Holocarboxylase synthetase (HCS) catalyzes the binding of the vitamin biotin to histones H3 and H4, thereby creating rare histone biotinylation marks in the epigenome. These marks co-localize with K9-methylated histone H3 (H3K9me), an abundant gene repression mark. The abundance of H3K9me marks in transcriptionally competent loci decreases when HCS is knocked down and when cells are depleted of biotin. Here we tested the hypothesis that the creation of H3K9me marks is at least partially explained by physical interactions between HCS and histone-lysine N-methyltransferases. Using a novel in silico protocol, we predicted that HCS-interacting proteins contain a GGGG(K/R)G(I/M)R motif. This motif, with minor variations, is present in the histone-lysine N-methyltransferase EHMT1. Physical interactions between HCS and the N-terminal, ankyrin and SET domains in EHMT1 were confirmed using yeast-two-hybrid assays, limited proteolysis assays and co-immunoprecipitation. The interactions were stronger between HCS and the N-terminus in EHMT1 compared with the ankyrin and SET domains, consistent with the localization of the HCS-binding motif in the EHMT1 N-terminus. HCS has the catalytic activity to biotinylate K161 within the binding motif in EHMT1. Mutation of K161 weakened the physical interaction between EHMT1 and HCS, but it is unknown whether this effect was caused by loss of biotinylation or loss of the motif. Importantly, HCS knockdown decreased the abundance of H3K9me marks in repeats, suggesting that HCS plays a role in creating histone methylation marks in these loci. We conclude that physical interactions between HCS and EHMT1 mediate epigenomic synergies between biotinylation and methylation events.

There is an urgent need for the identification of new, clinically useful biomarkers of CRC to enhance diagnostic and prognostic capabilities.

Hepatocellular carcinoma (HCC) is the third deadliest and sixth most common cancer in the world. Histone-lysine N-methyltransferase EHMT2 (also known as G9a) is a histone methyltransferase frequently overexpressed in many cancer types, including HCC. We showed that Myc-driven liver tumours have a unique H3K9 methylation pattern with corresponding G9a overexpression. This phenomenon of increased G9a was further observed in our c-Myc-positive HCC patient-derived xenografts. More importantly, we showed that HCC patients with higher c-Myc and G9a expression levels portend a poorer survival with lower median survival months. We demonstrated that c-Myc interacts with G9a in HCC and cooperates to regulate c-Myc-dependent gene repression. In addition, G9a stabilises c-Myc to promote cancer development, contributing to the growth and invasive capacity in HCC. Furthermore, combination therapy between G9a and synthetic-lethal target of c-Myc, CDK9, demonstrates strong efficacy in patient-derived avatars of Myc-driven HCC. Our work suggests that targeting G9a could prove to be a potential therapeutic avenue for Myc-driven liver cancer. This will increase our understanding of the underlying epigenetic mechanisms of aggressive tumour initiation and lead to improved therapeutic and diagnostic options for Myc-driven hepatic tumours.

Euchromatic histone-lysine N-methyltransferase 1 (EHMT1; G9a-like protein; GLP) and euchromatic histone-lysine N-methyltransferase 2 (EHMT2; G9a) are protein lysine methyltransferases that regulate gene expression and are essential for development and the ability of organisms to change and adapt. In addition to ankyrin repeats and the catalytic SET domain, the EHMT proteins contain a unique cysteine-rich region (CRR) that mediates protein-protein interactions and recruitment of the methyltransferases to specific sites in chromatin. We have determined the structure of the CRR from human EHMT2 by X-ray crystallography and show that the CRR adopts an unusual compact fold with four bound zinc atoms. The structure consists of a RING domain preceded by a smaller zinc-binding motif and an N-terminal segment. The smaller zinc-binding motif straddles the N-terminal end of the RING domain, and the N-terminal segment runs in an extended conformation along one side of the structure and interacts with both the smaller zinc-binding motif and the RING domain. The interface between the N-terminal segment and the RING domain includes one of the zinc atoms. The RING domain is partially sequestered within the CRR and unlikely to function as a ubiquitin ligase.

