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Leila J. Mady
B.Sc. 2007, New York University
Thesis Advisor: Sylvia Christakos, Ph.D.
Department of Biochemistry and Molecular Biology
Friday, May 18, 2012
10:00 A.M., MSB E609
Hormone dependent transcriptional regulation involves chromatin remodeling by coactivator proteins. Although roles of acetylation and phosphorylation in chromatin remodeling have been widely studied, recent findings have also indicated an important role for methylation. Little is known however about the exact role of methyltansferases and their regulation by different physiological signaling pathways. Using VDR transfected COS-7 and the rat 24(OH)ase promoter (-298/+74) we found that CARM1 (coactivator associated arginine methyltransferase 1) cooperates with the p160 coactivator GRIP1 to enhance 1,25(OH)2D3 induced transcription. A CARM1 mutant lacking methyltransferase activity failed to enhance 24(OH)ase activity in cooperation with GRIP1. In addition when the GRIP1 mutant ÄAD2, which lacks the binding site for CARM1, was used cooperative activation was also not observed. This result supports a role for GRIP1 as a primary coactivator or bridge to recruit the secondary coactivator, CARM1. Thus, the coactivator function of CARM1 requires methyltransferase activity and coexpression of GRIP1. CARM1 arginine specific methylation was also found to cooperate with another histone methyltransferase, G9a, to synergistically induce 24(OH)ase transcription. In order to determine the coactivator specificity of GRIP1 for CARM1, we tested another arginine methyltransferase PRMT2A. When PRMT2A was substituted for CARM1, cooperative transactivation with GRIP1 was not observed, suggesting a preferential role of CARM1 in VDR transactivation. Chromatin immunoprecipitation (ChIP) analysis indicated that dimethylation of histone H3 at arginine 17 [H3(Arg17)me2], which is mediated by CARM1, occurs in a 1,25(OH)2D3 dependent manner, further supporting the participation of CARM1 in VDR mediated gene activation. ChIP-sequencing analysis of the Cyp24A1 transcription locus revealed that H3(R17)me2 is associated with the VDR binding site in the 24(OH)ase promoter in kidney cells and that genome wide co-occurrence of VDR binding and H3(R17)me2 occurs in kidney cells in response to 1,25(OH)2D3. In AOKB50 cells transfected with the mouse 1á(OH)ase promoter, CARM1 was found to acts as a repressor of calcitonin and PRL/STAT5 mediated 1á(OH)ase transcription, supporting the role of CARM1 as a dual function coregulator. Co-expression of CEBPâ reversed the CARM1 mediated repression of calcitonin induction. These results describe the ability of CARM1 to act either as a coactivator (VDR mediated transcription) or corepressor (second messenger mediated transcription). Our findings suggest an interplay between acetylase and methylase in VDR mediated transcription and that CARM1 methylation may play a key role in the modulation of 1,25(OH)2D3 target genes.