Euchromatic Histone-lysine N-methyltransferase 2 Research Articles

Long non-coding RNA FPFSC promotes immature porcine Sertoli cell growth through modulating the miR-326/EHMT2 axis

Sertoli cells are indispensable for guaranteeing normal spermatogenesis and male fertility. Although a huge number of long non-coding RNAs (lncRNAs) are identified from developing porcine testicular tissues and have been predicted with crucial regulatory roles in spermatogenesis, their functions and regulatory mechanisms are still in infancy. Herein, we mainly explored the regulatory and functional roles of lncFPFSC in proliferation and apoptosis of immature porcine Sertoli cells. The results demonstrated that lncFPFSC was predominantly located in the cytoplasm of immature porcine Sertoli cells. lncFPFSC overexpression promoted cell cycle progression and cell proliferation, as well as inhibited cell apoptosis, whereas siRNA-induced lncFPFSC knockdown resulted in the opposite effects. Mechanistically, lncFPFSC acted as a sponge for miR-326. Overexpression of miR-326 inhibited cell proliferation and induced cell apoptosis, which further abolished the effects of lncFPFSC overexpression. The euchromatic histone-lysine N-methyltransferase 2 (EHMT2) gene was directly targeted by miR-326, and its mRNA and protein expressions were both negatively regulated by miR-326 in immature porcine Sertoli cells. Then, siRNA-induced EHMT2 knockdown resulted a similar effect of miR-326 inhibition. Collectively, lncFPFSC promoted proliferation and inhibited apoptosis in immature porcine Sertoli cells through modulating the miR-326/EHMT2 axis. This study expanded our understanding of non-coding RNAs in participating porcine spermatogenesis through deciding the fate of Sertoli cells, and the competing endogenous RNA (ceRNA) network, and provided new molecular markers to treat Sertoli cell disorder inducing male infertility.

Euchromatic Histone Lysine Methyltransferase 2 Inhibition Enhances Carfilzomib Sensitivity and Overcomes Drug Resistance in Multiple Myeloma Cell Lines.

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.

Cinobufagin induces FOXO1-regulated apoptosis, proliferation, migration, and invasion by inhibiting G9a in non-small-cell lung cancer A549 cells

Ethnopharmacological relevanceBufonis (VB), an animal drug called Chansu in China, is the product of the secretion of Bufo gargarizans Cantor or B. melanostictus Schneider. As a traditional Chinese medicine (TCM) for a long time, it has been widely used in the treatment of heart failure, ulcer, pain, and various cancers. Cinobufaginn (CNB), the cardiotonic steroid or bufalene lactone extracted from VB, has the effects of detoxification, detumescence, and analgesia. Aim of the studyThe present study aimed to define the effects of CNB on non-small-cell lung cancer (NSCLC) and identify the potential molecular mechanisms. Materials and methodsA549 cells were treated with cinobufagin and cell viability, apoptosis, migration, and invasion were then evaluated using Cell Counting Kit-8 (CCK8) assays, flow cytometry, and Transwell assays, respectively. Moreover, the levels of proliferating cell nuclear antigen (PCNA), cytokeratin8 (CK8), poly ADP-ribose polymerase (PARP), Caspase3, Caspase8, B-cell lymphoma/lewkmia-2(Bcl-2), Bcl2-Associated X(Bax), forkhead box O1 (FOXO1), and euchromatic histone-lysine N-methyltransferase2 (G9a, EHMT2) in A549 cells were evaluated using qRT–PCR and/or Western blot analysis (WB), Co-IP, immunofluorescence, and immunohistochemistry. An in vivo imaging system, TUNEL, Immunofluorescence, and immunohistochemistry were also used to detect proliferating cell nuclear antigen(PCNA), Ki67, E-Cadherin(E-Cad), FOXO1, and G9a in mouse xenograft model experiments. ResultsCNB suppressed cell proliferation, migration, and invasion but promoted apoptosis in A549 cells in a dose- and time-dependent manner, while cinobufagin had no cytotoxic effect on BEAS-2B cells. In vivo, cinobufagin inhibited the proliferation, migration, and invasion of A549 cells and promoted their apoptosis. The occurrence of the above phenomena was accompanied by an increase in FOXO1 expression and a decrease in G9a expression. In A549 cells, CNB did not reverse the changes in the proliferation, migration, invasion, and apoptosis of A549 cells after FOXO1 was successfully silenced. ConclusionOur study provides the first evidence that cinobufagin suppresses the malignant biological behaviours of NSCLC cells in vivo and in vitro and suggests that mechanistically, this effect may be achieved by inhibiting the expression of the histone methyltransferase G9a and activating the tumour suppressor gene FOXO1. Taken together, our findings provide important insights into the molecular mechanism underlying cinobufagin's anticancer activity, and suggest that cinobufagin could be a candidate for targeted cancer therapy.

