Nuclear receptors (NR) activate transcription by recruiting complexes of coactivators which locally remodel chromatin structure in the promoter and help to recruit and activate an RNA polymerase II complex. Multiple complexes of coactivators participate in transcription initiation, and some recruited coactivators may also facilitate the subsequent coupled steps of transcription elongation and RNA processing. There is clear evidence for physiological relevance of one coactivator complex, which includes the p160 family (GRIP1, SRC-1, and p/CIP), the protein acetyltransferases p300 and CBP, and the protein arginine methyltransferases CARM1 and PRMT1. The p160 coactivators bind directly to NRs and recruit the acetyltransferases and methyltransferases, which modify histones and other proteins in the transcription complex. Another coactivator, PGC-1, is an example of a tissue specific coactivator whose level of expression in liver, brown fat, and muscle plays a key role in controlling the expression of genes involved in processes such as gluconeogenesis and thermogenesis. The studies of these specific coactivators, in our lab and others, have been on the forefront in advancing our understanding of the complex mechanism of transcriptional regulation, but the specific mechanistic roles of the methyltransferases and their methylation of histones and other proteins is not clear. The proposed studies will focus on the roles of these methyltransferases as coactivators and their mechanism of synergistic cooperation with p300/CBP and with PGC-I. We will define: 1) the roles of arginine-specific methylation of histones H3 and H4 by CARM1 and PRMT1, respectively, in transcriptional activation, by using chromatin immunoprecipitation and by identifying proteins which preferentially bind to the methylated histones; 2) the functional domains of CARM1, their specific roles in coactivator function, and proteins that bind to critical domains of CARM1; 3) the mechanisms of CARM1-p300/CBP and PRMT1-PGC-1 coactivator synergy and the roles which methylation of p300/CBP by CARM1 and of PGC-1 by PRMT1 play in synergy; 4) the sub-nuclear locations and co-localization of SR and their coactivators, the assembly of SR and their coactivators onto stably integrated promoters of steroid responsive genes in vivo, and the role of coactivators in chromatin remodeling by SR on reconstituted chromatin in vitro.
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