Histone H3Lysine27Methylase And Demethylase Genes Expressinon Regulated By PI3K/AKT And MEK-ERK Pathway

Histone methylation is an important epigenetic modification, it involved in the regulation of gene expression. Histone H3K27me3is one of the epigenetic modification marks that can inhibit the transcription of target genes. The current study found that histone H3K27me3was regulated by the histone methylase and demethylase. Zester homologous enhancer2is the histone H3K27methyltransferase and EZH1is the homologous protein, both they are the members of the Polycomb family proteins. UTX and JMJD3are histone H3K27me3demethylase. It has been reported PI3K/AKT signaling pathway could regulate the expression of EZH2gene. But whether this signaling pathway could regulate the histone H3K27methylase and demethylase expression during skeletal muscle differentiation is not clear. In this study, the murine C2C12myoblast cell line was used to investigate that the PI3K/Akt signaling pathway regulate the histone H3K27the methylase EZH1/EZH2and demethylase UTX/JMJD3during the skeletal muscle differentiation. In addition, EZH2overexpression occurs in various malignancies and UTX and JMJD3were also be detected mutation or the expression of them was decreased. MEK/ERK signaling pathway was widespread in vivo,it involved in the regulation of cell growth, proliferation, differentiation apoptosis and so on. In same cancer cells, MER/ERK signaling pathway leads to EZH2overexpression. In order to further study this pathway role on regulation of histone methylase and demethylase. We use C2C12,C127, NIH3T3, HEPA1-6, RD five cell lines to investigate MER/ERK signaling pathway regulate the H3K27methylase EZH2/EZH1and demethylase UTX/JMJD3genes expression. The results are as follows:Western Blotting analysis showed Phosphorylated AKT gradually increased during the skeletal muscle differentiation process, EZH1and JMJD3protein expression did not change significantly,the UTX protein expression was increased and the expression of EZH2protein reduced.when C2C12cell differentiated after6d or7d,EZH2protein expression reached to a minimum value.The expression of Myogenin and MCK protein was increased significantly after PI3K/AKT activator treatment for24h during the C2C12cell differentiation. Chromatin immune precipitation (ChIP) and Quantitative PCR (Q-PCR) analysis showed that the enrichment of H3K27me3on the promoter regions of Myogenin and MCK genes and the enhancer region of MCK gene were decreased. It was opposite to the PI3K/AKT inhibitor treatment.UTX increased slightly after PI3K/AKT activator treatment for24h and UTX and EZH2both were decreased after PI3K/AKT inhibitor treatment for24h during the C2C12cell differentiation.EZHland JMJD3did not change significantly.RT-PCR and Western Bloting methods to detect the expression of EZH1, EZH2, UTX and JMJD3genes with the different concentrations of the MEK-ERK inhibitor U0126(0,10,20,40umol/L) in five cell lines C2C12C127NIH3T3HEPA1-6RD. The expression of H3K27mehylase genes EZH1and EZH2was decreased in the five cell lines compared to the control and the change of EZH2gene expression reached the significance leve(P<0.05) in C2C12, NIH3T3, HEPA1-6, RD four cell lines. The expression of UTX and JMJD3genes was increased and that of JMJD3gene reached the significance level(P<0.05).EZH2protein expression reduced, but in C127cell line it did not change. JMJD3protein expression increased except RD cell line.EZH1and UTX protein expressed both in the cytoplasm and nucleus. EZH2protein mainly expressed in the nucleus, JMJD3mainly expressed in the cytoplasm with Immunofluorescence detection in HEPA1-6cell and did not find EZH2, EZH1, UTX, JMJD3protein expression change after MEK/ERK inhibitor U0126treated for24h. Western Bloting analysis show in HEPA1-6and RD cell nucleus EZH1expression did not change significantly, EZH2protein expression was significantly reduced. In HEPA1-6cell nucleus, the UTX and JMJD3protein expression was elevated but it did not change significantly in RD cell line.Western Bloting to detect the protein expression in HEPA1-6cell with the MEK-ERK inhibitor U0126in different points of time, after treated for2h P-ERK1/2reduced and the subsequent expression level gradually increased, to24h it rises to the maximum, but still reduced compared with the control. EZHlhas no obvious change. The EZH2protein expression was decreased significantly after treated for8h, after24h the protein reduced to a minimum. JMJD3and UTX protein expression increased to a maximum after treated for4h, and subsequent weakening.HEPA1-6cell was interfered by si-RNA. EZH2and EZH1protein reduced, EZH1did not change significantly and The protein expression of UTX and JMJD3was significantly increased, UTX protein by siRNA-ERK1interference is not obvious.So we concluded that PI3K/AKT signaling pathway may be involved in the regulation of EZH2and UTX gene expression during the myoblast differentiation regulation, but there may be other signaling pathways joined in. MEK/ERK signaling pathway may be involved in the regulation of EZH2, UTX and JMJD3genes expression and has no obvious role for EZH1but this regulation has the cell specificity and time sequence.