Protein Arginine Methyltransferase (prmt) In Rd Cells Induced Differentiation Mechanism Of Action

The N-terminal tails of core histones in a nucleosome are covalently modified in vivo in a variety ways, such as acetylation, phosphorylation, methylation, and ubiquitination, etc. Histone modification plays a pivotal role in the regulation of eukaryotic gene transcription mainly by changing the interactions between nuclear proteins and DNA sequences in a nucleosome. Despite that the histone methylation has been known for over 35 years, its role in gene transcription remains unclear. In the past decade, several families of enzymes that act to methylate either arginine or lysine residues in the histones have been identified. Protein arginine methyltransferases (PRMTs) are a family of enzymes that share a highly conserved domain encompassing methyltransferase activity. PRMTs are capable of methylating a broad spectrum of substrates including histones, transcription factors and other proteins functions RNA processing, nucleo-cytoplasmic transport and DNA damage repair.Rhabdomyosarcoma, the most common pediatric soft tissue sarcoma, arises from skeletal muscle progenitor cell with high malignancy. RD is an embryonic rhabdomyosarcoma cell line that is taken as a model in this study, in which, despite the expression of MyoD and myogenin, is not efficient in terminal differentiation. TPA (12-O-tetradecanoyl-phorbol-13-acetate) is used here to induce RD cell differentiation and to investigate the functions of PRMTs therein.1. Expression of PRMTs during cell differentiationAfter treated with 100nM TPA, flowcytometry analyses show G2/M is increased and the cell cycle is arrested. Real-time RT-PCR, Western Blot experiments indicate PRMTs are expressed in RD cells and the expression of PRMTs increases during cell differentiation. The level of histone arginine methylation increases. Immunofluorescence analyses show RD cells acquire typical myogenic modality gradually after TPA inducing, such as a more elongated shape, more granule materials. The subcellular distributions of PRMTs are cytoplasm, then they enter nucleus after differentiation.2. The effect of PRMTs on the expression of myogenin during cell differentiation1) We construct PRMT-siRNA plasmids, PRMT5, PRMT6 expression plasmids [pCDNA6-PRMT5 (/PRMT6)-FLAG], and verify the availability of them.2) We use 20μM as the concentration of PRMT inhibitor AdOx in RD cells.3) Flowcytometry analyses show after AdOx treatment, the cells arrest at G2/M phase. The expression of myogenin is inhibited shows the cells fail to complete differentiation. Immunofluorescence analyses show RD cells did not acquire typical myogenic modality. The expression of PCAF mRNA and protein increases during cell differentiation and is restrained by AdOx. But the expression of Brgl, the core unit of SWI/SNF chromatin remodeling complex, does not change during differentiation or AdOx treatment. The analysis of promoter activity shows that TPA can activate the promoter activity of myogenin and AdoMet-mediated arginine methylation is indispensable for the full expression of myogenin. All the experiments of adenosine dialdehyde suggest that arginine methylation is necessary for (i) morphological differentiation, (ii) activation of essential transcription factors, and (iii) activation of myogenic relative factors.4) Transfecting RD cells with CARM1siRNA, we find (i) the expression and nuclear translocation of myogenin and PCAF protein is inhibited. (ii) RD cells do not acquire typical myogenic modality. (iii) The promoter activity of myogenin gene is obviously restrained. Overexpressing CARM1 makes the expression and nuclear translocation of myogenin, PCAF and Brgl obviously increase. In summary, protein arginine methyltransferase activity is necessary for the activation of critical transcription factors and cofactors involved in RD cells terminal differentiation.5) The assay of promoter activity of myogenin illuminates that Brgl can effectively increase myogenin promoter activity during RD cell differentiation and CARM1 is indispensable for this effect of Brg1. CARM1 may also cooperate with PCAF and p300 to regulate the expression of myogenin.3. The effect of PRMTs on the binding of chromatin modifiers on the promoter of myogenin during RD cell differetiation1) Chromatin Immunoprecipitation (ChIP) shows CARM1 can not bind the promoter of myogenin in RD cells. After induced by TPA for 3h, it begins to bind the myogenin promoter. The p38 inhibitor SB203580 can not restrain the binding of CARM1, which indicates the recruitment of CARM1 to the promoter is not p38-dependent.2) Histone H3 is acetylated at a low degree in RD cells, but the arginine methylation of histone H4 is very little. After induced by TPA, both the acetylation of H3 and arginine methylation of H4 is increased. They can be inhibited by SB or AdOx treatment.3) BAF60 and Brg1 can be recruited to the promoter of myogenin during RD cell differentiation, while they did not bind in normal RD cells. The binding is dependent on p38 and AdoMet-mediated methylation.4) P300 can bind the promoter of myogenin in RD cells. TPA inducement or SB treatment can not influence the binding, but AdOx can obviously inhibit it. PCAF can be recruited to the promoter during RD cell differentiation, whereas it can not bind the promoter in normal RD cells. The binding is p38-independent and depends on AdoMet-mediated methylation.5) MyoD can bind the promoter of myogenin in normal RD cells, and no matter TPA, SB or AdOx treatment can not influence the binding. PRMT5 can bind the myogenin promoter only when RD cells are induced by TPA. The binding is dependent on p38 and AdoMet-mediated methylation.6) RNA polymerization enzyme polⅡcan bind the promoter of myogenin at a low degree in RD cells, and the binding can be increased after TPA inducement. Both SB and AdOx treatment can partially influence the binding.In summary, PRMTs play an enhancing role in regulating the RD cells in TPA induced differentiation. Histone arginine methylated by PRMTs along with other histone modifications and the activation of transcription factors that jointly exert an increased expression of myogenin gene and lead the RD cells to differentiation. The regulatory functions of PRMTs in RD cell differentiation shed lights on novel target in drug development and its application to make the rhabdomyosarcoma reversal in the clinic.