Project/Summary Abstract Significant advances in our mechanistic understanding of progesterone receptor (PR) mediated transcription are inextricably tied to our structural knowledge of the core transcriptional complex through which PR along with its coregulators (Coregs) control target gene transcription. Pioneering the use of single-particle cryo- electron microscopy (cryo-EM) in steroid hormone nuclear receptor (NR) biology, we recently revealed the quaternary structure of the transcriptional complexes for the estrogen receptor (ERa) and androgen receptor (AR), which along with PR comprise the “NR triad” of sex steroid hormones that govern mammalian reproductive biology. Unlike ERa and AR, PR is composed of the PR-A and PR-B isoforms that mediate both distinct and overlapping progesterone transcriptional responses in physiological and pathophysiological contexts. Accordingly, a comprehensive understanding of the transcriptional response to progesterone can only occur through a structural characterization of the core transcriptional complexes of the homodimer and heterodimer configurations of these PR isoforms. Therefore, Aim 1 will use cryo-EM and complementary structural proteomic approaches to solve the quaternary structure of the core Coreg/transcriptional complex for the PR-A homodimer bound to DNA. Significantly, we have determined the core Coreg/transcriptional complex for the PR-B homodimer. Not only has this structural information provided critical insights into the unique assembly mechanisms by which PR-B recruits primary and secondary Coregs during transcriptional complex formation but reconfirms our ability to successfully execute the structural studies in Aims 1 and 2. Importantly, this technology presents a unique opportunity to investigate the contribution of the intrinsic disordered region of the activation function 1 (AF1) in PR during Coreg/transcriptional complex assembly. Despite its importance in PR mediated transactivation and its potential for therapeutic targeting, the AF1 domain (unlike the ordered AF2) is understudied due to its disordered topology. Using a PR AF1 mutant, Aim 2 will use cryo-EM and allied structural methods to assess the contribution of AF1’s structural disorder in the dynamic assembly mechanisms that underpin Coreg recruitment by PR-B to the nucleating transcriptional complex. This aim will be complemented by Aims 3 and 4, which will determine the transcriptional functional role of PR’s AF1 disordered structure using innovative cell-free/cell-based investigations and a CRISPR- engineered mouse model respectively. Collectively, these transformative investigations will: (i) furnish a much needed structural and mechanistic framework to explain the PR isoform-specific functional differences in physiological and pathophysiological contexts; (ii) define the structure-function relationship of AF1’s flexibility in dynamic interactions with multiple Coregs at the cell- and whole organism-level; and (iii) offer ...