The Role Of Histone Methyltransferase KMT2D On The Regulation Of Downstream Genes Transcription Of HIF-1? In Cardiomyocytes

Background:Histone-lysine N-methyltransferase 2D(KMT2D)is a major Histone 3 lysine 4(H3K4)methyltransferase.It is widely expressed in adult tissues and plays critical roles in regulating embryonic development,differentiation and metabolism.H3K4 methylation,a type of histone modification,marks genes activation by binding onto gene promoters or enhancers.Previous studies have reported that knockout of Kmt2d in murine results in severe cardiac developmental defects and embryonic death.Our previous study observed that myocardial infarct size was increased after myocardial ischemia(MI)in Kmt2d cardiac-conditional knockout mice compared to the wild-type mice,suggesting that KMT2D had a cardioprotective effect to MI.Hypoxia-inducible factor 1?(HIF-1?)is a key transcription factor regulating cellular responses to hypoxia.Under myocardial ischemia and hypoxia condition,HIF-1 a expressiori is up-regulated,which can change the expression of related genes and reduce myocardial injury.Recent studies have revealed that several factors selectively regulate expressions of HIF-1 target genes,and epigenetic modifications have been known as the main players of gene regulation.Previous studies have reported that histone methylation or acetylation could modulate HIF-1? stability and activity.However,it is unclear whether KMT2D is involved in the transcriptional regulation of HIF-1? for its target genes under hypoxic condition.Objective:To explore the role of histone-lysine N-methyltransferase 2D(KMT2D)in H9c2 cardiomyocytes against hypoxia and its effect on the regulation of downstream genes transcription of hypoxia-inducible factor-la(HIF-la).Methods:(1)Established a hypoxic culture system and determined the proteins level under hypoxic condition.Rat-derived cardiomyocytes H9c2 were cultured in a 1%O2 and 5%CO2 incubator at 37?,and cultured in hypoxic condition for 0 h,4 h,8 h,16 h,and 24 h to induce HIF-la protein accumulation.Nuclear and cytoplasmic proteins were extracted with NE-PERTM Nuclear and Cytoplasmic Extraction Reagents Kits.The expression levels of HIF-la,P300,KMT2D and H3K4mel at different time points were determined by western blotting.(2)Detected the transcriptional activity of HIF-1? downstream genes.To determine the mRNA expression levels of Vegf and Bnip3 by real-time quantitative PCR(qRT-PCR).(3)After hypoxia treatment of H9c2 cells for 16 h,the enrichment of H3K4mel and H3K27ac on the promoters of Hif-1? and Vegf were detected by ChIP-qPCR.(4)Explored the role of KMT2D in the transcriptional activity of HIF-1? downstream genes.Knockdown Kmt2d by small interfering RNA(siRNA),and then divide the cells into four groups:Negative siRNA(as negative control,NC)group,Negative siRNA+Hypoxia(NC+H)group,siKmt2d group,siKmt2d+Hypoxia(siKmt2d+H)group.Then siRNAs were transfected into H9c2 cells for 48 h and the cells were treated with hypoxia for 8h.The protein levels of H3K4me1 and HIF-1? were detected by western blotting,and the mRNA expression levels of Kmt2d,Vegf,Bnip3 and Glut1 were analyzed by qRT-PCR.(5)Treated H9c2 cells with histone demethylation LSD1 inhibitor(RN-1)and detected the expression of H3K4mel under hypoxic condition.The cells were treated with different concentration of LSD1 inhibitor and then selected the effective dose.With silenced Kmt2d for 48 h and added LSD1 inhibitor 24 h,the cells were hypoxia for 8 h,then the protein levels of H3K4me1 were detected by western blotting.Results:(1)The protein levels of HIF-1?/P300/KMT2D/H3K4mel in the H9c2 cells were enhanced under hypoxic condition.The accumulation of HIF-1? protein was induced,and reached the highest level at 8 h and then gradually decreased at 16 h and 24 h.P300 was significantly increased at 4 h and 8 h under hypoxic conditions.The KMT2D protein was achieved the highest level at 8 h and kept its high expression at both 16 h and 24h,while H3K4me1 protein level was significantly up-regulated at 16 h.(2)The transcription level of HIF-1? downstream genes were up-regulated.Vegf was significantly increased at 4 h after hypoxia treatment and reached the highest level at 24 h,and Bnip3 was up-regulated at 4 h and achieved the highest level at 16 h,but the expression was slightly decreased at 24 h.(3)ChIP-qPCR results indicated that an increase in the enrichment of H3K4mel on the promoter of Vegf after hypoxia for 16 h.The binding of H3K27ac on the promoter of Hif-1? and Vegf were increased.(4)After Kmt2d was silenced,the expression of HIF-1? downstream genes Vegf and Glut1 were affected.We observed the mRNA level of Kmt2d significantly down-regulated;compared to the NC+H group,the protein level of H3K4mel was reduced in the siKmt2d+H group.The protein level of HIF-la in the siKmt2d+H group was not upregulated compared to the siKmt2d group.The transcriptional levels of Vegfhad no difference between the NC group and the siKmt2d group,but it was decreased in the siKmt2d+H group compared to the NC+H group.However,there was no change in Bnip3 expression between the NC group and the siKmt2d group under normoxia or hypoxia.The transcription level of Glut1 between the NC group and the siKmt2d group did not change,whereas,the expression in the siKmt2d+H group was significantly up-regulated compared with the NC+H group.(5)Under normoxic conditions,4?M LSD1 inhibitor could increase the protein expression of H3K4me1,whereas,the level of H3K4me1 was not affected in the cell after siKmt2d knocked-down and treated with 4?M LSD 1 inhibitor(RN-1)under hypoxia for 8 h.Conclusion:Under hypoxic condition,the histone methyltransferase KMT2D regulates the transcriptional activity of HIF-1? downstream genes,and KMT2D may contributes to the selective regulation of HIF-1? downstream genes.