Reproductive Hormones - Biological and Molecular Actions

PROJECT SUMMARY Advancing our mechanistic understanding of androgen-dependent male reproductive function requires an in- depth structural understanding of the core transcriptional complex in which ligand-occupied androgen receptor (AR) in complex with steroid receptor coactivator-2 (SRC-2) and p300 enacts transcriptional initiation of specific androgen-responsive gene expression programs. Until recently, structural information on the assembly modes by which the nuclear receptor (NR) engages with primary and secondary coregulators (Coregs) was limited to studies based on NR and Coreg protein fragments, which provided a significantly incomplete and, in many cases, an erroneous perspective of NR/Coreg transcriptional complex formation. To address this limitation, my team recently used cryogenic electron microscopy (cryo-EM) and proteomics to solve the quaternary structure of a number of NR/Coreg transcriptional complexes related to cancer biology at a full-length structural level. Apart from providing essential insights into the structure and assembly mechanisms of these core transcriptional complexes, an important “take away” from these investigations was that the structure and stoichiometry of the transcriptional complex for one NR cannot be extrapolated to explain the structure of another NR, despite sharing a similar functional domain organization. With this mind, Specific Aim 1 studies will use breakthrough advances in cryo-EM to obtain the quaternary structure of the DNA-bound liganded AR dimer in complex with SRC-2 and p300. Advancing the applicability of cryo-EM further, structural investigations in Specific Aim 1 will also solve the 3-dimensional structure of the core AR/Coreg transcriptional assembly in complex with the nucleosome core particle, the next level of structural complexity to be studied if we are to significantly advance our understanding of AR-mediated transcriptional initiation in the context of chromatin. Studies in Specific Aim 2 will apply cryo-EM to determine the structure of an AR(N727K) transactivation mutant as an approach to elucidate (at a full-length structural level) the importance of allostery within the AR dimeric complex in N- and C-terminal cooperativity, a prerequisite for Coreg recruitment and transcriptional activation. The structural studies in Specific Aims 1 and 2 will be functionally complemented in Specific Aim 3 by versatile cell-free assays of AR or AR(N727K)/Coreg-mediated in vitro transcription and enhancer-promoter crosstalk at an AR target gene at the proteome level. Providing in vivo support for findings from Specific Aims 1-3, a uniquely engineered AR point mutant mouse in Specific Aim 4 will be phenotyped at the histological, cellular and molecular level to define the importance of AR N/C terminal domain cooperativity in male reproduction, in particular spermatogenesis. Collectively, our studies will furnish a structural, functional and physiological basis for AR-mediated transcription in the maintenance of male reproductive function. In keeping with NICHD priority goals, outcomes from these studies will also provide a conceptual framework for understanding abnormal responses to androgen that lead to androgen insensitivity.