Euchromatic Histone Methyltransferase Research Articles

EHMT1/2 inhibition promotes regression of therapy-resistant ovarian cancer tumors in a CD8 T cell-dependent manner.

Poly ADP-ribose polymerase inhibitors (PARPi) are first-line maintenance therapy for ovarian cancer and an alternative therapy for several other cancer types. However, PARPi-resistance is rising and there is currently an unmet need to combat PARPi-resistant tumors. Here, we created an immunocompetent, PARPi-resistant mouse model to test the efficacy of combinatory PARPi and euchromatic histone methyltransferase 1/2 inhibitor (EHMTi) in the treatment of PARPi-resistant ovarian cancer. We discovered that inhibition of EHMT1/2 resensitizes cells to PARPi. Markedly, we show that single EHMTi and combinatory EHMTi/PARPi significantly reduced PARPi-resistant tumor burden and that this reduction is partially dependent on CD8 T cells. Altogether, our results show a low-toxicity drug that effectively treats PARPi-resistant ovarian cancer in an immune-dependent manner, supporting its entry into clinical development and potential incorporation of immunotherapy. Implications: Targeting the epigenome of therapy-resistant ovarian cancer induces an anti-tumor response mediated in part through an anti-tumor immune response.

Ehmt2 inactivation in pancreatic epithelial cells shapes the transcriptional landscape and inflammation response of the whole pancreas.

Introduction: The Euchromatic Histone Methyl Transferase Protein 2 (EHMT2), also known as G9a, deposits transcriptionally repressive chromatin marks that play pivotal roles in the maturation and homeostasis of multiple organs. Recently, we have shown that Ehmt2 inactivation in the mouse pancreas alters growth and immune gene expression networks, antagonizing Kras-mediated pancreatic cancer initiation and promotion. Here, we elucidate the essential role of Ehmt2 in maintaining a transcriptional landscape that protects organs from inflammation. Methods: Comparative RNA-seq studies between normal postnatal and young adult pancreatic tissue from Ehmt2 conditional knockout animals (Ehmt2 fl/fl ) targeted to the exocrine pancreatic epithelial cells (Pdx1-Cre and P48 Cre/+ ), reveal alterations in gene expression networks in the whole organ related to injury-inflammation-repair, suggesting an increased predisposition to damage. Thus, we induced an inflammation repair response in the Ehmt2 fl/fl pancreas and used a data science-based approach to integrate RNA-seq-derived pathways and networks, deconvolution digital cytology, and spatial transcriptomics. We also analyzed the tissue response to damage at the morphological, biochemical, and molecular pathology levels. Results and discussion: The Ehmt2 fl/fl pancreas displays an enhanced injury-inflammation-repair response, offering insights into fundamental molecular and cellular mechanisms involved in this process. More importantly, these data show that conditional Ehmt2 inactivation in exocrine cells reprograms the local environment to recruit mesenchymal and immunological cells needed to mount an increased inflammatory response. Mechanistically, this response is an enhanced injury-inflammation-repair reaction with a small contribution of specific Ehmt2-regulated transcripts. Thus, this new knowledge extends the mechanisms underlying the role of the Ehmt2-mediated pathway in suppressing pancreatic cancer initiation and modulating inflammatory pancreatic diseases.

