Hormone-activated nuclear receptors (NR) bind to target gene promoters and recruit complexes of coactivators to help activate transcription. The p160 coactivator complex consists of a p160 protein, which binds directly to activated NRs, and several secondary coactivators. Our global strategy is to characterize the functional domains of coactivators and search for proteins that bind to their activation domains and mediate downstream events in the coactivator signaling pathway. By reiterative application of this process, we will find the ultimate targets of the p160 coactivators in the chromatin or basal transcription machinery. The p160 proteins were previously shown to have two C-terminal activation domains for signal transmission: AD1 binds CBP/p300, while AD2 binds CARM1. We recently identified a new activation domain in the N-terminal region of p160 coactivators (AD3) and identified two proteins which bind to the p160 N-terminus and cooperate synergistically with p160 proteins as coactivators for NRs: Coiled-coil Coactivator (CoCoA) and Flightless I (Flil). Here, we will determine the mechanisms by which each of these proteins cooperates with other NR coactivators and contributes to transcriptional activation by NRs. We will define the functional subdomains of CoCoA and Flil by defining their sites of interaction with NRs, p160 coactivators, and/or other known protein interaction partners. We will also define their activation domains, which are used to transmit the activating signal to the transcription machinery, and identify proteins that bind to the activation domains and are thus downstream in the activation signaling pathway. The involvement of CoCoA and Flil as coactivators with other classes of transcription factors will also be investigated. Finally, we will study the variable requirements for CoCoA and Flil and their various functional domains on multiple steroid hormone-regulated promoters (some hormone inducible and some hormone repressible) in the A549 cell line to investigate the relationship between mechanism of coactivator function and promoter architecture.
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