Euchromatin Histone Methyltransferase Research Articles

G9a-mediated methylation of ERα links the PHF20/MOF histone acetyltransferase complex to hormonal gene expression

The euchromatin histone methyltransferase 2 (also known as G9a) methylates histone H3K9 to repress gene expression, but it also acts as a coactivator for some nuclear receptors. The molecular mechanisms underlying this activation remain elusive. Here we show that G9a functions as a coactivator of the endogenous oestrogen receptor α (ERα) in breast cancer cells in a histone methylation-independent manner. G9a dimethylates ERα at K235 both in vitro and in cells. Dimethylation of ERαK235 is recognized by the Tudor domain of PHF20, which recruits the MOF histone acetyltransferase (HAT) complex to ERα target gene promoters to deposit histone H4K16 acetylation promoting active transcription. Together, our data suggest the molecular mechanism by which G9a functions as an ERα coactivator. Along with the PHF20/MOF complex, G9a links the crosstalk between ERα methylation and histone acetylation that governs the epigenetic regulation of hormonal gene expression.

A structured assessment of motor function and behavior in patients with Kleefstra syndrome

The present study aimed to further our understanding of Kleefstra syndrome, especially regarding motor function and behavioral characteristics. In total, four males and four females between two and 27 years of age with a genetically confirmed diagnosis of Kleefstra syndrome and their parents participated in this study. Four patients had 9q34.3 deletions that caused Euchromatin Histone Methyl Transferase 1 (EHMT1) haplo-insufficiency, and four patients harbored EHMT1 mutations. The motor function was evaluated via systematic observation. Standardized assessments such as the Vineland Adapted Behavior Scales II (VABS II), the Social Communication Questionnaire (SCQ) and the Child or Adult Behavior Checklist (CBCL, ABCL) were used for the behavioral assessment. All patients showed a delayed developmental status. Muscular hypotonia and its manifestations were present in all patients, regardless of their age. The mean values for all VABS II domains (communication, socialization, daily living skills, and motor skills) were significantly lower than the mean of the reference population (p < 0.001), but similar to other rare intellectual disabilities such as Smith-Magenis syndrome and Angelman syndrome. The results from the SCQ indicated that all patient values exceeded the cut-off value, suggesting the possibility of autism spectrum disorder. The behavioral and emotional problems assessed by CBCL and ABCL were less frequent. In conclusion, patients with Kleefstra syndrome present with a broad range of clinical problems in all age groups and are therefore in need of a multidisciplinary follow-up also after their transition into adulthood.

Reduced Euchromatin histone methyltransferase 1 causes developmental delay, hypotonia, and cranial abnormalities associated with increased bone gene expression in Kleefstra syndrome mice

Haploinsufficiency of Euchromatin histone methyltransferase 1 (EHMT1), a chromatin modifying enzyme, is the cause of Kleefstra syndrome (KS). KS is an intellectual disability (ID) syndrome, with general developmental delay, hypotonia, and craniofacial dysmorphisms as additional core features. Recent studies have been focused on the role of EHMT1 in learning and memory, linked to the ID phenotype of KS patients. In this study we used the Ehmt1+/− mouse model, and investigated whether the core features of KS were mimicked in these mice. When comparing Ehmt1+/− mice to wildtype littermates we observed delayed postnatal growth, eye opening, ear opening, and upper incisor eruption, indicating a delayed postnatal development. Furthermore, tests for muscular strength and motor coordination showed features of hypotonia in young Ehmt1+/− mice. Lastly, we found that Ehmt1+/− mice showed brachycephalic crania, a shorter or bent nose, and hypertelorism, reminiscent of the craniofacial dysmorphisms seen in KS. In addition, gene expression analysis revealed a significant upregulation of the mRNA levels of Runx2 and several other bone tissue related genes in P28 Ehmt1+/− mice. Runx2 immunostaining also appeared to be increased. The mRNA upregulation was associated with decreased histone H3 lysine 9 dimethylation (H3K9me2) levels, the epigenetic mark deposited by Ehmt1, in the promoter region of these genes. Together, Ehmt1+/− mice indeed recapitulate KS core features and can be used as an animal model for Kleefstra syndrome. The increased expression of bone developmental genes in the Ehmt1+/− mice likely contributes to their cranial dysmorphisms and might be explained by diminished Ehmt1-induced H3K9 dimethylation.