MLL5, a member of the histone-lysine N-methyltransferase family, has been implicated in the control of the cell cycle progression and survival. The aim of this study was to explore the relationship between the interaction of histone-lysine N-methyltransferase MLL5 gene polymorphism and CAD in a Chinese Han population.

Histone-lysine N-methyltransferase EZH2 (EZH2) is the principle component of the polycomb repressive complex 2 (PRC2)/embryonic ectoderm development protein-EZH2 complex, which promotes tumorigenesis by repressing transcription of tumor suppressor genes. EZH2 is considered a key marker in several types of cancer, such as colorectal and prostate cancer. However, the molecular mechanisms and clinical value of EZH2 in lung cancer have not yet been fully investigated. The aim of the present study was to investigate the functions of EZH2 in lung cancer progression and to determine whether treatment with an EZH2 inhibitor enhanced the chemosensitivity of lung cancer cells to cisplatin (CDDP). At the logarithmic growth phase, A549 cells were treated with a small interfering (si)RNA-EZH2, and cell viability was detected using an MTT assay. The degree of apoptosis and cell cycle were detected using flow cytometry. Cell migration and invasion were detected via wound healing and Transwell Matrigel assays. According to information from the Gene Expression Omnibus database, the results of the present study demonstrated that EZH2 was upregulated in lung cancer. Furthermore, overexpression of EZH2 was associated with poor patient prognosis, while EZH2 knockdown inhibited cell viability and migration, and enhanced apoptosis and chemosensitivity in a lung cancer cell line. EZH2 knockdown and treatment of A549 cells using EZH2 inhibitor elevated the inhibitory effects of CDDP on cell viability and apoptosis. Western blot and reverse transcription-quantitative PCR analyses were performed to assess the expression levels of relative protein and mRNA, respectively, in A549 cells treated with siRNA-EZH2 or with CDDP. Overall, the results of the present study demonstrated that high EZH2 expression was associated with poor prognosis, accompanied with a potential impairment of migration and viability in lung cancer cells. These findings suggest that EZH2 may act as a candidate molecular target for gene therapy, and treatment with EZH2 inhibitor may be used to increase chemosensitivity to CDDP agents in lung cancer.

Congenital heart defects are the most common birth defect and have a clear genetic component, yet genomic structural variations or gene mutations account for only a third of the cases. Epigenomic dynamics during human heart organogenesis thus may play a critical role in regulating heart development. However, it is unclear how histone mark H3K36me3 acts on heart development. Here we report that histone-lysine N-methyltransferase SETD2, an H3K36me3 methyltransferase, is a crucial regulator of the mouse heart epigenome. Setd2 is highly expressed in embryonic stages and accounts for a predominate role of H3K36me3 in the heart. Loss of Setd2 in cardiac progenitors results in obvious coronary vascular defects and ventricular non-compaction, leading to fetus lethality in mid-gestation, without affecting peripheral blood vessel, yolk sac, and placenta formation. Furthermore, deletion of Setd2 dramatically decreased H3K36me3 level and impacted the transcriptional landscape of key cardiac-related genes, including Rspo3 and Flrt2. Taken together, our results strongly suggest that SETD2 plays a primary role in H3K36me3 and is critical for coronary vascular formation and heart development in mice.

