The role of the histone methyltransferase SET domain bifurcated 1 during palatal development.

The histone methyltransferase SET domain bifurcated 1 (SETDB1) catalyzes the trimethylation of lysine 9 of histone H3, thereby regulating gene expression. In this study, we used conditional knockout mice, where Setdb1 was deleted only in neural crest cells (Setdb1fl/fl,Wnt1-Cre + mice), to clarify the role of SETDB1 in palatal development. Setdb1fl/fl,Wnt1-Cre + mice died shortly after birth due to a cleft palate with full penetration. Reduced palatal mesenchyme proliferation was seen in Setdb1fl/fl,Wnt1-Cre + mice, which might be a possible mechanism of cleft palate development. Quantitative RT-PCR and in situ hybridization showed that expression of the Pax9, Bmp4, Bmpr1a, Wnt5a, and Fgf10 genes, known to be important for palatal development, were markedly decreased in the palatal mesenchyme of Setdb1fl/fl,Wnt1-Cre + mice. Along with these phenomena, SMAD1/5/9 phosphorylation was decreased by the loss of Setdb1. Our results demonstrated that SETDB1 is indispensable for palatal development partially through its proliferative effect. Taken together with previous reports that PAX9 regulates BMP signaling during palatal development which implies that loss of Setdb1 may be involved in the cleft palate development by decreasing SMAD-dependent BMP signaling through Pax9.

Histone methyltransferase DOT1L mediates the TGF-ß1/Smad3 signaling pathway through epigenetic modification of SYK in myocardial infarction.

Myocardial infarction (MI) represents the most critical condition in coronary artery disease, and the fibrotic process, detrimental to optimal recovery, often sustains. In the present work, we assessed whether suppression of disruptor of telomeric silencing 1-like (DOT1L) could alleviate fibrosis in vivo and cardiac fibroblast (CFS) proliferation in vitro, and elucidated the possible mechanism involved in these events. After left coronary artery ligation, we found that the MI mice exhibited a decrease in cardiac function, along with evident MI and myocardial fibrosis. In addition, AngII increased CFS viability and migration, and enhanced the expression of fibrotic proteins. Inhibition of DOT1L ameliorated proliferation and fibrosis in CFS. Furthermore, DOT1L promoted the expression of spleen tyrosine kinase (SYK) by increasing the H3K79me2 modification of the SYK promoter. SYK upregulation reversed the inhibitory effect of DOT1L knockdown on CFS proliferation and fibrosis by activating the TGF-ß1/Smad3 signaling. SYK also mitigated the ameliorative effect of DOT1L knockdown on myocardial injury and fibrosis caused by MI in vivo. In conclusion, these data indicated that DOT1L depletion might be a promising therapeutic target for fibrosis in MI.

Functions of SMYD proteins in biological processes: What do we know? An updated review.

BACKGROUND: Epigenetic modifiers, such as methyltransferases, play crucial roles in the regulation of many biological processes, including development, cancer and multiple physiopathological conditions. SUMMARY: The Su(Var)3-9, Enhancer-of-zeste and Trithorax (SET) and Myeloid, Nervy, and DEAF-1 (MYND) domain-containing (SMYD) protein family consists of five members in humans and mice (i.e. SMYD1, SMYD2, SMYD3, SMYD4 and SMYD5), which are known or predicted to have methyltransferase activity on histone and non-histone substrates. The abundance of information concerning SMYD2 and SMYD3 is of note, whereas the other members of the SMYD family have not been so thoroughly studied CONCLUSION: Here we review the literature regarding SMYD proteins published in the last five years, including basic molecular biology mechanistic studies using in vitro systems and animal models, as well as human studies with a more translational or clinical approach.

The disordered regions of the methyltransferase SETD2 govern its function by regulating its proteolysis and phase separation.

