Recruitment on co-activators to the promoters of target genes is essential for nuclear receptors (NRs) to function as transcription factors. One of the goals of this Bridging Project is to catalog the factors present in the various NR co-activator complexes. This information will be a cornerstone for future work on ONR, because the one of the final steps towards their characterization, after identifying ligands, is to define their relationship with the various co-activators. It is also becoming clear that there are tissue specific effects of NR ligands, very possibly due to different complements of co-activator and co-repressor complexes present in different cell types. We will test this hypothesis by analyzing the composition of co-activator complexes in different tissues. A corollary of tissue specific variation of co-activator complexes is the fact that cell types themselves are not static entities but undergo differentiation processes and respond to various hormonal signals etc. We will therefore determine if co-activator complexes undergo changes in composition during basic differentiation processes and in response to specific cellular signals. Since phosphorylation is known to be an important regulatory mechanism for many signaling proteins, we will identify the key regulatory phosphorylation sites of a number of important co-activators and evaluate their mechanistic importance in co- activator function. Finally, the downstream effect of co-activators is thought to be regulation of the expression of target genes in response to various dependent and independent pathways. To understand the role of co-activators in these processes we will be required to catalog co- activator specific target genes. We will analyze the manner in which the tissue-specific composition of co-regulator complexes contributes to distinct gene regulation profiles in different tissues and across differentiation processes. We will utilize proteomic technologies and genomic tools to identify and characterize co-activator interacting proteins, and the gene networks they regulate. This study will provide us with an insight into as yet undiscovered metabolic and signaling pathways and regulatory gene networks in which co-activators, and the receptors with which they interact, participate.
The Specific Aims are: 1. To identify specific co-activator protein complex in HeLa cells. 2) To analyze the composition of co-activator complexes in cell-specific, differentiation status-identify co-activator target genes using chromatin immunoprecipitation assays.
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