Histone lysine methyltransferases (HMT) comprise a subclass of epigenetic regulators; dysregulation of these enzymes affects gene expression, which may lead to tumorigenesis. Here, we performed an integrated analysis of 50 HMTs in bladder cancer and found intrinsic links between copy number alterations, mutations, gene expression levels, and clinical outcomes. Through integrative analysis, we identified six HMT genes (PRDM9,ASH1L,SETD3,SETD5,WHSC1L1, and KMT2D) that may play a key role in the development and progression of bladder cancer. Of these six HMTs, histone lysine N-methyltransferase 2D (KMT2D) exhibited the highest mutation rate in bladder cancer. Our comparison of the mRNA and miRNA expression profiles of mutated and wild-type KMT2D suggested that two signaling pathways (FOX1-miR-1224-5p-DLK1 and HIF/GATA5-miR-133a-3p-DRD5) may mediate the tumor suppressive effect of the KMT2D mutation. In summary, our findings indicate that mutations in HMT genes, especially KMT2D mutation, may play a role in the development of bladder cancer.

Proteasome inhibitors (PIs) are extensively used for the therapy of multiple myeloma. However, patients continuously relapse or are intrinsically resistant to this class of drugs. In addition, adverse toxic effects such as peripheral neuropathy and cardiotoxicity could arise. Here, to identify compounds that can increase the efficacy of PIs, we performed a functional screening using a library of small-molecule inhibitors covering key signaling pathways. Among the best synthetic lethal interactions, the euchromatic histone-lysine N-methyltransferase 2 (EHMT2) inhibitor UNC0642 displayed a cooperative effect with carfilzomib (CFZ) in numerous multiple myeloma (MM) cell lines, including drug-resistant models. In MM patients, EHMT2 expression correlated to worse overall and progression-free survival. Moreover, EHMT2 levels were significantly increased in bortezomib-resistant patients. We demonstrated that CFZ/UNC0642 combination exhibited a favorable cytotoxicity profile toward peripheral blood mononuclear cells and bone-marrow-derived stromal cells. To exclude off-target effects, we proved that UNC0642 treatment reduces EHMT2-related molecular markers and that an alternative EHMT2 inhibitor recapitulated the synergistic activity with CFZ. Finally, we showed that the combinatorial treatment significantly perturbs autophagy and the DNA damage repair pathways, suggesting a multi-layered mechanism of action. Overall, the present study demonstrates that EHMT2 inhibition could provide a valuable strategy to enhance PI sensitivity and overcome drug resistance in MM patients.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, painful disease with a 5-year survival rate of only 9%. Recent evidence indicates that distinct epigenomic landscapes underlie PDAC progression, identifying the H3K9me pathway as important to its pathobiology. Here, we delineate the role of Euchromatic Histone-lysine N-Methyltransferase 2 (EHMT2), the enzyme that generates H3K9me, as a downstream effector of oncogenic KRAS during PDAC initiation and pancreatitis-associated promotion. EHMT2 inactivation in pancreatic cells reduces H3K9me2 and antagonizes Kras G12D -mediated acinar-to-ductal metaplasia (ADM) and Pancreatic Intraepithelial Neoplasia (PanIN) formation in both the Pdx1-Cre and P48 Cre/+ Kras G12D mouse models. Ex vivo acinar explants also show impaired EGFR-KRAS-MAPK pathway-mediated ADM upon EHMT2 deletion. Notably, Kras G12D increases EHMT2 protein levels and EHMT2-EHMT1-WIZ complex formation. Transcriptome analysis reveals that EHMT2 inactivation upregulates a cell cycle inhibitory gene expression network that converges on the Cdkn1a/p21-Chek2 pathway. Congruently, pancreas tissue from Kras G12D animals with EHMT2 inactivation have increased P21 protein levels and enhanced senescence. Furthermore, loss of EHMT2 reduces inflammatory cell infiltration typically induced during Kras G12D -mediated initiation. The inhibitory effect on Kras G12D -induced growth is maintained in the pancreatitis-accelerated model, while simultaneously modifying immunoregulatory gene networks that also contribute to carcinogenesis. This study outlines the existence of a novel KRAS-EHMT2 pathway that is critical for mediating the growth-promoting and immunoregulatory effects of this oncogene in vivo, extending human observations to support a pathophysiological role for the H3K9me pathway in PDAC.