Human gut-microbiome-derived propionate coordinates proteasomal degradation via HECTD2 upregulation to target EHMT2 in colorectal cancer

The human microbiome plays an essential role in the human immune system, food digestion, and protection from harmful bacteria by colonizing the human intestine. Recently, although the human microbiome affects colorectal cancer (CRC) treatment, the mode of action between the microbiome and CRC remains unclear. This study showed that propionate suppressed CRC growth by promoting the proteasomal degradation of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) through HECT domain E3 ubiquitin protein ligase 2 (HECTD2) upregulation. In addition, EHMT2 downregulation reduced the H3K9me2 level on the promoter region of tumor necrosis factor α-induced protein 1 (TNFAIP1) as a novel direct target of EHMT2. Subsequently, TNFAIP1 upregulation induced the apoptosis of CRC cells. Furthermore, using Bacteroides thetaiotaomicron culture medium, we confirmed EHMT2 downregulation via upregulation of HECTD2 and TNFAIP1 upregulation. Finally, we observed the synergistic effect of propionate and an EHMT2 inhibitor (BIX01294) in 3D spheroid culture models. Thus, we suggest the anticancer effects of propionate and EHMT2 as therapeutic targets for colon cancer treatment and may provide the possibility for the synergistic effects of an EHMT2 inhibitor and microbiome in CRC treatment.

EHMT2 (G9a) activation in mantle cell lymphoma and its associated DNA methylation and gene expression.

Objective:The function of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) has been studied in several cancers; however, little is known about its role in mantle cell lymphoma (MCL). Thus, this study aimed to characterize the significance and function of EHMT2 in MCL.Methods:EHMT2 expression in MCL and reactive hyperplasia (RH) were investigated by immunohistochemistry. Genome-wide analysis of DNA methylation was performed on EHMT2 + MCL samples. The function of EHMT2 was determined by CCK8, flow cytometry, and western blot assays. Gene expression profile analysis was performed before and after EHMT2 knockdown to search for EHMT2-regulated genes. Co-immunoprecipitation (Co-IP) experiments were conducted to identify the proteins interacting with EHMT2.Results:EHMT2 was expressed in 68.57% (24/35) of MCLs but not in any RHs. Genome-wide analysis of DNA methylation on EHMT2 + MCLs revealed that multiple members of the HOX, FOX, PAX, SOX, and CDX families were hypermethylated or hypomethylated in EHMT2 + MCLs. BIX01294, a EHMT2 inhibitor, inhibited MCL cell growth and stalled cells in the G1 phase. Additionally, BIX01294 downregulated the expressions of cell cycle proteins, cyclin D1, CDK4, and P21, but upregulated the expressions of apoptosis-related proteins, Bax and caspase-3. Co-IP experiments revealed that EHMT2 interacted with UHRF1, HDAC1, and HDAC2 but not with HDCA3. After EHMT2 knockdown, multiple genes were regulated, including CD5 and CCND1, mostly enriched in the Tec kinase signaling pathway. In addition, several genes (e.g., MARCH1, CCDC50, HIP1, and WNT3) were aberrantly methylated in EHMT2 + MCLs.Conclusions:For the first time, we determined the significance of EHMT2 in MCL and identified potential EHMT2-regulated genes.

Epigenetic treatment of behavioral and physiological deficits in a tauopathy mouse model.