SETD2 is an important methyltransferase that methylates crucial substrates such as histone H3, tubulin, and STAT1 and also physically interacts with transcription and splicing regulators such as Pol II and various hnRNPs. Of note, SETD2 has a functionally uncharacterized extended N-terminal region, the removal of which leads to its stabilization. How this region regulates SETD2 half-life is unclear. Here we show that SETD2 consists of multiple long disordered regions across its length that cumulatively destabilize the protein by facilitating its proteasomal degradation. SETD2 disordered regions can reduce the half-life of the yeast homolog Set2 in mammalian cells as well as in yeast, demonstrating the importance of intrinsic structural features in regulating protein half-life. In addition to the shortened half-life, by performing fluorescence recovery after photobleaching assay we found that SETD2 forms liquid droplets in vivo, another property associated with proteins that contain disordered regions. The phase-separation behavior of SETD2 is exacerbated upon the removal of its N-terminal segment and results in activator-independent histone H3K36 methylation. Our findings reveal that disordered region-facilitated proteolysis is an important mechanism governing SETD2 function.

High Yap and Mll1 promote a persistent regenerative cell state induced by Notch signaling and loss of p53.

Specified intestinal epithelial cells reprogram and contribute to the regeneration and renewal of the epithelium upon injury. Mutations that deregulate such renewal processes may contribute to tumorigenesis. Using intestinal organoids, we show that concomitant activation of Notch signaling and ablation of p53 induce a highly proliferative and regenerative cell state, which is associated with increased levels of Yap and the histone methyltransferase Mll1. The induced signaling system orchestrates high proliferation, self-renewal, and niche-factor-independent growth, and elevates the trimethylation of histone 3 at lysine 4 (H3K4me3). We demonstrate that Yap and Mll1 are also elevated in patient-derived colorectal cancer (CRC) organoids and control growth and viability. Our data suggest that Notch activation and p53 ablation induce a signaling circuitry involving Yap and the epigenetic regulator Mll1, which locks cells in a proliferative and regenerative state that renders them susceptible for tumorigenesis.

Setd1a Plays Pivotal Roles for the Survival and Proliferation of Retinal Progenitors via Histone Modifications of Uhrf1.

Purpose: The trimethylation of histone H3 at lysine 4 (H3K4me3) facilitates transcriptional gene activation, and Setd1a is the methyltransferase specific to H3K4. H3K4me3 has been reported to regulate rod photoreceptor differentiation; however, the roles H3K4me3 plays in retinal progenitor cell (RPC) proliferation and differentiation during early retinal development remain unclear. Methods: Using an in vitro retinal explant culture system, we suppressed the expression of Setd1a by introducing shSetd1a. We examined the expression level and H3K4me3 level of genes by RNA Sequencing and ChIP assay, respectively. Results: We found that Setd1a depletion resulted in increased apoptosis and proliferation failure in late RPCs. Expression of wild-type SETD1A, but not SETD1A that lacked the catalytic SET domain, reversed the shSetd1a-induced phenotype. RNA Sequencing revealed that proliferation-related genes were downregulated upon shSetd1a expression. Based on publicly available H3K4me3-ChIP sequencing data of retinal development, we identified Uhrf1 as a candidate target gene of Setd1a. The expression of shSetd1a led to a decrease in Uhrf1 transcript levels and reduced H3K4me3 levels at the Uhrf1 locus. Increased apoptosis and the suppression of proliferation in late RPCs were observed in retinal explants expressing shUhrf1, similar to the outcomes observed in shSetd1a-expressing retinas. The overexpression of UHRF1 did not rescue shSetd1a-induced apoptosis, but reversed the suppression of proliferation. Conclusions: These results indicate that Setd1a contributes to the survival and proliferation of retinal cells by regulating histone methylation, Setd1a regulates Uhrf1 expression, and these two molecules cooperate to regulate RPC survival and proliferation.

DOT1L Regulates Thermogenic Adipocyte Differentiation and Function via Modulating H3K79 Methylation.