Longitudinal imaging in Kleefstra syndrome—Brief report and literature review

AbstractBackgroundKleefstra syndrome (KS) is a rare genetic condition affecting the euchromatic histone methyltransferase 1 (EHMT1) gene, typically presenting with developmental delay, generalized hypotonia, distinctive facial dysmorphisms, and neuropsychiatric anomalies.MethodsWe collected longitudinal magnetic resonance imaging (MRI) data of a child with KS and cerebrospinal fluid (CSF) dissection over 16 years and reviewed the literature focusing on articles reporting MRI findings in KS based on a PubMed search.ResultsAn 18‐year‐old female with KS presented with developmental delay at age 18 months and seizures at age 10 years. At age 23 months, MRI showed ventriculomegaly. Follow‐up MRI nine years later showed CSF dissection from the right lateral ventricle, which progressed similarly in the contralateral side 3 years later and stabilized over time. To date, only 20 articles report MRI findings from 49 patients with KS. The brain imaging findings range from normal (about 25%) to various abnormalities, including ventriculomegaly, white matter signal abnormalities, and dysmorphic corpus callosum and brainstem structures. CSF dissection was not found in any cases with white matter or other abnormalities.ConclusionsCSF dissection may be a rare neuroimaging finding of KS. Its etiology is unclear, but we speculate that epigenetic mutations could indirectly affect the development and integrity of ependymal cell lining along the ventricles. Alternatively, the long‐standing hydrocephalus may have caused the development of pseudodiverticula and subsequent CSF dissection.

The role of the gut microbiota in patients with Kleefstra syndrome.

Kleefstra Syndrome (KS) is a rare monogenetic syndrome, caused by haploinsufficiency of the euchromatic histone methyl transferase 1 (EHMT1) gene, an important regulator of neurodevelopment. The clinical features of KS include intellectual disability, autistic behavior and gastrointestinal problems. The gut microbiota, an important modifier of the gut-brain-axis, may constitute an unexplored mechanism underlying clinical KS variation. We investigated the gut microbiota composition of 23 individuals with KS (patients) and 40 of their family members, to test whether (1) variation in the gut microbiota associates with KS diagnosis and (2) variation within the gut microbiota relates with KS syndrome symptoms. Both alpha and beta diversity of patients were different from their family members. Genus Coprococcus 3 was lower in abundance in patients compared to family members. Moreover, abundance of genus Merdibacter was lower in patients versus family members, but only in participants reporting intestinal complaints. Within the patient group, behavioral problems explained 7% of beta diversity variance. Also, within this group, we detected higher levels of Atopobiaceae - uncultured and Ruminococcaceae Subdoligranulum associated with higher symptom severity. These significant signatures in the gut microbiota composition in patients with KS suggest that microbiota differences are part of the KS phenotype.

Abstract A021: Lysine methylation in EHMT1/GLP acts as a molecular switch to reprogram transcription networks to drive prostate cancer progression

Abstract Background: Prostate Cancer (PCa) progresses to a metastatic form of cancer called Castration-Resistant Prostate Cancer (CRPC) after treatment with androgen deprivation therapies (ADTs). Epigenetic reprogramming through altered expression and activity of histone modifier proteins is one major mechanism of tumor resistance. In particular, histone lysine methyltransferases (KMTs) and demethylases (KDMs) are important epigenetic targets in CRPC. In this study, we have focused on studying the function and regulation of Euchromatic Histone Methyltransferase 1 protein (EHMT1/GLP), which is a well-known H3K9 methyltransferase and functions in repressing gene transcription. EHMT family genes are altered in ~2-3% of primary PCa and ~10% of CRPC (primarily gene amplification) and their expression levels are significantly increased in CRPC, suggesting these proteins may have oncogenic activities in driving CRPC progression. Methods: We conducted an integrated analysis of ChIP-seq and RNA-seq analysis in LNCaP PCa cells to characterize the transcription program of EHMT1. We then performed proteomics analyses in VCaP and LNCaP PCa cells to identify post-translational modifications of EHMT1 and functionally validated the findings by generating loss-of-function mutations. We performed a histone demethylase assay to discover if EHMT1 is a substrate of LSD1 (a member of the KDM family). Moreover, we also assessed the effects of EHMT1 silencing or inhibition (in multiple PCa cell lines) on cell proliferation and metastasis in vitro using cell cycle analysis and transwell migration assay, and tumor growth and metastasis in vivo using mouse subcutaneous injection and zebrafish embryo injection approaches. Results: Our data showed that EHMT1 can transcriptionally activate multiple oncogenic pathways, including E2F and MYC signaling. Proteomic analyses revealed that EHMT1 is methylated in PCa cell lines, with dual-lysine methylation occurring at the K450/K451 sites. The histone demethylase assay showed that methylated K450, but not K451 is a potential substrate of LSD1. By generating the K450R, K451R, and K450/451R mutants, we showed that the K450/451R mutant, but not any single lysine mutants, can greatly expand EHMT1 chromatin binding independent of its H3K9 methyltransferase activity. This mutant also significantly induced EHMT1 oncogenic activity by activating E2F and MYC pathways. Moreover, EHMT1 silencing, or inhibition can significantly suppress tumor growth and metastasis. Conclusion: We have demonstrated that EHMT1 can function to activate oncogenic transcriptional programs in PCa by an H3K9-independent mechanism, possibly mediated through dual-lysine demethylation at K450/451 sites. Our data also suggest that targeting EHMT1 in CRPC may be a potential therapeutic strategy to suppress tumor growth and metastasis. Citation Format: Anna Besschetnova, Wanting Han, Mingyu Liu, Yanfei Gao, Muqing Li, Zifeng Wang, Maryam Labaf, Susan Patalano, Kavita Venkataramani, Rachel Muriph, Jill Macoska, Kellee Siegfried-Harris, Jason Evans, Steven Balk, Shuai Gao, Dong Han, Changmeng Cai. Lysine methylation in EHMT1/GLP acts as a molecular switch to reprogram transcription networks to drive prostate cancer progression. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A021.

