Role Of Histone Methyltransferase G9a Research Articles

Oxidative stress-induced aberrant G9a activation disturbs RE-1-containing neuron-specific genes expression, leading to degeneration in human SH-SY5Y neuroblastoma cells.

Oxidative stress-induced neurodegeneration is one of several etiologies underlying neurodegenerative disease. In the present study, we investigated the functional role of histone methyltransferase G9a in oxidative stress-induced degeneration in human SH-SY5Y neuroblastoma cells. Cell viability significantly decreased on H2O2 treatment; however, treatment with the G9a inhibitor BIX01294 partially attenuated this effect. The expression of neuron-specific genes also decreased in H2O2-treated cells; however, it recovered on G9a inhibition. H2O2-treated cells showed high levels of H3K9me2 (histone H3 demethylated at the lysine 9 residue), which is produced by G9a activation; BIX01294 treatment reduced aberrant activation of G9a. H3K9me2 occupancy of the RE-1 site in neuron-specific genes was significantly increased in H2O2-treated cells, whereas it was decreased in BIX01294-treated cells. The differentiation of H2O2-treated cells also recovered on G9a inhibition by BIX01294. Consistent results were observed when used another G9a inhibitor UCN0321. These results demonstrate that oxidative stress induces aberrant activation of G9a, which disturbs the expression of neuron-specific genes and progressively mediates neuronal cell death. Moreover, a G9a inhibitor can lessen aberrant G9a activity and prevent neuronal damage. G9a inhibition may therefore contribute to the prevention of oxidative stress-induced neurodegeneration.

Inhibition of histone methyltransferase G9a attenuates liver cancer initiation by sensitizing DNA-damaged hepatocytes to p53-induced apoptosis

While the significance of acquired genetic abnormalities in the initiation of hepatocellular carcinoma (HCC) has been established, the role of epigenetic modification remains unknown. Here we identified the pivotal role of histone methyltransferase G9a in the DNA damage-triggered initiation of HCC. Using liver-specific G9a-deficient (G9aΔHep) mice, we revealed that loss of G9a significantly attenuated liver tumor initiation caused by diethylnitrosamine (DEN). In addition, pharmacological inhibition of G9a attenuated the DEN-induced initiation of HCC. After treatment with DEN, while the induction of γH2AX and p53 were comparable in the G9aΔHep and wild-type livers, more apoptotic hepatocytes were detected in the G9aΔHep liver. Transcriptome analysis identified Bcl-G, a pro-apoptotic Bcl-2 family member, to be markedly upregulated in the G9aΔHep liver. In human cultured hepatoma cells, a G9a inhibitor, UNC0638, upregulated BCL-G expression and enhanced the apoptotic response after treatment with hydrogen peroxide or irradiation, suggesting an essential role of the G9a-Bcl-G axis in DNA damage response in hepatocytes. The proposed mechanism was that DNA damage stimuli recruited G9a to the p53-responsive element of the Bcl-G gene, resulting in the impaired enrichment of p53 to the region and the attenuation of Bcl-G expression. G9a deletion allowed the recruitment of p53 and upregulated Bcl-G expression. These results demonstrate that G9a allows DNA-damaged hepatocytes to escape p53-induced apoptosis by silencing Bcl-G, which may contribute to the tumor initiation. Therefore, G9a inhibition can be a novel preventive strategy for HCC.

Radiation-induced H3K9 methylation on E-cadherin promoter mediated by ROS/Snail axis : Role of G9a signaling during lung epithelial-mesenchymal transition

Lung cancer patients who have undergone radiotherapy developed severe complications such as pneumonitis and fibrosis. Upon irradiation, epithelial cells acquire mesenchymal phenotype via a process called epithelial to mesenchymal transition (EMT), which plays a vital role in organ fibrosis. Several mechanisms have been studied on EMT, however, the correlation between radiation-induced EMT and epigenetic changes are not well known. In the present study, we investigated the role of histone methyltransferase G9a on radiation-induced EMT signaling. There was an increase in total global histone methylation level in irradiated epithelial cells. Western blot analysis on irradiated cells showed an increased expression of H3K9me2/3. The pre-treatment of G9a inhibitor enhanced E-cadherin expression and decreased the mesenchymal markers like N-cadherin, vimentin in the radiated group. Surprisingly, radiation-induced ROS generation and pERK1/2 levels were also inhibited by G9a inhibitor BIX01294, which is showing its antioxidant potential. The ChIP-qPCR analysis on the E-cadherin promoter suggested that G9a and Snail might have formed complex to enrich suppressive marker H3K9me2/3. On the whole, our present study suggested that 1] ROS could modify H3K9 methylation via G9a and promote radiation-induced lung EMT in Beas2B and A549 cells 2] E-cadherin promoter enrichment with heterochromatin mark H3K9me2 expression upon irradiation could be modified by regulating G9a methyltransferase.

