Regulating Mechanism Of Carm1in P19Cells Neural Differentiation

Epigenetics studies all heritable changes in gene expression and functions without a change in DNA sequence, including chromatin remodeling, histone modification, DNA methylation and so on. Methylation at arginines has come to the attention of cell biologists through a series of genetic and molecular biology experiments that implicated the important roles of arginine methylation in signaling and transcription activation. Arginine is a positively charged amino acid that can mediate hydrogen bonding and amino-aro-matic interactions. The nitrogens of arginine within polypeptides can be modified to contain methyl groups in posttranslation, a process termed arginine methylation. One or two methyl groups was added to the guanidine nitrogen atoms of arginine in the process of protein arginine methylation. There are three main forms of methylated arginine in eukaryotes:ω-NG,monomethylarginines(MMA);ω-NG,NG-asymmetric dimethylarginines (aDMA);ω-NG,N’G-symmetric dimethylarginines (sDMA). PRMTs are classified as either Type Ⅰ or Type Ⅱ enzymes. Type Ⅰ PRMTs (PRMT1, PRMT2, PRMT3, PRMT4, and PRMT6) generate the production of aDNA, whereas Type Ⅱ PRMTs(PRMT5and PRMT7) catalyze the production of sDNA.Carml (Coactivator-associated Arginine methylase, also referred to as PRMT4) belongs to Type Ⅰ PRMTs and has many different substances including histone and non-histone. Carml plays an important role in transcriptional coactivator activity and pre-mRNA splice. Recently, more and more attention is paid to the function of Carml in development and differentiation. Gene targeting of Carml in mice has been performed, and knock-out mice, which are smaller than their wild-type littermates, die just after birth. An enzyme-dead knock-in of Carml cells and mice have defects similar to those seen in their knock-out counterparts involving the time of embryo lethality, T cell development, adipocyte differentiation, and transcriptional coactivator activity. It seems that Carml requires its enzymatic activity for all of its known celluar function.1. Detection of Carml mRNA expression in P19cells neural differentiationTeratocarcinoma P19cells were cultured in medium with0.5μM RA for4days and then in medium without RA for another4days. The morphology of P19neurons is similar to cultured brain cells. P19neurons have small cell bodies with long, elaborate processes similar to axons and dendrites in appearance. Western blot showed that TuJ1increased gradually during the period of P19cells neural differentiation. Quantitative Real-time RT-PCR analysis of Carml mRNA expression in RA induced P19different time points, the result showed the Carml mRNA level change significantly in early differentiation.2. Carml plays an important part in P19cells neural differentiationGenerate P19Carml Stable knockdown Cell Lines and then induce them with RA. Quantitative Real-time RT-PCR analysis and Immunostaining detect the early neural mark gene Mash1. The result showed Mash1mRNA level was influenced by Carml. At the same time, immunostaining analyzed the late neural mark gene TuJ1and the result showed that TuJ1was reduced dramatically in P19Carml Stable knockdown Cell Lines compared with control group.3. Detection of the dimethylation degree of Sox2in P19Carml Stable knockdown Cell LinesQuantitative Real-time RT-PCR analysis and western blot showed the mRNA and protein level of Oct4,Sox2,Nanog did not change siginificantly. But the dimethylation degree of Sox R113decreased obviously.4. Analysis of the dimethylation degree of H3R2in P19Carml Stable knockdown Cell LinesWestern blot analyzed the dimethylation degree of H3R17and H3R2in P19Carml Stable knockdown Cell Lines and the result showed H3R17me2did not change, however H3R2me2decreased dramatically in P19Carml Stable knockdown Cell Lines.5. Test of Carml-responsive, ERα-target gene for expression in P19Carml Stable knockdown Cell LinesEstrogen-respomsive gene expression was aberrant in Carm-/-fibroblasts and embryos, thus emphasizing the characteristic of Carml as a transcriptional coactivator. To assess Carml-responsive estrogen-respomsive gene expression in P19Carml Stable knock down Cell Lines by Quantitative Real-time RT-PCR analysis, the result showed that Stc2was reduced dramatically in P19Carml Stable knockdown Cell Lines.Generally speaking, Carm1plays an important part in P19cells neural differentiation. The mechanism of Carm1in P19cells neural differentiation includes:1. Carml methylates main pluripotency factors such as Sox2and regulates the P19cells neural differentiation.2. Carml regulate H3R2me2of some gene involving neural mark genes or pluripotency genes to function in P19cells neural differentiation.3. Carml regulate the Carm1-resposive ERα-target gene to influence P19cells neural differentiation.