We propose to systematically purify cocativator complexes from HeLa cells by using antibodies specifically designed for this application and mass spectrometry based proteomics. The coactivators we have selected for this Specific Aim have been prioritized into three groups. The identification of components of the various co-activator complexes will provide us with an important foundation from which to develop a detailed mechanistic approach to elucidating coactivator function.

2. To analyze the composition of coactivator complexes in cell-specific-, differentiation status- and cellular signaling-dependent contexts.

Several key studies suggest that efficient target gene regulation at different promoters may be related to differing coactivator complex composition. These results have led us to hypothesize that coregulator composition might also vary on a cell-to-cell basis on the basis of a cell differentiation status and in response to different cellular signals. In order to test whether tissue specific configurations of coactivator complexes exist, we propose to purify and identify the group 1 coactivator complexes in cell lines derived from representative nuclear receptor (NR) target tissues that express varying levels of coactivators, e.g. MCF-7, T47D, 3T3 fibroblasts, P19 and LNCaP cells. Coactivator complex composition may also undergo alterations during differentiation processes. Accordingly, we will sample coactivator complex composition in the PPAR-gamma ligand-induced differentiation of 3T3 fibroblasts into adipocytes, and the retinoid-induced differentiation of embryonic carcinoma cells into neurons.

3. To identify basal phosphorylation sites of coactivators.

Despite the fact that it is well known that many, if not all co-activators are phosphoproteins, the global role of phosphorylation in regulation of coactivator function, and the function of their cognate receptors, is not yet understood. Having established the identity of the proteins with which coactivators exist in stable complexes, and made some inference as to their function in these complexes, we will proceed to identify the critical regulatory residues.through which kinases regulate their function.

We propose to identify the basal phosphorylation sites in key coactivators using new mass spectrometry-based technology. Characterizing the basal phosphorylation sites of coactivators from different cell types will implicate their cognate kinases in the regulation of these coactivators and, will shed light on the phosphorylation codes responsible for regulating coactivator function in the basal and active states. Moreover, information collected in this aim will set the stage for studying activated phosphorylation in response to a variety of physiological stimulus, including nuclear receptors, growth factors and cytokines.

4. To identify coactivator target genes using chromatin immunoprecipitation assays.

While evidence suggests that coactivators are recruited to target genes by NRs, the factors (if any), which influence selective patterns of target gene regulation by individual coactivators, are not yet known. In an effort to determine the extent to which coactivators influence NR activation of target genes, chromatin immunoprecipitation (ChIP) of group 1 coactivators from cells will be performed, and libraries of associated gene fragments will be created, sequenced, annotated and mined. We anticipate that bioinformatic interrogation of data from multiple cell types will generate considerable insight into the role of coactivators in influencing selective patterns of target gene regulation by these NR coactivators. The recruitment of coactivators to NRs is in many cases ligand-dependent, and so to identify coactivator target genes we will use various ligand treatments and cell types as our model systems, including (i) TZD treatment of 3T3 fibroblasts; (ii) retinoid treatment of P19 cells and (iii) steroid hormone treatment of breast cancer cells. We will focus on the coactivator having the highest expression levels and the most dynamic composition variations across the systems being studied.

For more information on projects in this Bridging Strand, see also Strand B, Project 2 (Mitchell Lazar) and Strand B, Project 3 (Ming Jer-Tsai and Donald McDonnell).