Metallothionein 1 h tumour suppressor activity in prostate cancer is mediated by euchromatin methyltransferase 1

Metallothioneins (MTs) are a group of metal binding proteins thought to play a role in the detoxification of heavy metals. Here we showed by microarray and validation analyses that MT1h, a member of MT, is down-regulated in many human malignancies. Low expression of MT1h was associated with poor clinical outcomes in both prostate and liver cancer. We found that the promoter region of MT1h was hypermethylated in cancer and that demethylation of the MT1h promoter reversed the suppression of MT1h expression. Forced expression of MT1h induced cell growth arrest, suppressed colony formation, retarded migration, and reduced invasion. SCID mice with tumour xenografts with inducible MT1h expression had lower tumour volumes as well as fewer metastases and deaths than uninduced controls. MT1h was found to interact with euchromatin histone methyltransferase 1 (EHMT1) and enhanced its methyltransferase activity on histone 3. Knocking down of EHMT1 or a mutation in MT1h that abrogates its interaction with EHMT1 abrogated MT1h tumour suppressor activity. This demonstrates tumour suppressor activity in a heavy metal binding protein that is dependent on activation of histone methylation.

Hippocampal dysfunction in the Euchromatin histone methyltransferase 1 heterozygous knockout mouse model for Kleefstra syndrome

Euchromatin histone methyltransferase 1 (EHMT1) is a highly conserved protein that catalyzes mono- and dimethylation of histone H3 lysine 9, thereby epigenetically regulating transcription. Kleefstra syndrome (KS), is caused by haploinsufficiency of the EHMT1 gene, and is an example of an emerging group of intellectual disability (ID) disorders caused by genes encoding epigenetic regulators of neuronal gene activity. Little is known about the mechanisms underlying this disorder, prompting us to study the Euchromatin histone methyltransferase 1 heterozygous knockout (Ehmt1(+/-)) mice as a model for KS. In agreement with the cognitive disturbances observed in patients with KS, we detected deficits in fear extinction learning and both novel and spatial object recognition in Ehmt1(+/-) mice. These learning and memory deficits were associated with a significant reduction in dendritic arborization and the number of mature spines in hippocampal CA1 pyramidal neurons of Ehmt1(+/-) mice. In-depth analysis of the electrophysiological properties of CA3-CA1 synapses revealed no differences in basal synaptic transmission or theta-burst induced long-term potentiation (LTP). However, paired-pulse facilitation (PPF) was significantly increased in Ehmt1(+/-) neurons, pointing to a potential deficiency in presynaptic neurotransmitter release. Accordingly, a reduction in the frequency of miniature excitatory post-synaptic currents (mEPSCs) was observed in Ehmt1(+/-) neurons. These data demonstrate that Ehmt1 haploinsufficiency in mice leads to learning deficits and synaptic dysfunction, providing a possible mechanism for the ID phenotype in patients with KS.

A Small-Molecule Probe of the Histone Methyltransferase G9a Induces Cellular Senescence in Pancreatic Adenocarcinoma

Post-translational modifications of histones alter chromatin structure and play key roles in gene expression and specification of cell states. Small molecules that target chromatin-modifying enzymes selectively are useful as probes and have promise as therapeutics, although very few are currently available. G9a (also named euchromatin histone methyltransferase 2 (EHMT2)) catalyzes methylation of lysine 9 on histone H3 (H3K9), a modification linked to aberrant silencing of tumor-suppressor genes, among others. Here, we report the discovery of a novel histone methyltransferase inhibitor, BRD4770. This compound reduced cellular levels of di- and trimethylated H3K9 without inducing apoptosis, induced senescence, and inhibited both anchorage-dependent and -independent proliferation in the pancreatic cancer cell line PANC-1. ATM-pathway activation, caused by either genetic or small-molecule inhibition of G9a, may mediate BRD4770-induced cell senescence. BRD4770 may be a useful tool to study G9a and its role in senescence and cancer cell biology.

Familial Kleefstra syndrome due to maternal somatic mosaicism for interstitial 9q34.3 microdeletions

The Kleefstra syndrome (Online Mendelian Inheritance in Man 607001) is caused by a submicroscopic 9q34.3 deletion or by intragenic euchromatin histone methyl transferase 1 (EHMT1) mutations. So far only de novo occurrence of mutations has been reported, whereas 9q34.3 deletions can be either de novo or caused by complex chromosomal rearrangements or translocations. Here we give the first descriptions of affected parent-to-child transmission of Kleefstra syndrome caused by small interstitial deletions, approximately 200 kb, involving part of the EHMT1 gene. Additional genome-wide array studies in the parents showed the presence of similar deletions in both mothers who only had mild learning difficulties and minor facial characteristics suggesting either variable clinical expression or somatic mosaicism for these deletions. Further studies showed only one of the maternal deletions resulted in significantly quantitative differences in signal intensity on the array between the mother and her child. But by investigating different tissues with additional fluorescent in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA) analyses, we confirmed somatic mosaicism in both mothers. Careful clinical and cytogenetic assessments of parents of an affected proband with an (interstitial) 9q34.3 microdeletion are merited for accurate estimation of recurrence risk.