Euchromatic histone-lysine N-methyltransferase 2 (G9a/Ehmt2) is the main enzyme responsible for the apposition of H3K9 di-methylation on histones. Due to its dual role as an epigenetic regulator and in the regulation of non-histone proteins through direct methylation, G9a has been implicated in a number of biological processes relevant to cell fate control. Recent reports employing in vitro cell lines indicate that Ehmt2 methylates MyoD to repress its transcriptional activity and therefore its ability to induce differentiation of activated myogenic cells.

Genomic changes during human linage evolution contribute to the expansion of the cerebral cortex to allow more advanced thought processes. The hominoid-specific gene TBC1D3 displays robust capacity of promoting the generation and proliferation of neural progenitors (NPs), which are thought to contribute to cortical expansion. However, the underlying mechanisms remain unclear. Here, we found that TBC1D3 interacts with G9a, a euchromatic histone lysine N-methyltransferase, which mediates dimethylation of histone 3 in lysine 9 (H3K9me2), a suppressive mark for gene expression. TBC1D3 displayed an inhibitory role in G9a's histone methyltransferase activity. Treatment with G9a inhibitor markedly increased NP proliferation and promoted human cerebral organoid expansion, mimicking the effects caused by TBC1D3 up-regulation. By contrast, blockade of TBC1D3/G9a interaction to disinhibit G9a caused up-regulation of H3K9me2, suppressed NP proliferation, and impaired organoid development. Together, this study has demonstrated a mechanism underlying the role of a hominoid-specific gene in promoting cortical expansion.

α-Synuclein (αS) is a protein linked to Parkinson's disease (PD) and related neurodegenerative disorders. It is mostly localized within synapses, but αS has also been suggested to play a role in the nucleus. We used transgenic Drosophila and inducible SH-SY5Y neuroblastoma cells to investigate the effects of αS on chromatin with a particular focus on histone modifications. Overexpression of αS in male flies as well as in retinoic acid pre-treated neuroblastoma cells led to an elevation of histone H3K9 methylations, mostly mono- (H3K9me1) and di- (H3K9me2). The transient increase of H3K9 methylation in αS-induced SH-SY5Y cells was preceded by mRNA induction of the euchromatic histone lysine N-methyltransferase 2 (EHMT2). EHMT2 and H3K9me2 can function within the REST complex. Chromatin immunoprecipitation (ChIP) analyses of selected candidate, REST regulated genes showed significantly increased H3K9me2 promoter occupancy of genes encoding the L1CAM cell adhesion molecule and the synaptosomal-associated protein SNAP25, whose reduced expression levels were confirmed by RT-qPCR in αS induced cells. Treatment with EHMT inhibitor UNC0638 restored the mRNA levels of L1CAM and SNAP25. Thus, αS overexpression enhances H3K9 methylations via ΕΗΜΤ2 resulting in elevated H3K9me2 at the SNAP25 promoter, possibly affecting SNARE complex assembly and hence synaptic vesicle fusion events regulated by αS.

Prostate cancer (PCa) is the most common type of cancer in men. Destruction of or blocking lipid metabolism impairs the growth, proliferation, and survival of tumor cells. Recent studies on hepatic steatosis suggest that kinase tethers histone-lysine N-methyltransferase 2D (KMT2D) to peroxisome proliferator-activated receptor gamma (PPARγ), transactivating its target genes. Here, to determine a therapeutic approach that may interfere with PCa lipid metabolism, the interaction mechanism of KMT2D and PPARγ was verified in PCa.