Brown and beige adipocytes are characterized as thermogenic adipocytes and have great potential for treating obesity and associated metabolic diseases. In this article, we identify a conserved mammalian lysine 79 of histone H3 (H3K79) methyltransferase, disruptor of telomeric silencing-1 like (DOT1L), as a new epigenetic regulator that controls thermogenic adipocyte differentiation and function. We show that deletion of DOT1L in thermogenic adipocytes potently protects mice from diet-induced obesity, improves glucose homeostasis, alleviates hepatic steatosis, and facilitates adaptive thermogenesis in vivo. Loss of DOT1L in primary preadipocytes significantly promotes brown and beige adipogenesis and thermogenesis in vitro. Mechanistically, DOT1L epigenetically regulates the brown adipose tissue-selective gene program by modulating H3K79 methylation, in particular H3K79me2 modification. Thus, our study demonstrates that DOT1L exerts an important role in energy homeostasis by regulating thermogenic adipocyte differentiation and function.

Multilevel Regulation of ß-Catenin Activity by SETD2 Suppresses the Transition from Polycystic Kidney Disease to Clear Cell Renal Cell Carcinoma.

Patients with polycystic kidney disease (PKD) are at a high risk of developing renal cell carcinoma (RCC). However, little is known about genetic alterations or changes in signaling pathways during the transition from PKD to RCC. SET domain-containing 2 (SETD2) is a histone methyltransferase, which catalyzes tri-methylation of H3K36 (H3K36me3) and has been identified as a tumor suppressor in clear cell renal cell carcinoma (ccRCC), but the underlying mechanism remains largely unexplored. Here we report that knockout of SETD2 in a c-MYC-driven PKD mouse model drove the transition to ccRCC. SETD2 inhibited ß-catenin activity at transcriptional and posttranscriptional levels by competing with ß-catenin for binding promoters of target genes and maintaining transcript levels of members of the ß-catenin destruction complex. Thus, SETD2 deficiency enhanced the epithelial-to-mesenchymal transition and tumorigenesis through the hyperactivation of Wnt/ß-catenin signaling. Our findings reveal previously unrecognized roles of SETD2-mediated competitive DNA binding and H3K36me3 modification in regulating Wnt/ß-catenin signaling during the transition from PKD to ccRCC. The novel autochthonous mouse models of PKD and ccRCC will be useful for preclinical research into disease progression. SIGNIFICANCE: These findings characterize multiple mechanisms by which SETD2 inhibits ß-catenin activity during the transition of polycystic kidney disease to renal cell carcinoma, providing a potential therapeutic strategy for high-risk patients. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/13/3554/F1.large.jpg.

SETD7 mediates the vascular invasion in articular cartilage and chondrocytes apoptosis in osteoarthriis.

The pathological characteristics of osteoarthritis are cartilage matrix degradation, chondrocytes apoptosis, and low-grade inflammation of the joint. Recent studies have shown that blood vessels grow from the subchondral bone to the articular cartilage. However, the relationship among inflammation, angiogenesis, and chondrocyte apoptosis is still unclear. We found that chondrocytes could secrete chemokines and VEGF to promote the migration of vascular endothelial cells in response to TNF-α stimulation. The invasion of blood vessels leads to increased oxygen tension in the local environment, which increased the expression of SETD7 in chondrocytes by activating the JAK-STAT5 pathway. The bond of phosphorylated STAT5 and the specific locus in the promoter of SETD7 directly increased the transcription of SETD7. On the one hand, SETD7-regulated chemokine expression by forming a positive loop; on the other hand, SETD7-mediated chondrocyte apoptosis by inhibiting the nuclear localization of HIF-1α. In this study, we discovered a novel function of chondrocytes as mediators of inflammation and angiogenesis. Our study demonstrates that SETD7 is a potential molecular target to prevent OA development and progression.

