how alteration or mutation of the PRC2 core or axillary component promotes oncogenesis, how post-translational modification regulates functionality of EZH2 and PRC2, and how PRC2 and other epigenetic pathways crosstalk. Lastly, we will briefly touch on advances in targeting EZH2 and PRC2 dependence as cancer therapeutics.

De novo methylation of histone H3K23 by the methyltransferases EHMT1/GLP and EHMT2/G9a

Epigenetic modifications to histone proteins serve an important role in regulating permissive and repressive chromatin states, but despite the identification of many histone PTMs and their perceived role, the epigenetic writers responsible for generating these chromatin signatures are not fully characterized. Here, we report that the canonical histone H3K9 methyltransferases EHMT1/GLP and EHMT2/G9a are capable of catalyzing methylation of histone H3 lysine 23 (H3K23). Our data show that while both enzymes can mono- and di-methylate H3K23, only EHMT1/GLP can tri-methylate H3K23. We also show that pharmacologic inhibition or genetic ablation of EHMT1/GLP and/or EHMT2/G9a leads to decreased H3K23 methylation in mammalian cells. Taken together, this work identifies H3K23 as a new direct methylation target of EHMT1/GLP and EHMT2/G9a, and highlights the differential activity of these enzymes on H3K23 as a substrate.

Neuroinflammation regulates the balance between hippocampal neuron death and neurogenesis in an ex vivo model of thiamine deficiency

BackgroundThiamine (vitamin B1) is a cofactor for enzymes of central energy metabolism and its deficiency (TD) impairs oxidative phosphorylation, increases oxidative stress, and activates inflammatory processes that can lead to neurodegeneration. Wernicke–Korsakoff syndrome (WKS) is a consequence of chronic TD, which leads to extensive neuronal death, and is associated with neuropathological disorders, including cognitive deficits and amnesia. The hippocampus is one of the brain areas most affected by WKS. B1 replacement may not be enough to prevent the irreversible cognitive deficit associated with WKS.Materials and methodsAn organotypic hippocampal slice culture (OHC) model was developed to investigate, using immunofluorescence and confocal microscopy and transcriptome analysis, the molecular mechanisms underlying the neurodegeneration associated with TD. The effect of anti-inflammatory pharmacological intervention with resveratrol (RSV) was also assessed in B1-deprived OHCs.ResultsIn OHCs cultured without B1, neuronal density decayed after 5 days and, on the 7th day, the epigenetic markings H3K4me3 and H3K9me3 were altered in mature neurons likely favoring gene transcription. Between the 7th and the 14th day, a pulse of neurogenesis was observed followed by a further massive neuron loss. Transcriptome analysis at day nine disclosed 89 differentially expressed genes in response to B1 deprivation. Genes involved in tryptophan metabolism and lysine degradation KEGG pathways, and those with Gene Ontology (GO) annotations related to the organization of the extracellular matrix, cell adhesion, and positive regulation of synaptic transmission were upregulated. Several genes of the TNF and FoxO signaling pathways and with GO terms related to inflammation were inhibited in response to B1 deprivation. Nsd1, whose product methylates histone H3 lysine 36, was upregulated and the epigenetic marking H3K36me3, associated with negative regulation of neurogenesis, was increased in neurons. Treating B1-deprived OHCs with RSV promoted an earlier neurogenesis pulse.ConclusionNeuroregeneration occurs in B1-deficient hippocampal tissue during a time window. This phenomenon depends on reducing neuroinflammation and, likely, on metabolic changes, allowing acetyl-CoA synthesis from amino acids to ensure energy supply via oxidative phosphorylation. Thus, neuroinflammation is implicated as a major regulator of hippocampal neurogenesis in TD opening a new search space for treating WKS.