Efficient synthesis of a novel euchromatic histone methyl transferase 2 (G9a) inhibitor

An efficient synthetic route was set up to prepare in good scale M-108, a novel bicycle derivative recently identified as a potent and selective G9a inhibitor, potentially useful as anti-fibroadipogenic agent. In particular, a facile three-steps sequential transformation of a substituted cis-cinnamate ethyl ester intermediate allowed to obtain the corresponding cyclopropyl nitrile derivative in high yield, which was smoothly transformed into the title racemate and then resolved by chiral HPLC.

Kleefstra syndrome: Recurrence in siblings due to a paternal mosaic mutation

Kleefstra syndrome (KS) is a rare autosomic dominant genetic disorder caused by euchromatic histone methyltransferase 1 (EHMT1) alterations. Patients mainly present with moderate to severe intellectual disability, a severe delay in/or absence of speech, autism spectrum disorder, childhood hypotonia, neuropsychiatric anomalies, and distinctive dysmorphic features. Here, we report the cases of a male and a female, two younger siblings of three, with asymptomatic parents. An EHMT1 new mutation was identified. Both presented with a typical core phenotype. Some specific features were noted, such as macrocephaly (previously reported) and enuresis (not yet described). Parental analysis identified the mutation in the mosaic state in the father. Reverse phenotyping enabled us to highlight the pauci phenotype features of inguinal hernia, azoospermia, and possible behavioral disorders. This allowed us to adapt his follow-up and genetic counseling for the family. Our three reported cases provide a new description of KS with an intragenic EHMT1 mutation, whereas in the literature most reported cases have EHMT1 deletions. Moreover, in the areas of next-generation sequencing and trio techniques with parental segregation, it is important to remain cautious about disregarding variants based on an autosomal recessive hypothesis.

Cell shape-based chemical screening reveals an epigenetic network mediated by focal adhesions.

Adapter proteins CRK and CRKL participate in a variety of signaling pathways, including cell adhesion, and fate regulation of mammalian cells. However, the molecular functions of CRK/CRKL in epigenetic regulation remain largely unknown. Here, we developed a pipeline to evaluate cell morphology using high-content image analysis combined with chemical screening of kinase and epigenetic modulators. We found that CRK/CRKL modulates gene regulatory networks associated with cell morphology through epigenetic alteration in mouse embryonic fibroblasts. Integrated epigenome and transcriptome analyses revealed that CRK/CRKL is involved in super-enhancer activity and upregulation of Cdt1, Rin1, and Spp1 expression for the regulation of cell morphology. Screening of a library of 80 epigenetic inhibitors showed that histone H3 modifiers, euchromatic histone methyltransferase 2 and mitogen- and stress-activated kinase 1, may be important for CRK/CRKL-mediated morphological changes. Taken together, our results indicate that CRK/CRKL plays a critical role in gene regulatory networks through epigenetic modification. DATABASES: Chromatin immunoprecipitation sequencing and RNA sequencing data were deposited in the DNA Data Bank of Japan under DRA011080 and DRA011081 accession numbers, respectively.