The histone H3 lysine 9 methyltransferase G9a/GLP complex activity is required for long-term consolidation of spatial memory in mice.

The G9a/G9a-like protein (GLP) histone lysine dimethyltransferase complex and downstream histone H3 lysine 9 dimethylation (H3K9me2) repressive mark have recently emerged as key transcriptional regulators of gene expression programs necessary for long-term memory (LTM) formation in the dorsal hippocampus. However, the role for hippocampal G9a/GLP complex in mediating the consolidation of spatial LTM remains largely unknown. Using a water maze competition task in which both dorsal hippocampus-dependent spatial and striatum-dependent cue navigation strategies are effective to solve the maze, we found that pharmacological inhibition of G9a/GLP activity immediately after learning disrupts long-term consolidation of previously learned spatial information in male mice, hence producing cue bias on the competition test performed 24 h later. Importantly, the inhibition of hippocampal G9a/GLP did not disrupt short-term memory retention. Immunohistochemical analyses revealed increases in global levels of permissive histone H3K9 acetylation in the dorsal hippocampus and dorsal striatum at 1 h post-training, which persisted up to 24 h in the hippocampus. Conversely, H3K9me2 levels were either unchanged in the dorsal hippocampus or transiently decreased at 15 min post-training in the dorsal striatum. Finally, the inhibition of G9a/GLP activity further increased global levels of H3K9 acetylation while decreasing H3K9me2 in the hippocampus at 1 h post-training. However, both marks returned to vehicle control levels at 24 h. Together, these findings support the possibility that G9a/GLP in the dorsal hippocampus is required for the transcriptional switch from short-term to long-term spatial memory formation.

The Essential Function of SETDB1 in Homologous Chromosome Pairing and Synapsis during Meiosis.

SETDB1 is a histone-lysine N-methyltransferase critical for germline development. However, its function in early meiotic prophase I remains unknown. Here, we report that Setdb1 null spermatocytes display aberrant centromere clustering during leptotene, bouquet formation during zygotene, and subsequent failure in pairing and synapsis of homologous chromosomes, as well as compromised meiotic silencing of unsynapsed chromatin, which leads to meiotic arrest before pachytene and apoptosis of spermatocytes. H3K9me3 is enriched in centromeric or pericentromeric regions and is present in many sites throughout the genome, with a subset changed in the Setdb1 mutant. These observations indicate that SETDB1-mediated H3K9me3 is essential for the bivalent formation in early meiosis. Transcriptome analysis reveals the function of SETDB1 in repressing transposons and transposon-proximal genes and in regulating meiotic and somatic lineage genes. These findings highlight a mechanism in which SETDB1-mediated H3K9me3 during early meiosis ensures the formation of homologous bivalents and survival of spermatocytes.

Histone Methyltransferase SETDB1: A Common Denominator of Tumorigenesis with Therapeutic Potential.

Epigenetic regulation of gene expression has been ultimately linked to cancer development, with posttranslational histone modifications representing attractive targets for disease monitoring and therapy. Emerging data have demonstrated histone lysine (K) methylation by methyltransferase SETDB1 as a common denominator of gene regulation in several cancer types. SETDB1 reversibly catalyzes the di- and trimethylation of histone 3 (H3) K9 in euchromatic regions of chromosomes, inhibiting gene transcription within these regions and promoting a switch from euchromatic to heterochromatic states. Recent studies have implicated aberrant SETDB1 activity in the development of various types of cancers, including brain, head and neck, lung, breast, gastrointestinal, ovarian, endometrial and prostate cancer, mesothelioma, melanoma, leukemias, and osteosarcoma. Although its role has not been fully elucidated in every case, most data point toward a pro-oncogenic potential of SETDB1 via the downregulation of critical tumor-suppressive genes. Less commonly, however, SETDB1 can also acquire a tumor-suppressive role, depending on cancer type and stage. Here we provide an updated overview of the cellular and molecular effects underlying SETDB1 activity in cancer development and progression along with current targeting strategies in different cancer types, with promising effects either as a standalone therapy or in conjunction with other therapeutic agents.

