Mechanism of p160 nuclear coactivators

University Of Southern California, Los Angeles CA

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Abstract

The nuclear receptor (NR) family includes the receptors for steroid, thyroid, retinoid, and vitamin D hormones; NRs are hormone-regulated transcription factors which bind enhancer elements and recruit coactivators to activate target genes by modulating local chromatin structure and recruiting RNA polymerase II to the promoter. The current study focuses on the three p160 coactivators (p160 CoAs) (SRC-1, GRIP1, and p/CIP) which bind to AF-2 and (in some cases) AF-1 activation domains of NRs. The p160 CoAs also recruit secondary coactivators such as p300/CBP, p/CAF, and CARM1 which act synergistically with p160 CoAs by acetylating or methylating histones and other proteins in the transcription machinery. Many studies now indicate that the p160 CoAs play central roles in NR function. The p160 CoAs also serve as coactivators for other transcription factors, such as MEF-2C and myogenin which promote muscle cell differentiation. We propose to focus on the role of the N-terminal region of GRIP1, which is the most highly conserved but least understood region of p160 CoAs. By mutational analysis, we will map the functionally important subregions of the N-terminal domain, using both transiently transfected and stably integrated reporter genes and chromatin immunoprecipitation (ChIP) assays to study the coactivator function of GRIP1 with NRs, MEF-2C, and myogenin. The yeast 2-hybrid system will be used to identify p160- interacting proteins (p160-IP) which bind the N-terminal region of GRIP1. We will characterize the functions of p160-IPs as coactivators that may collaborate with GRIP1 or as transcriptional activators which use GRIP1 as a coactivator. We have recently developed transient transfection conditions under which NR function depends on co-expression of three different coactivators (GRIP1 + CARM1 + p/CAF); these conditions will be used to characterize the mechanism of synergy, with a focus on the nature of the interaction between GRIP1 and p/CAF. The role of GRIP1 as a coactivator for MEF-2C and myogenin will be studied in transient transfections; and the role of various GRIP1 domains in muscle cell differentiation will be studied in cultured C2C12 cells which require GRIP1 for differentiation into myotubes in culture.