We showed previously that the histone lysine methyltransferase (HKMT) H3K27me3 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 (PRC2) and is required for the maintenance of HIV-1 latency in Jurkat T cells. Here we show, by using chromatin immunoprecipitation experiments, that both PRC2 and euchromatic histone-lysine N-methyltransferase 2 (EHMT2), the G9a H3K9me2-3 methyltransferase, are highly enriched at the proviral 5' long terminal repeat (LTR) and rapidly displaced upon proviral reactivation. Clustered regularly interspaced short palindromic repeat(s) (CRISPR)-mediated knockout of EZH2 caused depletion of both EZH2 and EHMT2, but CRISPR-mediated knockout of EHMT2 was selective for EHMT2, consistent with the failure of EHMT2 knockouts to induce latent proviruses in this system. Either (i) knockout of methyltransferase by short hairpin RNA in Jurkat T cells prior to HIV-1 infection or (ii) inhibition of the enzymes with drugs significantly reduced the levels of the resulting silenced viruses, demonstrating that both enzymes are required to establish latency. To our surprise, inhibition of EZH2 (by GSK-343 or EPZ-6438) or inhibition of EHMT2 (by UNC-0638) in the Th17 primary cell model of HIV latency or resting memory T cells isolated from HIV-1-infected patients receiving highly active antiretroviral therapy, was sufficient to induce the reactivation of latent proviruses. The methyltransferase inhibitors showed synergy with interleukin-15 and suberanilohydroxamic acid. We conclude that both PRC2 and EHMT2 are required for the establishment and maintenance of HIV-1 proviral silencing in primary cells. Furthermore, EZH2 inhibitors such as GSK-343 and EPZ-6438 and the EHMT2 inhibitor UNC-0638 are strong candidates for use as latency-reversing agents in clinical studies.IMPORTANCE Highly active antiretroviral therapy (HAART) reduces the circulating virus to undetectable levels. Although patients adhering to the HAART regimen have minimal viremia, HIV persists because of the existence of latent but replication-competent proviruses in a very small population of resting memory CD4+ T cells (~1 in 106 cells). Latency remains the major obstacle to a functional cure for HIV infection, since the persistent reservoir almost invariably rebounds within 2 to 8 weeks when HAART is interrupted. In latently infected cells, the HIV genome is stably integrated into the host chromosome but transcriptionally repressed because of epigenetic silencing mechanisms. We demonstrate here that multiple histone lysine methyltransferases play a critical role in both the establishment and maintenance of proviral silencing in cells obtained from well-suppressed patients. Drugs that inhibit these enzymes are available from oncology applications and may find a use in reversing latency as part of a reservoir reduction strategy.

To investigate the function of histone-lysine N-methyltransferase 2D (KMT2D) on the methylation of H3 lysine 4 (H3K4) in the progression of Ovarian cancer (OV). KMT2D, ESR1 and H3K4me expressions in surgical resected tumors and tumor adjacent tissues of OV from 198 patients were determined using immunohistochemistry (IHC). Human OV cell lines including SKOV3, HO-8910 cells and normal ovarian epithelial cell line IOSE80 were employed for in vitro experiment, and BALB/C female nude mice were used for in vivo study. qRT-PCR and Western blotting were implemented for measuring the KMT2D, ESR1, PTGS2, STAT3, VEGFR2, H3K4me and ELF3 levels. Chromatin immunoprecipitation (ChIP) analysis was used for studying the binding between ESR1 and H3K4me. Edu staining assay was executed to determine cell viability, and colony formation and cell invasion assay. The immunofluorescence method was utilized for the visualization of protein expression and distribution in cells. In this study, KMT2D, ESR1 and H3K4me were found upregulated in OV progression. Mutated H3K4me could inhibit the proliferation, colony formation and invasion ability of OV cells. Mutated H3K4me could also hinder the ESR1 in SKOV3 expressions and HO-8910 cells, which would further mediate PTGS2/STAT3/VEGF pathway. In vivo studies also demonstrated that mutated H3K4me inhibited OV progression via targeting ESR1. All the ChIP-PCR analysis indicated the moderator effect of H3K4me on ESR1. Our findings indicated that ESR1 played an important role in the OV progression. Besides, H3K4me could promote cell proliferation and inhibit apoptosis of OV cells. Meanwhile, it could also targets the ESR1 production to enhance the migration and invasion of OV cells, which was through the activation of ESR1-ELF3-PTGS2-STAT3-VEGF cascade signaling pathway.