Epigenetic abnormality is implicated in neurodegenerative diseases associated with cognitive deficits, such as Alzheimer's disease (AD). A common feature of AD is the accumulation of neurofibrillary tangles composed of hyperphosphorylated tau. Transgenic mice expressing mutant P301S human tau protein develop AD‐like progressive tau pathology and cognitive impairment. Here, we show that the euchromatic histone‐lysine N‐methyltransferase 2 (EHMT2) is significantly elevated in the prefrontal cortex (PFC) of P301S Tau mice (5–7 months old), leading to the increased repressive histone mark, H3K9me2, which is reversed by treatment with the selective EHMT inhibitor UNC0642. Behavioral assays show that UNC0642 treatment induces the robust rescue of spatial and recognition memory deficits in P301S Tau mice. Concomitantly, the diminished PFC neuronal excitability and glutamatergic synaptic transmission in P301S Tau mice are also normalized by UNC0642 treatment. In addition, EHMT inhibition dramatically attenuates the hyperphosphorylated tau level in PFC of P301S Tau mice. Transcriptomic analysis reveals that UNC0642 treatment of P301S Tau mice has normalized a number of dysregulated genes in PFC, which are enriched in cytoskeleton and extracellular matrix organization, ion channels and transporters, receptor signaling, and stress responses. Together, these data suggest that targeting histone methylation enzymes to adjust gene expression could be used to treat cognitive and synaptic deficits in neurodegenerative diseases linked to tauopathies.

Inactivation of the Euchromatic Histone-Lysine N-Methyltransferase 2 Pathway in Pancreatic Epithelial Cells Antagonizes Cancer Initiation and Pancreatitis-Associated Promotion by Altering Growth and Immune Gene Expression Networks.

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 KrasG12D-mediated acinar-to-ductal metaplasia (ADM) and Pancreatic Intraepithelial Neoplasia (PanIN) formation in both the Pdx1-Cre and P48Cre/+ KrasG12D mouse models. Ex vivo acinar explants also show impaired EGFR-KRAS-MAPK pathway-mediated ADM upon EHMT2 deletion. Notably, KrasG12D 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 KrasG12D animals with EHMT2 inactivation have increased P21 protein levels and enhanced senescence. Furthermore, loss of EHMT2 reduces inflammatory cell infiltration typically induced during KrasG12D-mediated initiation. The inhibitory effect on KrasG12D-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.

Abstract 4180: Therapeutic targeting of STAT3 in nasopharyngeal carcinoma

Abstract Cancer stem cells (CSCs) have been reported to associate with resistance, metastasis, recurrence, and related to treatment failure in Nasopharyngeal Carcinoma (NPC). Signal transducer and activator of transcription 3 (STAT3) was associated with CSCs. When activated at tyrosine-705 (Y705) and serine-727 (S727) by phosphorylation, STAT3 promotes tumour inflammation. In CSCs, STAT3 was activated independently of upstream modulator, further increases resistance towards chemotherapy. Hence, we aim to investigate the potential of STAT3 as a therapeutic target in NPC. Immunohistochemistry revealed STAT3 activation in 100% primary NPC tissues (P-STAT3 Y705, n=27). Comparable STAT3 status was observed in patient derived xenografts and cell line established xenografts. Western blot analysis of NPC cell lines (C17, NPC43 and C666-1) substantiated P-STAT3 (Y705 and S727) activation status. STAT3 targeting were examined by specific small interfering RNA (siRNA) and cancer stemness inhibitor Napabucasin, both were shown to knockdown STAT3 activation. Napabucasin reduced cell viability, induced cell death, decelerated NPC cell migration, and inhibited STAT3 phosphorylation. Notably, chemoresistant NPC cell line (C666-1 FR) despite showing 300% higher resistance towards standard chemodrug 5-fluorouracil (5-FU), showed comparable sensitivity towards Napabucasin with reference to its parental cells C666-1. This demonstrates feasibility of STAT3 targeted therapy as alternative treatment strategy in overcoming chemoresistance of NPC. Interestingly, despite significant STAT3 knockdown by siRNA approach, minimal effect on cell proliferation and cell death was observed. In addition, Napabucasin was showed to impair NPC stemness properties including colony and spheroid formation. NPC spheroids were observed to have elevated STAT3 and classical cancer stemness markers (ALDH1A1, CD133) comparing the three-dimensional culture to their corresponding cells in two-dimensional culture. ALDH1A1 and CD133 were downregulated when treated with Napabucasin and STAT3 siRNA. RNA-sequencing and real-time qPCR (RT-qPCR) analysis showed significant downregulation of cancer stemness gene, Euchromatic histone-lysine N-methyltransferase 2 (EHMT2), upon Napabucasin treatment, but not with STAT3 siRNA. This work first reports that Napabucasin downregulates EHMT2 in NPC. qPCR analysis also demonstrated overexpressed EHMT2 in NPC spheroids, suggesting EHMT2 role in regulating NPC cancer stemness. Indeed, when treated with Napabucasin, EHMT2 was downregulated in spheroids. These observations indicate specificity of Napabucasin in controlling NPC cancer stemness. Congruent to in vitro data, Napabucasin suppressed tumor growth in vivo, diminished P-STAT3 (Y705) activation, decreased proliferation (Ki67), and elevated apoptosis (cleaved caspase-3). In summary, this work demonstrated promising therapeutic potential of Napabucasin targeting STAT3 and cancer stemness in NPC. Citation Format: Shinyee Hui, Kwok Wai Lo, Alyssa Ming-Ting Liu, Marcus Hung-Nam Law, Linda Wing-Chi Ng, Peter Tin-Chung Li, Siu Tim Cheung. Therapeutic targeting of STAT3 in nasopharyngeal carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4180.