Histone methyltransferase ATX1 dynamically regulates fiber secondary cell wall biosynthesis in Arabidopsis inflorescence stem.

Secondary wall thickening in the sclerenchyma cells is strictly controlled by a complex network of transcription factors in vascular plants. However, little is known about the epigenetic mechanism regulating secondary wall biosynthesis. In this study, we identified that ARABIDOPSIS HOMOLOG of TRITHORAX1 (ATX1), a H3K4-histone methyltransferase, mediates the regulation of fiber cell wall development in inflorescence stems of Arabidopsis thaliana. Genome-wide analysis revealed that the up-regulation of genes involved in secondary wall formation during stem development is largely coordinated by increasing level of H3K4 tri-methylation. Among all histone methyltransferases for H3K4me3 in Arabidopsis, ATX1 is markedly increased during the inflorescence stem development and loss-of-function mutant atx1 was impaired in secondary wall thickening in interfascicular fibers. Genetic analysis showed that ATX1 positively regulates secondary wall deposition through activating the expression of secondary wall NAC master switch genes, SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1) and NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1). We further identified that ATX1 directly binds the loci of SND1 and NST1, and activates their expression by increasing H3K4me3 levels at these loci. Taken together, our results reveal that ATX1 plays a key role in the regulation of secondary wall biosynthesis in interfascicular fibers during inflorescence stem development of Arabidopsis.

Deletion of Histone Methyltransferase G9a Suppresses Mutant Kras-driven Pancreatic Carcinogenesis.

BACKGROUND/AIM: The entire mechanisms by which epigenetic modifiers contribute to the development of pancreatic cancer remain unknown. Although the histone methyltransferase G9a is a promising target in human cancers, its role in pancreatic carcinogenesis has been under-studied. The aim of the study was to examine the role of G9a in pancreatic carcinogenesis by a gene-targeting mouse model. MATERIALS AND METHODS: We established pancreas-specific G9aflox/flox mice and crossed them with Ptf1aCre/; KrasG12D/+ (KC) mice, which spontaneously develop pancreatic cancer. The phenotypes of the resulting KC mice with G9a deletion were examined. We analyzed transcriptomic data by microarray and genome-wide chromatin accessibility by transposase-accessible chromatin using sequencing. We established pancreatic organoids from KC mice. RESULTS: G9a deficiency impaired the progression of pancreatic intraepithelial neoplasia (PanIN) and prolonged the survival of KC mice. The number of phosphorylated Erk-positive cells and Dclk1-positive cells, which are reported to be essential for the progression of PanIN, were decreased by G9a deletion. UNC0638, an inhibitor of G9a, suppressed the growth of organoids and increased global chromatin accessibility, especially around the regions including the protein phosphatase 2A genes. CONCLUSION: Thus, our study suggested the functional interaction of G9a, Dclk1 and Mapk pathway in the Kras-driven pancreatic carcinogenesis. The inhibition of G9a may suppress the initiation of oncogenic Kras-driven pancreatic carcinogenesis.

MLL5, a histone modifying enzyme, regulates androgen receptor activity in prostate cancer cells by recruiting co-regulators, HCF1 and SET1.

