| Epigenetics is the study of stable alternation in gene expression and function without changing DNA sequence and leading to inheritable phenotype, mainly including DNA methylation, histone modification, chromatin remodeling and non-coding RNA-mediated regulation. Histone modifications including histone methylation, acetylation, phosphorylation and ubiquitination play crucial roles in gene activation and inactivation.Mashl, one of bHLH protein, play a crucial role in neuronal differentiation. To investigate the epigenetic regulation especially histone modification of Mash1 gene during neurogenesis, RA (all-trans retinoic acid)-induced P19 embryonal carcinoma cells were used as a neuronal differentiation model.1. The establishment of RA-incduced neuronal differentiation of P19 cell modelP19 cells were exposed to 0.5μM RA, cells began to aggregate. After 4 days, P19 cells cultured in medium without RA for another 3 or 4 days to differentiate into neuron-like cells. Western blotting assay showed that neuronal marker geneβ-Tubulin increased gradually with RA induced. These results indicated that RA-incduced neural differentiation of P19 cells model was established.2. Analysis of neuronal marker gene Mashl expressionQuantitative RT-PCR and Western blotting assay showed that Mashl greatly increased after 2 days of RA treatment at mRNA and protein levels. Immunofluoresense assay showed that Mashl increased after RA induction. Simultaneously, RNA polymeraseⅡ(RNA PolⅡ) was enriched on the promoter of Mashl analyzed by chromatin immunoprecipitation assay (ChIP). These results suggested that the expression of Mashl gene was activated by RA induction in P19 cells.3. Alternation of histone modifications in Mash1 gene region with RA treatment in P19 cellsCovalent modifications of histones are key regulations of chromatin dynamics, and play pivotal roles in genes regulation. The N-terminal tail of core histones, as well as positions in the globular domain, can carry post-translational modifications such as acetylation, phosphorylation, ubiquitination, methylation, SUMOylation and ADP-ribosylation. Such modifications can alter DNA-histone interactions within and between nucleosomes and affect gene activation.ChIP assays were performed to investigate changes of histone modifications in Mash1 gene region during RA treatment. It was found that acetylation of histone H3K9 and H3K14 significantly increased, especially in the 4th day. Methylation is another important modification of histones, which is considered pivotal in epigenetic regulation. We analysed the methylation of histone H3K9me3, H3K4me3 and H3K27me3 in P19 cells during RA treatment by ChIP assays. It was found that H3K9me3 had no significant change. H3K4me3 increased steadily, especially in the proximal promoter region and exon region of the gene. H3K27me3 decreased gradually especially after 3rd day of RA treatment. These results suggested histone modifications play privotal roles in Mashl activation.4. Histone lysine H3K27 demethylase Jmjd3 enhanced Mashl expression.The mRNA and protein expression of Jmjd3, histone lysine H3K27 demethylase increased after RA treated while H3K27me3 level of Mashl, neuronal differentiation marker gene, decreased dramatically. Overexpression of Jmjd3 enhanced Mashl expression. Knockdown of Jmjd3 inhibited Mashl expression. Dual-luciferase reporter assay showed that Jmjd3 enhanced Mashl promoter activity. ChIP assay suggested remarkably elevated recruitment of Jmjd3 at the proximal promoter of Mash1 on the 3rd day of RA treatment led to an increased promoter activity of Mashl. Demethylase mutant of Jmjd3 abolished Mashl activation. These results suggested elevation of Jmjd3 expression and its demethylase activity was required to enhance the expression and promoter activity of Mashl in RA-induced neuronal differentiation of P19 cells. Demethylase activity of Jmjd3 was the prerequisite in RA induced Mashl expression.
|
PDF Full Text Request