G9a Promotes Invasion and Metastasis of Non-Small Cell Lung Cancer through Enhancing Focal Adhesion Kinase Activation via NF-κB Signaling Pathway.

Potential roles of euchromatic histone methyltransferase 2 (EHMT2 or G9a) in invasion and metastasis are not well understood in non-small cell lung cancer (NSCLC). Here, we investigated the effect and underlying mechanisms of G9a and therapeutic implications of targeting G9a in the invasion and metastasis of NSCLC. Overexpression of G9a significantly enhanced in vitro proliferation and invasion, while knockdown of G9a drastically suppressed in vivo growth and metastasis of A549 and H1299 NSCLC cells. Knockdown or inhibition of G9a significantly decreased the expression of focal adhesion kinase (FAK) protein and activation of FAK pathway. In addition, defactinib, a potent FAK inhibitor, partially abolished the G9a-enhanced invasion in these NSCLC cells. Furthermore, targeting G9a was found to suppress NF-κB transcriptional activity in NSCLC cells through stabilizing NF-κB inhibitor alpha (IκBα), while an NF-κB inhibitor Parthenilide partially abolished the G9a-enhanced FAK activation, which suggests that G9a-enhanced invasion and activation of FAK is mediated by elevated NF-κB activity. Notably, a strong positive correlation between the IHC staining of G9a and phosphorylated FAK proteins was identified in H1299 xenografts and 159 cases of NSCLC tissues (R = 0.408). IMPLICATIONS: The findings of this study strongly demonstrate that G9a may promote invasion and metastasis of NSCLC cells by enhancing FAK signaling pathway via elevating NF-κB transcriptional activity, indicating potential significance and therapeutic implications of these pathways in the invasion and metastasis of NSCLCs that overexpress G9a protein.

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.

Context-Dependent Requirement of Euchromatic Histone Methyltransferase Activity during Reprogramming to Pluripotency.

SummaryMethylation of histone 3 at lysine 9 (H3K9) constitutes a roadblock for cellular reprogramming. Interference with methyltransferases or activation of demethylases by the cofactor ascorbic acid (AA) facilitates the derivation of induced pluripotent stem cells (iPSCs), but possible interactions between specific methyltransferases and AA treatment remain insufficiently explored. We show that chemical inhibition of the methyltransferases EHMT1 and EHMT2 counteracts iPSC formation in an enhanced reprogramming system in the presence of AA, an effect that is dependent on EHMT1. EHMT inhibition during enhanced reprogramming is associated with rapid loss of H3K9 dimethylation, inefficient downregulation of somatic genes, and failed mesenchymal-to-epithelial transition. Furthermore, transient EHMT inhibition during reprogramming yields iPSCs that fail to efficiently give rise to viable mice upon blastocyst injection. Our observations establish novel functions of H3K9 methyltransferases and suggest that a functional balance between AA-stimulated enzymes and EHMTs supports efficient and less error-prone iPSC reprogramming to pluripotency.

Abstract 3842: Histone methyltransferase G9a promotes migration, invasion of non-small cell lung cancer through enhancing FAK and NF-kB signal pathways