Histone methyltransferase G9a protects against acute liver injury through GSTP1

Acute liver injury is commonly caused by bacterial endotoxin/lipopolysaccharide (LPS), and by drug overdose such as acetaminophen (APAP). The exact role of epigenetic modification in acute liver injury remains elusive. Here, we investigated the role of histone methyltransferase G9a in LPS- or APAP overdose-induced acute liver injury. Under d-galactosamine sensitization, liver-specific G9a-deficient mice (L-G9a−/−) exhibited 100% mortality after LPS injection, while the control and L-G9a+/− littermates showed very mild mortality. Moreover, abrogation of hepatic G9a or inhibiting the methyltransferase activity of G9a aggravated LPS-induced liver damage. Similarly, under sublethal APAP overdose, L-G9a−/− mice displayed more severe liver injury. Mechanistically, ablation of G9a inhibited H3K9me1 levels at the promoters of Gstp1/2, two liver detoxifying enzymes, and consequently suppressed their transcription. Notably, treating L-G9a−/− mice with recombinant mouse GSTP1 reversed the LPS- or APAP overdose-induced liver damage. Taken together, we identify a novel beneficial role of G9a-GSTP1 axis in protecting against acute liver injury.

Essential Role of Histone Methyltransferase G9a in Rapid Tolerance to the Anxiolytic Effects of Ethanol.

Tolerance to ethanol-induced anxiolysis promotes alcohol intake, thus contributing to alcohol use disorder development. Recent studies implicate histone deacetylase-mediated histone H3K9 deacetylation in regulating neuropeptide Y expression during rapid ethanol tolerance to the anxiolytic effects of ethanol. Furthermore, the histone methyltransferase, G9a, and G9a-mediated H3K9 dimethylation (H3K9me2) have recently emerged as regulators of addiction and anxiety; however, their role in rapid ethanol tolerance is unknown. Therefore, we investigated the role of G9a-mediated H3K9me2 in neuropeptide Y expression during rapid ethanol tolerance. Adult male rats were administered one injection of n-saline followed by single acute ethanol injection (1 g/kg) 24 hours later (ethanol group) or 2 injections (24 hours apart) of either n-saline (saline group) or ethanol (tolerance group). Anxiety-like behaviors and global and Npy-specific G9a and H3K9me2 levels in the amygdala were measured. Effects of G9a inhibitor (UNC0642) treatment on behavioral and epigenetic measures were also examined. Acute ethanol produced anxiolysis and decreased global H3K9me2 and G9a protein levels in the central and medial nucleus of the amygdala as well as decreased occupancy levels of H3K9me2 and G9a near a putative binding site for cAMP-response element binding protein on the Npy gene. Two identical doses of ethanol produced no behavioral or epigenetic changes relative to controls, indicating development of rapid ethanol tolerance. Interestingly, treatment with UNC0642, before the second ethanol dose reversed rapid ethanol tolerance, decreased global H3K9me2 and increased neuropeptide Y levels in the central and medial nucleus of the amygdala. These results implicate amygdaloid G9a-mediated H3K9me2 mechanisms in regulating rapid tolerance to the anxiolytic effects of ethanol via neuropeptide Y expression regulation.

Abstract 419: The role of histone methyltransferase G9a in chemotherapeutic drugs-induced DNA damage

Abstract Head and neck squamous cell carcinoma (HNSCC) is one of the serious life-threatening diseases in the world. In addition to surgery, the conventional treatments for oral cancer includes radiation and chemotherapy, which usually act by induction of DNA damage. However, approximately one-third of treated patients will experience local or regional recurrence. G9a is a histone methyltransferase responsible for mono- or di-methylation of lysine 9 in histone H3. Our recent work has demonstrated that G9a level is highly correlated with aggressiveness and poor prognosis of lung, ovarian and colon cancer patients in Taiwanese population. Here, we found that G9a expression in HNSCC is upregulated and positively correlated with poor prognosis. Moreover, knockdown of G9a resulted in decrease cell survival and cologenic capacity following treating with chemotherapeutic drug, hydroxyurea and etoposide. Futhermore, depletion of G9a caused sustained phosphorylation of γ-H2AX after etoposide treatment. Together, We suggest that G9a may contribute to HNSCC proliferation by improvement of DNA double break repair. Our study paves the way for exploring the blockade of G9a overexpression as a novel approach for the prevention and treatment of HNSCC. Citation Format: Chia-Wen Liu. The role of histone methyltransferase G9a in chemotherapeutic drugs-induced DNA damage. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 419. doi:10.1158/1538-7445.AM2014-419

Role of Histone Methyltransferase G9a in CpG Methylation of the Prader-Willi Syndrome Imprinting Center

Imprinted genes in mammals are often located in clusters whose imprinting is subject to long range regulation by cis-acting sequences known as imprinting centers (ICs). The mechanisms by which these ICs exert their effects is unknown. The Prader-Willi syndrome IC (PWS-IC) on human chromosome 15 and mouse chromosome 7 regulates imprinted gene expression bidirectionally within an approximately 2-megabase region and shows CpG methylation and histone H3 Lys-9 methylation in somatic cells specific for the maternal chromosome. Here we show that histone H3 Lys-9 methylation of the PWS-IC is reduced in mouse embryonic stem (ES) cells lacking the G9a histone H3 Lys-9/Lys-27 methyltransferase and that maintenance of CpG methylation of the PWS-IC in mouse ES cells requires the function of G9a. We show by RNA fluorescence in situ hybridization (FISH) that expression of Snrpn, an imprinted gene regulated by the PWS-IC, is biallelic in G9a -/- ES cells, indicating loss of imprinting. By contrast, Dnmt1 -/- ES cells lack CpG methylation of the PWS-IC but have normal levels of H3 Lys-9 methylation of the PWS-IC and show normal monoallelic Snrpn expression. Our results demonstrate a role for histone methylation in the maintenance of parent-specific CpG methylation of imprinting regulatory regions and suggest a possible role of histone methylation in establishment of these CpG methylation patterns.