Hypoxic-ischemic encephalopathy (HIE) is recognized as the main cause of neonatal death, and efficient treatment strategies remain limited. This study aims to investigate the mechanism of sevoflurane (SF) post-treatment in alleviating HIE in rats. The HIE rat model and oxygen-glucose deprivation (OGD) cell model were established, and adeno-associated virus (AAV)-histone-lysine N-methyltransferase EHMT2 (G9a) was transfected after SF treatment. The learning and memory ability and the levels of nerve growth factor (NGF)/brain-derived neurotrophic factor (BDNF) were evaluated and determined. The levels of G9a/histone H3 lysine 9 dimethylation (H3K9me2) and the enrichment level of H3K9me2 in the promoter region of BDNF gene were analyzed. After SF post-treatment, the neurons in cerebral cortex, the learning and memory skills and the contents of NGF/BDNF were increased, while the apoptosis and G9a/H3K9me2 levels were reduced. After overexpression of G9a in vitro/vivo, the enrichment levels of H3K9me2 in the promoter region of BDNF were increased, the levels of BDNF were decreased, the neurons were damaged and the learning and memory abilities of HIE rats were impaired. The conclusion is that SF post-treatment can promote the expression of BDNF by inhibiting H3K9me2 on the BDNF gene promoter and inhibiting G9a, thus alleviating HIE in rats.

Frequent truncation mutations of the histone lysine N-methyltransferase KMT2C have been detected by whole exome sequencing studies in various cancers, including malignancies of the prostate. However, the biological consequences of these alterations in prostate cancer have not yet been elucidated.

FOXA1 is a pioneer factor that binds to enhancer regions that are enriched in H3K4 mono- and dimethylation (H3K4me1 and H3K4me2). We performed a FOXA1 rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) screen in ERα-positive MCF-7 breast cancer cells and found histone-lysine N-methyltransferase (MLL3) as the top FOXA1-interacting protein. MLL3 is typically thought to induce H3K4me3 at promoter regions, but recent findings suggest it may contribute to H3K4me1 deposition. We performed MLL3 chromatin immunoprecipitation sequencing (ChIP-seq) in breast cancer cells, and MLL3 was shown to occupy regions marked by FOXA1 occupancy and H3K4me1 and H3K4me2. MLL3 binding was dependent on FOXA1, indicating that FOXA1 recruits MLL3 to chromatin. MLL3 silencing decreased H3K4me1 at enhancer elements but had no appreciable impact on H3K4me3 at enhancer elements. We propose a mechanism whereby the pioneer factor FOXA1 recruits the chromatin modifier MLL3 to facilitate the deposition of H3K4me1 histone marks, subsequently demarcating active enhancer elements.

Post-translational modifications (PTMs) are reversible chemical modifications that can modulate protein structure and function. Methylation and acetylation are two such PTMs with integral and well-characterized biological roles, including modulation of chromatin structure; and unknown or poorly understood roles, exemplified by the influence of these PTMs on transcription factor structure and function. The need for biological insights into the function of these PTMs motivates the development of a nondestructive and label-free method that enables pursuit of molecular mechanisms. Here, we present a protocol for implementing nuclear magnetic resonance (NMR) methods that allow for unambiguous detection of methylation and acetylation events and demonstrate their utility by observing these marks on histone H3 tail as a model system. We leverage strategic isotopic enrichment of cofactor and peptide for visualization by [1H, 13C]-HSQC and 13C direct-detect NMR measurements. Finally, we present 13C-labeling schemes that facilitate one-dimensional NMR experiments, which combine reduced measurement time relative to two-dimensional spectroscopy with robust filtering of background signals that would otherwise create spectral crowding or limit detection of low-abundance analytes.