Epigenetic Regulation of Learning and Memory by Drosophila EHMT/G9a

Author SummaryEpigenetic regulators can affect gene transcription through modification of DNA and histones, which together form chromatin. The importance of such regulators for cognition is increasingly appreciated, but only few key factors have been identified so far. Excellent candidates are histone modifiers that are involved in intellectual disability, such as EHMT1, implicated in Kleefstra Syndrome. Here, we characterized the neuronal function of EHMT in Drosophila. Flies that lack EHMT are viable but show highly selective defects in specific aspects of neuronal development and function, including learning and memory. Genome-wide analysis of EHMT-mediated histone methylation revealed that EHMT targets the majority of all currently known Drosophila learning and memory genes. It also targets genes known to be involved in the other aspects of behavior and neuronal development that are compromised in EHMT mutants. Remarkably, EHMT mutant memory deficits can be reversed in adulthood, suggesting that epigenetic influences on cognition are not always permanent. Our results provide novel insights into the epigenetic control of cognition in health and disease.

Reduced exploration, increased anxiety, and altered social behavior: Autistic-like features of euchromatin histone methyltransferase 1 heterozygous knockout mice

The 9q34.3 subtelomeric deletion syndrome is a newly defined mental retardation syndrome, caused by haplo-insufficiency of the euchromatin histone methyltransferase 1 (EHMT1) gene. Patients also have childhood hypotonia, facial dysmorphisms, delay in reaching developmental milestones, and behavioral problems like aggressive outbursts, hypoactivity, or autistic-like features. Male and female heterozygous Ehmt1 knockout mice (Ehmt1+/−, aged 1–20 months, kept on a C57BL/6J background), were used to investigate whether they mimic the patients behavioral characteristics by comparing their behavior to wildtype littermates. The Ehmt1+/− mice revealed reduced activity and exploration, with increased anxiety compared to wildtype mice when exposed to novel environments in the open field, object exploration, marble burying, light-dark box, mirrored chamber and T-maze tests. They also demonstrated diminished social play when encountering a mouse from a different litter, and a delayed or absent response to social novelty when exposed to a stranger mouse. However, no differences in phenotyper home cage locomotor activity or rotarod motor function were observed between Ehmt1+/− and wildtype mice. Together, these results indicate that the hypoactivity and the autistic-like features of 9q34.3 subtelomeric deletion syndrome patients are recapitulated in this Ehmt1+/− mouse model, and that the hypoactivity is apparently not caused by any motor dysfunction. Together, these observations make it plausible that the Ehmt1+/− mouse is a faithful mammalian model for the autistic-like behavioral features of patients with the 9q34.3 subtelomeric deletion syndrome.

Loss-of-Function Mutations in Euchromatin Histone Methyl Transferase 1 ( EHMT1) Cause the 9q34 Subtelomeric Deletion Syndrome

A clinically recognizable 9q subtelomeric deletion syndrome has recently been established. Common features seen in these patients are severe mental retardation, hypotonia, brachycephaly, flat face with hypertelorism, synophrys, anteverted nares, cupid bow or tented upper lip, everted lower lip, prognathism, macroglossia, conotruncal heart defects, and behavioral problems. The minimal critical region responsible for this 9q subtelomeric deletion (9q-) syndrome has been estimated to be <1 Mb and comprises the euchromatin histone methyl transferase 1 gene (EHMT1). Previous studies suggested that haploinsufficiency for EHMT1 is causative for 9q subtelomeric deletion syndrome. We have performed a comprehensive mutation analysis of the EHMT1 gene in 23 patients with clinical presentations reminiscent of 9q subtelomeric deletion syndrome. This analysis revealed three additional microdeletions that comprise the EHMT1 gene, including one interstitial deletion that reduces the critical region for this syndrome. Most importantly, we identified two de novo mutations--a nonsense mutation and a frameshift mutation--in the EHMT1 gene in patients with a typical 9q- phenotype. These results establish that haploinsufficiency of EHMT1 is causative for 9q subtelomeric deletion syndrome.