Abstract Overexpression of euchromatic histone methyltransferase 2 (EHMT2 or G9a) is associated with carcinogenesis and poor prognosis in a number of cancer types. In this study, we investigated the effect of G9a on invasion of non-small cell lung cancer (NSCLC) cells and the mechanism underlying cancer cell invasion. Knockdown of G9a was shown to drastically suppress in vitro cellular proliferation, migration, and invasion of A549 and H1299 NSCLC cells, while overexpression of G9a significantly enhanced the proliferation and invasion of these NSCLC cells. Additionally, knockdown of G9a led to significant decrease in expression of focal adhesion kinase (FAK) gene and activation of FAK pathway. UNC0638, a selective inhibitor of G9a, significantly suppressed both the proliferation, migration and invasion of these NSCLC cells, and concurrently led to a dramatic drop of global demethylation of H3K9 (H3K9me2) and suppressed the activation of FAK pathway. In addition, Defactinib, a potent FAK inhibitor, partially abolished the enhanced invasion by overexpression of G9a in these NSCLC cells. Furthermore, G9a was proved to regulate nuclear NF-κB transcriptional activity in NSCLC cells, and NF-κB inhibitor partially abolished the activated FAK signaling pathway by overexpressed G9a, which indicates that G9a-enhanced invasion and activation of FAK pathway may be mediated by NF-κB transcriptional activity. Notably, immunohistochemistry analysis further revealed that activation of FAK pathway was associated with overexpressed G9a in about 150 cases of NSCLC tissues. Altogether, these data have strongly demonstrated that G9a may promote invasion of NSCLC cells by upregulating FAK protein and pathway via enhancing NF-κB transcriptional activity, indicating a therapeutic implication of targeting these two pathways in NSCLC with overexpressed G9a. Citation Format: Ting Sun, Keqiang Zhang, Rajendra P. Pangeni, Wendong Li, Dan J. Raz. Histone methyltransferase G9a promotes migration, invasion of non-small cell lung cancer through enhancing FAK and NF-kB signal pathways [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 3842.

Kleefstra syndrome and epilepsy

Kleefstra syndrome is a rare autosomal dominant genetic disorder caused by haploinsufficiency of the EHMT1 (Euchromatic Histone MethylTransferase 1). Patients with Kleefstra syndrome have following most common symptoms: moderate or severe intellectual deficiency, absence of speech, significant diffuse muscular hypotonia, micro-brachycephaly, congenital defects of heart, kidneys, genitourinary tract and recognizable dysmorphic features of face. The article presents 2 similar clinical cases of Kleefstra syndrome in combination with epilepsy. Both patients, along with a typical clinical picture of the underlying disease, have serial epileptic spasms with an onset after first year of life, modified hypsarrhythmia with tendency to synchronization on the electroencephalogram, pharmacoresistant epilepsy. This indicates that Kleefstra syndrome can include epilepsy as one of symptoms of the disease.

OR14-03 The Transcriptional Coactivation Function of EHMT2 Restricts Chronic Glucocorticoid Exposure Induced Insulin Resistance

Glucocorticoids are required for metabolic adaptations during times of stress. However, chronic glucocorticoid exposure is associated with metabolic disorders such as insulin resistance. Glucocorticoids mainly convey their signals through an intracellular glucocorticoid receptor (GR). GR is a transcription factor that requires interactions with transcriptional coregulators to modulate the transcription of GR primary target genes, which in turn regulate specific aspects of physiology. Euchromatic Histone Methyltransferase 2 (Ehmt2) is a transcriptional coregulator for GR that can act as a corepressor or a coactivator. We found that glucocorticoid-induced insulin resistance was exacerbated when Ehmt2 levels were reduced in the liver. Intriguingly, this phenotype resulted from the transactivation function of Ehmt2. This is because a mutation at the lysine 182 automethylation site, which is required for the coactivation but not the corepression function of Ehmt2, results in similar exacerbated GC-induced insulin resistance. These results suggest that Ehmt2 coactivation dependent GR primary target genes restrict the extent of glucocorticoid-induced insulin resistance. Gene expression analysis identified Dusp4 (a.k.a. Mkp-2) as an Ehmt2 coactivation dependent GR-activated gene, which when overexpressed in liver, attenuated glucocorticoid-induced insulin resistance. Thus, we have identified a novel GR-Ehmt2-Dusp4 axis that plays a key role in controlling the extent of the development of insulin resistance. Notably, the classical view of how GC induce hepatic insulin resistance is that GR activates genes that inhibit insulin signaling and enhance hepatic gluconeogenesis. Our study, however, provides a revolutionary concept in which the extent of GC-induced insulin resistance is controlled by the balance of GR-activated genes that promote insulin sensitivity or insulin resistance.