In prostate cancer, the androgen receptor (AR) transcription factor is a major regulator of cell proliferation and metastasis. To identify new AR regulators, we focused on Mixed lineage leukemia 5 (MLL5), a histone-regulating enzyme, because significantly higher MLL5 expression was detected in prostate cancer tissues than in matching normal tissues. When we expressed shRNAs targeting MLL5 gene in prostate cancer cell line, the growth rate and AR activity were reduced compared to those in control cells, and migration ability of the knockdown cells was reduced significantly. To determine the molecular mechanisms of MLL5 on AR activity, we proved that AR physically interacted with MLL5 and other co-factors, including SET-1 and HCF-1, using an immunoprecipitation method. The chromatin immunoprecipitation analysis showed reduced binding of MLL5, co-factors, and AR enzymes to AR target gene promoters in MLL5 shRNA-expressing cells. Histone H3K4 methylation on the AR target gene promoters was reduced, and H3K9 methylation at the same site was increased in MLL5 knockdown cells. Finally, xenograft tumor formation revealed that reduction of MLL5 in prostate cancer cells retarded tumor growth. Our results thus demonstrate the important role of MLL5 as a new epigenetic regulator of AR in prostate cancer. [BMB Reports 2020; 53(12): 634-639].

Histone H3.3 G34 mutations promote aberrant PRC2 activity and drive tumor progression.

A high percentage of pediatric gliomas and bone tumors reportedly harbor missense mutations at glycine 34 in genes encoding histone variant H3.3. We find that these H3.3 G34 mutations directly alter the enhancer chromatin landscape of mesenchymal stem cells by impeding methylation at lysine 36 on histone H3 (H3K36) by SETD2, but not by the NSD1/2 enzymes. The reduction of H3K36 methylation by G34 mutations promotes an aberrant gain of PRC2-mediated H3K27me2/3 and loss of H3K27ac at active enhancers containing SETD2 activity. This altered histone modification profile promotes a unique gene expression profile that supports enhanced tumor development in vivo. Our findings are mirrored in G34W-containing giant cell tumors of bone where patient-derived stromal cells exhibit gene expression profiles associated with early osteoblastic differentiation. Overall, we demonstrate that H3.3 G34 oncohistones selectively promote PRC2 activity by interfering with SETD2-mediated H3K36 methylation. We propose that PRC2-mediated silencing of enhancers involved in cell differentiation represents a potential mechanism by which H3.3 G34 mutations drive these tumors.

Knockout of SETDB1 gene using the CRISPR/cas-9 system increases migration and transforming activities via complex regulations of E-cadherin, ß-catenin, STAT3, and Akt.

SETDB1 HMTase participates in various cellular processes via epigenetic transcriptional regulation. SETDB1 expression is downregulated by anticancer drug treatment in cancer cells, but we still need to verify the functional significance on SETDB1 downregulation. CRISPR/cas9 is a useful technology for doing a knockout (KO) of a target gene. It is widely used to examine the function of genes. In this study, we prepared SETDB1-KO from A549 human lung cancer cells using the CRISPR/Cas9 system, and we compared molecular changes between the A549 cells and the SETDB1-KO cells. The SETDB1-KO cell proliferation rate was slightly decreased as compared to the A549 cells, but there was no large difference in sensitivity with doxorubicin treatment. Instead, the migration activity and transforming activity were dramatically increased in SETDB-KO cells. Using a western blot analysis and an immunostaining experiment, we confirmed that SETDB1-KO downregulates the expression of E-cadherin and ß-catenin. A qPCR and an RT-PCR analysis suggested that SETDB1 transcriptionally regulates E-cadherin and ß-catenin. Moreover, E-cadherin expression was also detected in the cytoplasmic region of SETDB1-KO cells, indicating that functional localization of E-cadherin might be changed in SETDB1-KO cells. On the other hand, total levels of STAT3 and Akt were increased in the SETDB1-KO cells, but activation of STAT3 (pSTAT3) was not induced in doxorubicin-treated SETDB1-KO cells. SETDB1 overexpression into SETDB1-KO cells restores the expression of E-cadherin, ß-catenin, STAT3, and Akt, suggesting that those proteins are tightly regulated by SETDB1. Collectively, we suggest that complex regulations on E-cadherin, ß-catenin, STAT3, and Akt are correlated with the increased migration and transforming activity of SETDB1-KO cells.