EHMT2 Inhibition Induces Cell Death in Human Non-Small Cell Lung Cancer by Altering the Cholesterol Biosynthesis Pathway.

Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer. Besides genetic and environmental factors, epigenetic alterations contribute to the tumorigenesis of NSCLC. Epigenetic changes are considered key drivers of cancer initiation and progression, and altered expression and activity of epigenetic modifiers reshape the epigenetic landscape in cancer cells. Euchromatic histone-lysine N-methyltransferase 2 (EHMT2) is a histone methyltransferase and catalyzes mono- and di-methylation at histone H3 lysine 9 (H3K9me1 and H3K9me2, respectively), leading to gene silencing. EHMT2 overexpression has been reported in various types of cancer, including ovarian cancer and neuroblastoma, in relation to cell proliferation and metastasis. However, its role in NSCLC is not fully understood. In this study, we showed that EHMT2 gene expression was higher in NSCLC than normal lung tissue based on publicly available data. Inhibition of EHMT2 by BIX01294 (BIX) reduced cell viability of NSCLC cell lines via induction of autophagy. Through RNA sequencing analysis, we found that EHMT2 inhibition significantly affected the cholesterol biosynthesis pathway. BIX treatment directly induced the expression of SREBF2, which is a master regulator of cholesterol biosynthesis, by lowering H3K9me1 and H3K9me2 at the promoter. Treatment of a cholesterol biosynthesis inhibitor, 25-hydroxycholesterol (25-HC), partially recovered BIX-induced cell death by attenuating autophagy. Our data demonstrated that EHMT2 inhibition effectively induced cell death in NSCLC cells through altering cholesterol metabolism-dependent autophagy.

Nanodiamond-Mediated Delivery of a G9a Inhibitor for Hepatocellular Carcinoma Therapy.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer with high mortality but limited therapeutic options. Epigenetic regulations including DNA methylation and histone modification control gene expressions and play a crucial role during tumorigenesis. G9a, also known as EHMT2 (euchromatic histone-lysine N-methyltransferase 2), is a histone methyltransferase predominantly responsible for dimethylation of histone H3 lysine 9 (H3K9). G9a has been shown to play a key role in promoting tumor progression. Recent studies have identified that G9a is a critical mediator of HCC pathogenesis. UNC0646 is a G9a inhibitor that has shown potent in vitro efficacy. However, due to its water insolubility, the in vivo efficacy of UNC0646 is not satisfactory. In this study, nanodiamonds (NDs) were utilized as a drug delivery platform to improve in vivo delivery of this small-molecule inhibitor. Our results showed that ND-UNC0646 complexes could be rapidly synthesized by physical adsorption, meanwhile possessing favorable drug delivery properties and was able to improve the dispersibility of UNC0646 in water, therefore making it amenable for intravenous administration. The release profile of UNC0646 from ND-UNC0646 was demonstrated to be pH-responsive. Moreover, ND-UNC0646 maintained the biological functionality of UNC0646, with higher efficacy in reducing H3K9 methylation as well as enhanced invasion suppressive effects. Most importantly, increased in vivo efficacy was demonstrated using an orthotopic HCC mouse model, which paves the way of translating this small-molecule inhibitor toward HCC treatment. Our work demonstrates the potential of NDs in the clinical application for HCC treatment.

EHMT2 promotes the development of prostate cancer by inhibiting PI3K/AKT/mTOR pathway.