CDK2 regulates the NRF1/Ehmt1 axis during meiotic prophase I

Meiosis generates four genetically distinct haploid gametes over the course of two reductional cell divisions. Meiotic divisions are characterized by the coordinated deposition and removal of various epigenetic marks. Here we propose that nuclear respiratory factor 1 (NRF1) regulates transcription of euchromatic histone methyltransferase 1 (EHMT1) to ensure normal patterns of H3K9 methylation during meiotic prophase I. We demonstrate that cyclin-dependent kinase (CDK2) can bind to the promoters of a number of genes in male germ cells including that of Ehmt1 through interaction with the NRF1 transcription factor. Our data indicate that CDK2-mediated phosphorylation of NRF1 can occur at two distinct serine residues and negatively regulates NRF1 DNA binding activity in vitro. Furthermore, induced deletion of Cdk2 in spermatocytes results in increased expression of many NRF1 target genes including Ehmt1 We hypothesize that the regulation of NRF1 transcriptional activity by CDK2 may allow the modulation of Ehmt1 expression, therefore controlling the dynamic methylation of H3K9 during meiotic prophase.

CBFA2T2 is required for BMP-2-induced osteogenic differentiation of mesenchymal stem cells

Bone morphogenetic protein (BMP) signaling is one of the essential pathways involved in osteogenic differentiation of mesenchymal stem cells (MSCs) and regulation of bone formation. While BMP-2 has been approved for clinic use, the underlying mechanisms remain not fully understood. In this study, we found co-repressor CBFA2T2 (core-binding factor, runt domain, alpha subunit 2, translocated to, 2) expression was significantly upregulated in response to BMP-2 treatment during osteogenic differentiation of human dental pulp stem cells (hDPSCs) and mouse bone marrow stromal cells (mBMSCs). siRNA-mediated knockdown of CBFA2T2 blunted the BMP-2-induced allkaline phosphatase (ALP) activity, mineralization of extracelluar matrix (ECM), and expression of osteogenic related genes in both hDPSCs and mBMSCs. Mechanistically, knockdown of CBFA2T2 promoted expression of euchromatic histone methyltransferase 1 (EHMT1) in mBMSCs, which further led to upregulation of H3K9me2 levels at promoter of runt related transcription factor 2 (Runx2), the master regulator of osteogenesis. Collectively, our findings indicate that CBFA2T2 is required for BMP-2-induced osteogenic differentiation of MSCs through inhibition of EHMT1-mediated histone methylation at Runx2 promoter.

Pharmacological and transcriptional inhibition of the G9a histone methyltransferase suppresses proliferation and modulates redox homeostasis in human microvascular endothelial cells

Epigenetic mechanisms, including histone post-translational modifications, are central regulators of cell cycle control. The euchromatic G9a histone methyltransferase (G9a HMT) is a key enzyme catalyzing histone H3 methylation on lysines 9 and 27, and its dysregulation has been linked to uncontrolled proliferation of tumor cells. Here, we have investigated the effect of G9a HMT silencing on cell proliferation of microvascular endothelial cells, a process necessary to sustain tumor growth through the formation of the vascular capillary network.Inhibition of G9a HMT activity in human microvascular endothelial cells (HMEC-1) was performed either pharmacologically, by treatment of cells with BIX-01294 or chaetocin, or transcriptionally, using shRNA. Cell viability and proliferation were examined using the resazurin reduction assay, flow cytometry and immunostaining of phosphorylated checkpoint kinase 1 (pSer317Chk1). Expression of cell cycle- and redox homeostasis-related genes was determined by quantitative PCR. Reactive oxygen species production was measured by oxidation of the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate and the cell’s total antioxidant capacity by using the ABTS assay.Inhibition of G9a HMT activity by BIX-01294 treatment or by shRNA attenuated the proliferation of HMEC-1, nuclear localization of phosphorylated Chk1, and induced cell cycle arrest in G1 phase. Transcriptional analysis demonstrated increased gene expression of the cyclin-dependent kinase (CDK) inhibitor p21, and also of Rb1, in BIX-01294 treated cells. Decreased proliferation rate was accompanied by enhanced antioxidant potential of HMEC-1 cells, as demonstrated by reduced production of reactive oxygen species, increased total antioxidant capacity and expression of the antioxidant enzymes catalase and superoxide dismutase 1.Collectively, our results demonstrate of the central role of G9a HMT in the promotion of endothelial cells proliferation, and suggest that endothelial G9a HMT may be a target in the treatment of vascular proliferative disorders and tumor neovascularization.