EHMT1 regulates Parvalbumin-positive interneuron development and GABAergic input in sensory cortical areas.

Mutations in the Euchromatic Histone Methyltransferase 1 (EHMT1) gene cause Kleefstra syndrome, a rare form of intellectual disability (ID) with strong autistic traits and sensory processing deficits. Proper development of inhibitory interneurons is crucial for sensory function. Here we report a timeline of Parvalbumin-positive (PV+) interneuron development in the three most important sensory cortical areas in the Ehmt1+/- mouse. We find a hitherto unreported delay of PV+ neuron maturation early in sensory development, with layer- and region-specific variability later in development. The delayed PV+ maturation is also reflected in a delayed maturation of GABAergic transmission in Ehmt1+/- auditory cortex, where we find a reduced GABA release probability specifically in putative PV+ synapses. Together with earlier reports of excitatory impairments in Ehmt1+/- neurons, we propose a shift in excitatory-inhibitory balance towards overexcitability in Ehmt1+/- sensory cortices as a consequence of early deficits in inhibitory maturation.

The histone methyltransferase DOT1L prevents antigen-independent differentiation and safeguards epigenetic identity of CD8+ T cells.

Cytotoxic T cell differentiation is guided by epigenome adaptations, but how epigenetic mechanisms control lymphocyte development has not been well defined. Here we show that the histone methyltransferase DOT1L, which marks the nucleosome core on active genes, safeguards normal differentiation of CD8+ T cells. T cell-specific ablation of Dot1L resulted in loss of naïve CD8+ T cells and premature differentiation toward a memory-like state, independent of antigen exposure and in a cell-intrinsic manner. Mechanistically, DOT1L controlled CD8+ T cell differentiation by ensuring normal T cell receptor density and signaling. DOT1L also maintained epigenetic identity, in part by indirectly supporting the repression of developmentally regulated genes. Finally, deletion of Dot1L in T cells resulted in an impaired immune response. Through our study, DOT1L is emerging as a central player in physiology of CD8+ T cells, acting as a barrier to prevent premature differentiation and controlling epigenetic integrity.

Euchromatin histone methyltransferase II (EHMT2) regulates the expression of ras-related GTP binding C (RRAGC) protein.

Dimethylation of the histone H3 protein at lysine residue 9 (H3K9) is mediated by euchromatin histone methyltransferase II (EHMT2) and results in transcriptional repression of target genes. Recently, chemical inhibition of EHMT2 was shown to induce various physiological outcomes, including endoplasmic reticulum stress-associated genes transcription in cancer cells. To identify genes that are transcriptionally repressed by EHMT2 during apoptosis, and cell stress responses, we screened genes that are upregulated by BIX-01294, a chemical inhibitor of EHMT2. RNA sequencing analyses revealed 77 genes that were upregulated by BIX-01294 in all four hepatic cell carcinoma (HCC) cell lines. These included genes that have been implicated in apoptosis, the unfolded protein response (UPR), and others. Among these genes, the one encoding the stress-response protein Ras-related GTPase C (RRAGC) was upregulated in all BIX-01294-treated HCC cell lines. We confirmed the regulatory roles of EHMT2 in RRAGC expression in HCC cell lines using proteomic analyses, chromatin immune precipitation (ChIP) assay, and small guide RNA-mediated loss-of-function experiments. Upregulation of RRAGC was limited by the reactive oxygen species (ROS) scavenger N-acetyl cysteine (NAC), suggesting that ROS are involved in EHMT2-mediated transcriptional regulation of stress-response genes in HCC cells. Finally, combined treatment of cells with BIX-01294 and 5- Aza-cytidine induced greater upregulation of RRAGC protein expression. These findings suggest that EHMT2 suppresses expression of the RRAGC gene in a ROS-dependent manner and imply that EHMT2 is a key regulator of stress-responsive gene expression in liver cancer cells. [BMB Reports 2020; 53(11): 576-581].