To investigate the expression characteristics and the potential role of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) in the clinical pathology and prognosis of prostate cancer (PCa). Quantitative Real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of EHMT2 in 55 pairs of PCa tissues and adjacent normal tissues. The relationship between EHMT2 expression and the pathological features, as well as the prognosis of PCa patients was analyzed. EHMT2 expression in PCa cells was determined by qRT-PCR as well. In addition, EHMT2 knockdown model was constructed by transfection of the small interfering RNA in PCa cell lines PC-3 and DU-145. The regulatory effects of EHMT2 on the behaviors of PCa cells were evaluated through cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), colony formation assay and flow cytometry. Finally, we detected the protein levels of relative genes in PI3K/AKT/mTOR pathway through Western blot. QRT-PCR results showed that the expression level of EHMT2 in PCa tissues was markedly higher than that in adjacent normal tissues. Compared with PCa patients with low expression of EHMT2, those with high expression of EHMT2 had higher pathological grade and lower overall survival. EHMT2 was also highly expressed in PCa cell lines. Knockdown of EHMT2 inhibited the proliferative potential and induced apoptosis of PCa cells. Western blot results revealed that PCa cells with EHMT2 knockdown presented downregulated p-PI3K, p-AKT and p-mTOR. EHMT2 was highly expressed in PCa, and its expression is closely correlated with tumor stage and poor prognosis of PCa patients. EHMT2 promoted the malignant progression of PCa by inhibiting PI3K/AKT/mTOR pathway.

Inhibition of G9a promoted 5-fluorouracil (5-FU) induced gastric cancer cell apoptosis via ROS/JNK signaling pathway in vitro and in vivo.

A histone methyltransferase G9a, encoded by euchromatic histone-lysine N-methyltransferase 2 (EHMT2), is up-regulated in various cancers, and is involved in their poor prognosis. In the study reported here, the abnormal expression of G9a in gastric cancer it was investigated in vitro and in vivo. Furthermore, the expression of G9a was revealed to have a negative correlation with chemotherapy response in gastric cancer patients. Next, the effect of G9a knockdown on fluorouracil (5-FU) induced cell apoptosis in gastric cancer cells was focused on. The results demonstrated that G9a knockdown significantly activated the expression level of phospho c-Jun N-terminal kinase (p-JNK) and increased the intracellular reactive oxygen species (ROS) levels in the gastric cancer cells. Inhibition of the ROS/JNK signaling partial reversed the effect of G9a knockdown on 5-FU treated gastric cancer cells. Down-regulation of G9a enhanced the sensitivity of 5-FU to the gastric cancer cells in vitro and in vivo, which was involved in the activation of the ROS/JNK signaling pathway. These results demonstrated that G9a could play a critical role in the sensitivity of chemotherapy for gastric cancer and might be a novel method for treating gastric cancer in the clinic.

The novel prognostic marker, EHMT2, is involved in cell proliferation via HSPD1 regulation in breast cancer.

Molecular classifications of breast cancer (BRC), such as human epidermal growth factor receptor 2 (HER2), luminal A and luminal B, have been developed to reduce unnecessary treatment by dividing patients with BRC into low- and high-risk progression groups. However, these methods do not cover all of the pathological characteristics of BRC, and investigations into novel prognostic/therapeutic markers are thus continually required. In this study, we identified the overexpression of the histone methyltransferase, euchromatic histone-lysine N-methyltransferase 2 (EHMT2) in BRC samples (n=1,222) and normal samples (n=113) derived from the TCGA portal by performing a BRC tissue microarray. EHMT2 overexpression was clearly associated with a poor prognosis in multiple cohorts of patients with BRC (total, n=1,644). Furthermore, the knockdown of EHMT2 expression affected cell apoptosis via the downregulation and re-localization of heat shock protein family D (Hsp60) member 1 (HSPD1). In addition, a statistically significant positive correlation between EHMT2 and HSPD1 expression was revealed in the clinical cohorts. On the whole, the findings of this study may assist the development of novel therapeutic strategies and provide a prognostic marker (EHMT2) for patients with BRC.

Multiple Histone Lysine Methyltransferases Are Required for the Establishment and Maintenance of HIV-1 Latency.

ABSTRACTWe 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.

α-Synuclein enhances histone H3 lysine-9 dimethylation and H3K9me2-dependent transcriptional responses.

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.

Chromatin remodeling factor LSH affects fumarate hydratase as a cancer driver.

