The long-term goal of our work is to understand the mechanism of transcriptional regulation by steroid hormone receptors and their dysregulation during disease. We seek to reveal how specific coactivators and corepressors translate the hormonal signals into promoter-dependent transcriptional outputs. Our work centers on the mechanism of gene activation by the glucocorticoid receptor (GR). GR harbors two major transcriptional activation functions (AF1 and AF2) that are differentially utilized in individual promoter and cellular contexts. In contrast to the C-terminal AF2 function, little is known regarding the coregulators that interact with, and mediate the functions of the N-terminal AF1. This region plays key roles in important therapeutic effects of glucocorticoids such as the induction of thymocyte apoptosis. We recently generated specific mutations in AF1 that pinpoint the critical residues responsible for function. We have used them in complementary approaches to discover AF1 coregulators. In a genetic approach, we isolated two extragenic suppressor yeast strains where the function of deficient AF1 mutants is restored. The genes responsible are likely to encode conserved factors intimately involved in AF1 function. Through an independent functional approach, we identified the 150kDa subunit of the DRIP/TRAP/Mediator complex as a protein that interacts with a transcriptionally competent AF1.
In Aim 1, we propose to define the nature and structural basis for the interaction of DRIP 150 with GR AF1. We plan to map the minimal regions necessary in each protein and identify key residues through random and targeted mutagenesis coupled with screening in yeast. We will explore the structural basis of the interaction through a biophysical characterization of the complex.
In Aim 2, we will ascertain the functional consequences of AF1 binding to DRIP 150 and define the effector regions of DRIP 150 involved in transcriptional activation. Using multiple approaches, we will test the hypothesis that AF1 recruitment of the DRIP complex determines whether GR activation of a given promoter requires participation of AF1.
In Aim 3, we propose to identify novel conserved AF1 coregulators by cloning the genes responsible for the extragenic suppression in the yeast strains we have isolated. We will then identify mammalian counterparts and begin to ascertain their role in AF1 function using some of the assays outlined in Aims 1 and 2.
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