The Mechanism Of Gene Transcription Facilitated By Histone Arginine Methyltransferase CARM1

Protein Arginine Methyltransferase4(PRMT4), is also referred to as Coactivitor-Associated Arginine Methyltransferase1(CARM1). It can catalyze methylation at Arg-2, Arg-17and Arg-26in the N-terminal region and Arg-128, Arg-129, Arg-131and Arg-134in the C-terminus of hisone H3. It can also methylate histone H2A. Two of the N-terminal sites of histone H3, Arg-17and Arg-26, are the major sites for methylation, and the methylation of these sites affect gene transcription. CARM1acts as a transcriptional coactivator in the context of NR-mediated transcriptional activation. Under hormone stimulation, the association of the primary coactivator p160with NR leads to the recruitment of secondary coactivators PRMT1, CBP/p300and CARM1. Methylation of H4R3by PRMT1has been shown to stimulate acetylation of H3K14by CBP/p300. Acetylation in turn enhances CARM1methylation of H3R17, a modification that associated with transcriptional activation. In addition, CARM1can also associate with chromatin remodeling complex and stimulate its ATPase activity. Together, these processes lead to the activation of gene transcription.The mechanism that CARMl facilitates gene transcription after methylation at H3R17and H3R26is not clear yet. This thesis is aiming to address the mechanism of gene transcription facilitated by CARM1.In effort to identify effectors that bind specifically to R17and R26methylated H3peptide, we found that R17and R26methylation significantly reduced the binding of nuclear proteins compared with unmodified histone H3peptide in an in vitro pull-down assay. Through mass spectrometry analysis, we identified proteins, whose binding were significantly affected, including MTA1、TIF1α and TIF1γ which belong to transcriptional corepressors. These results were verified by autoradiography and Western blot. These works suggest that CARM1may promote gene transcription by reducing the recruitment of corepressors to histones in vivo.To test if CARM1-mediated histone methylation affects association of corepressors with chromatin, we made use of CARM1knockout cells. Chromatin and histones of CARM1(+/+) MEF cells and CARMl (-/-) MEF cells were extracted, resolved by SDS-PAGE, and followed by Western blot to check the states of corepressors. We found that the chromatin and histones prepared from CARM1(-/-) MEF cells contained more corepressors compared with chromatin and histones from CARM1(+/+) MEF cells.We also tested if overexpression of CARM1would reduce its association of corepressors with chromatin. We successfully established Flp-In. T-Rex CARMlinducible stable cell line. The cells were treated with doxycyline hyclate for6h to induce CARMl overexpression. Chromatin was prepared from induced and uninduced cells and subjected to Western blot for associated corepressors. We found that the binding of corepressors with chromatin of induced cells was reduced comparing to uninduced cells.In conclusion, through the experiments in vitro and in vivo, we show the evidence that CARM1may facilitate transcription by R17and R26methylation-mediated disassociation of corepressors from chromatin.