Cancer metabolism and epigenetic alteration are two critical mechanisms for tumorigenesis and cancer progression; however, the dynamic interplay between them remains poorly understood. As reported in the article entitled “Chromatin remodeling factor LSH drives cancer progression by suppressing the activity of fumarate hydratase,” which was recently published in Cancer Research, our group examined the physiological role of lymphocyte-specific helicase (LSH) in nasopharyngeal carcinoma (NPC) by focusing on cancer progression and the tricarboxylic acid cycle. We found that LSH was overexpressed in NPC, and its expression associated with Epstein-Barr virus infection. We also found that LSH directly suppressed fumarate hydratase (FH), a key component of the tricarboxylic acid cycle, in combination with euchromatic histone-lysine N-methyltransferase 2 (EHMT2), also known as G9a. Depletion of FH promoted epithelial-mesenchymal transition (EMT). Moreover, LSH controlled expression of tricarboxylic acid cycle intermediates that promote cancer progression, including EMT, through activation by inhibitor of nuclear factor kappa-B kinase alpha (IKKα), a chromatin modifier and transcriptional activator. Our study showed that LSH plays a critical role in cancer progression, which has important implications for the development of novel strategies to treat NPC.

Effect of BIX-01294 on H3K9me2 levels and the imprinted gene Snrpn in mouse embryonic fibroblast cells.

Histone H3 lysine 9 dimethylation (H3K9me2) hypermethylation is thought to be a major influential factor in cellular reprogramming, such as somatic cell nuclear transfer (SCNT) and induction of pluripotent stem cells (iPSCs). The diazepin-quinazolin-amine derivative (BIX-01294) specifically inhibits the activity of histone methyltransferase EHMT2 (euchromatic histone-lysine N-methyltransferase 2) and reduces H3K9me2 levels in cells. The imprinted gene small nuclear ribonucleoprotein N (Snrpn) is of particular interest because of its important biological functions. The objective of the present study was to investigate the effect of BIX-01294 on H3K9me2 levels and changes in Snrpn DNA methylation and histone H3K9me2 in mouse embryonic fibroblasts (MEFs). Results showed that 1.3 μM BIX-01294 markedly reduced global levels of H3K9me2 with almost no cellular toxicity. There was a significant decrease in H3K9me2 in promoter regions of the Snrpn gene after BIX-01294 treatment. A significant increase in methylation of the Snrpn differentially methylated region 1 (DMR1) and slightly decreased transcript levels of Snrpn were found in BIX-01294-treated MEFs. These results suggest that BIX-01294 may reduce global levels of H3K9me2 and affect epigenetic modifications of Snrpn in MEFs.

EHMT2 inhibitor BIX-01294 induces apoptosis through PMAIP1-USP9X-MCL1 axis in human bladder cancer cells.

BIX-01294, an euchromatic histone-lysine N-methyltransferase 2 (EHMT2) inhibitor, has been reported to induce apoptosis in human neuroblastoma cells and inhibit the proliferation of bladder cancer cells. However, the definite mechanism of the apoptosis mediated by BIX-01294 in bladder cancer cells remains unclear. In the present study, we found that BIX-01294 induced caspase-dependent apoptosis in human bladder cancer cells. Moreover, our data show BIX-01294 stimulates endoplasmic reticulum stress (ER stress) and up-regulated expression of PMAIP1 through DDIT3 up-regulation. Furthermore, down-regulation of the deubiquitinase USP9X by BIX-01294 results in downstream reduction of MCL1 expression, leading to apoptosis eventually. Thus, our findings demonstrate PMAIP1-USP9X-MCL1 axis may contribute to BIX-01294-induced apoptosis in human bladder cancer cells.

BIX-01294 induces autophagy-associated cell death via EHMT2/G9a dysfunction and intracellular reactive oxygen species production

