The long-term stated goal of this proposal is to understand how auxiliary factors modulate transcriptional regulation by the thyroid hormone receptor (TR). This investigator has isolated a distinct group of novel proteins termed TRAPs (thyroid receptor associated proteins). In vitro transcription assays showed that the TR-TRAP complex markedly activates transcription from promoter templates containing TR-binding sites. He has cloned one of the TRAPs (TRAP 220) and found it directly interacts with TR in vivo in a T3-dependent manner. This protein shares significant sequence homology with PBP, a putative co-activator of peroxisome proliferator-activated receptors (PPARs), and RB18A, a novel p53-like co-regulatory protein. The investigator hypothesizes that TRAP 220, alone or in concert with other TRAP subunits, functions as a positive co-activator for gene-specific transcription.
The aims of the proposal are: (1) Characterize TRAP 220 function in terms of ligand- induced interactions with TR and other nuclear hormone receptors (NRs) using deletion and site-directed mutagenesis to define interaction motifs of both TRAP 220 and other NRs using various protein-protein interaction studies and DNA mobility shift assays. (2) Examine the functional relevance of TRAP 220 as a transcriptional co-activator for hormone-dependent activation by TR and other NRs in co-transfection assays and in vitro transcription analyses. (3) Identify the specific cofactors which physically and functionally interact with TRAP 220. He proposes to screen the TR-TRAP complex by far western analysis using TRAP 220 as a probe to identify TRAP 220-interacting factors and a panel of recently cloned TRAP subunits will be screened for TRAP 220 interactions using protein-protein interaction assays. Identified cofactors will be further assayed for in vivo and functional associations with TRAP 220.
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