EHMT1/GLP; Biochemical Function and Association with Brain Disorders

The gene EHMT1 that encodes the Euchromatic Histone Methyltransferase-1, also known as GLP (G9a-like protein), has been associated with a number of neurodevelopmental and neurodegenerative disorders. GLP is a member of the euchromatic lysine histone methyltransferase family, along with EHMT2 or G9A. As its name implies, Ehmt1/GLP is involved in the addition of methyl groups to histone H3 lysine 9, a generally repressive mark linked to classical epigenetic process such as genomic imprinting, X-inactivation, and heterochromatin formation. However, GLP also plays both a direct and indirect role in regulating DNA-methylation. Here, we discuss what is currently known about the biochemical function of Ehmt1/GLP and its association, via various genetic studies, with brain disorders.

A Novel Kleefstra Syndrome-associated Variant That Affects the Conserved TPLX Motif within the Ankyrin Repeat of EHMT1 Leads to Abnormal Protein Folding

Kleefstra syndrome (KS) (Mendelian Inheritance in Man (MIM) no. 610253), also known as 9q34 deletion syndrome, is an autosomal dominant disorder caused by haploinsufficiency of euchromatic histone methyltransferase-1 (EHMT1). The clinical phenotype of KS includes moderate to severe intellectual disability with absent speech, hypotonia, brachycephaly, congenital heart defects, and dysmorphic facial features with hypertelorism, synophrys, macroglossia, protruding tongue, and prognathism. Only a few cases of de novo missense mutations in EHMT1 giving rise to KS have been described. However, some EHMT1 variants have been described in individuals presenting with autism spectrum disorder or mild intellectual disability, suggesting that the phenotypic spectrum resulting from EHMT1 alterations may be quite broad. In this report, we describe two unrelated patients with complex medical histories consistent with KS in whom next generation sequencing identified the same novel c.2426C>T (p.P809L) missense variant in EHMT1. To examine the functional significance of this novel variant, we performed molecular dynamics simulations of the wild type and p.P809L variant, which predicted that the latter would have a propensity to misfold, leading to abnormal histone mark binding. Recombinant EHMT1 p.P809L was also studied using far UV circular dichroism spectroscopy and intrinsic protein fluorescence. These functional studies confirmed the model-based hypotheses and provided evidence for protein misfolding and aberrant target recognition as the underlying pathogenic mechanism for this novel KS-associated variant. This is the first report to suggest that missense variants in EHMT1 that lead to protein misfolding and disrupted histone mark binding can lead to KS.

40: Inhibition of euchromatic histone methyltransferase 1 and 2 sensitizes chronic myeloid leukemia cells to interferon treatment

H3K9 methylation is one of the essential histone post-translational modifications for heterochromatin formation and transcriptional repression. Recently, several studies have demonstrated that H3K9 methylation negatively regulates the type I interferon response. We report the application of EHTM1 and EHMT2 specific chemical inhibitors to sensitize CML cell lines to interferon and imatinib treatments. Inhibition of EHMT1 and EHMT2 with BIX01294 enhances the cytotoxicity of IFNa2a in all four CML cell lines tested but not three non-CML cell lines. Additionally, BIX01294 augments IFNa2a- and imatinib-mediated apoptosis in CML cells. Moreover, our data suggest that the expression level of EHMT1 and EHMT2 inversely correlates with the type I interferon response in CML cell lines. Our study sheds light on the role of EHMT1 and EHMT2 as potential targets in improving the efficacy of standard treatments of CML.