We screened a chemical library in MCF-7 cells stably expressing green fluorescent protein (GFP)-conjugated microtubule-associated protein 1 light chain 3 (LC3) (GFP-LC3-MCF-7) using cell-based assay, and identified BIX-01294 (BIX), a selective inhibitor of euchromatic histone-lysine N-methyltransferase 2 (EHMT2), as a strong autophagy inducer. BIX enhanced formation of GFP-LC3 puncta, LC3-II, and free GFP, signifying autophagic activation. Inhibition of these phenomena with chloroquine and increasement in punctate dKeima ratio (550/438) signal indicated that BIX activated autophagic flux. BIX-induced cell death was suppressed by the autophagy inhibitor, 3-methyladenine, or siRNA against BECN1 (VPS30/ATG6), ATG5, and ATG7, but not by caspase inhibitors. Moreover, EHMT2 siRNA augmented GFP-LC3 puncta, LC3-II, free GFP, and cell death, implying that inhibition of EHMT2 caused autophagy-mediated cell death. Treatment with EHMT2 siRNA and BIX accumulated intracellular reactive oxygen species (ROS). BIX augmented mitochondrial superoxide via NADPH oxidase activation. In addition, BIX increased hydrogen peroxide and glutathione redox potential in both cytosol and mitochondria. Treatment with N-acetyl-L-cysteine (NAC) or diphenyleneiodonium chloride (DPI) decreased BIX-induced LC3-II, GFP-LC3 puncta, and cell death, indicating that ROS instigated autophagy-dependent cell death triggered by BIX. We observed that BIX potentiated autophagy-dependent and caspase-independent cell death in estrogen receptor (ESR)-negative SKBr3 and ESR-positive MCF-7 breast cancer cells, HCT116 colon cancer cells, and importantly, in primary human breast and colon cancer cells. Together, the results suggest that BIX induces autophagy-dependent cell death via EHMT2 dysfunction and intracellular ROS accumulation in breast and colon cancer cells, therefore EHMT2 inhibition can be an effective therapeutic strategy for cancer treatment.

122 SUPPRESSION OF EPIGENETIC MODIFIERS ALTERS THE BOVINE EMBRYONIC DEVELOPMENTAL PROGRAM DURING IN VITRO CULTURE

During early bovine embryogenesis, the regular establishment of DNA methylation and histone modification patterns is essential for proper gene expression and continuation of embryonic development. Epigenome patterns established during this period, if improperly maintained, can lead to developmental anomalies and may partially explain the lower pregnancy rates of in vitro-produced embryos. We hypothesised that the suppression of translation of the genes euchromatic histone-lysine N-methyltransferase 2 (EHMT2), DNA methyltransferase 3A (DNMT3A), absent, small, or homeotic-like (ASH2L), and SET domain, bifurcated 1 (SETDB1) would provide insightful information on the importance of these genes during early embryonic development in an in vitro setting. In order to define the roles of these genes, small interfering RNA (siRNA) targeting the gene of interest were synthesised and target verified in bovine cell culture using quantitative real-time RT-PCR (RT-qPCR). We acquired matured bovine oocytes from commercial suppliers, followed by IVF by standard laboratory procedures. Eighteen hours post IVF, cumulus cells were removed and zygotes separated into 3 different treatment groups: non-injected controls (CNTL), non-targeting siRNA injected controls (siNULL), and injection with siRNA targeting the gene of interest (si “gene target”). Each siRNA was mixed with a green fluorescent dextran at a concentration of 20 μM and ~100 pL injected cytoplasmically. The green fluorescent dextran was used to give visual confirmation that zygotes were indeed injected. Post-injection, fluorescent embryos were separated and cultured in Bovine Evolve (Zentih Biotech) medium supplemented with 4 mg mL–1 of BSA (Probumin, Millipore). Cleavage rates were monitored on Day 2, and only cleaved embryos were cultured further. On Day 8 post-IVF, embryos were morphologically examined and numbers of blastocysts recorded. Mean development rates between siNULL and targeting siRNA were compared using a t-test statistic. Over the course of these experiments the mean blastocyst rate for CNTL zygotes was 34.5% ± 2.6 s.e.m. (n = 1647). None of the zygotes injected with siEHMT2 (n = 1184) or siSETDB1 (n = 361) reached the blastocyst stage and these rates differed from the siNULL rate (21.0% ± 2.5 s.e.m., n = 1587; P < 0.05). Morphologically, embryos from both groups developed to the morula stage before they exhibited fragmentation. Injection of siDNMT3A also resulted in significant loss of viability at the 8-cell stage and few zygotes injected (n = 1057) developed to blastocyst (2.1% ± 0.5 s.e.m.; P < 0.001). Inhibiting gene expression of ASH2L showed little variation in blastocyst rate from our siNULL embryos (31.3% ± 2.0 s.e.m., n = 466 v. 34.8% ± 1.9 s.e.m., n = 418, respectively, P > 0.2). It is unknown at this time if inhibition of ASH2L translation will have effects later in development. Ongoing experiments analysing DNA methylation and histone modifications through immunocytochemistry and global gene expression via RT-qPCR will further explore the establishment and maintenance of these genes in the embryonic epigenome.