Nothing short of defining the three-dimensional (3D) structure of the nuclear receptor (NR) and coregulator (CoReg) containing transcriptional complex and its order of assembly will significantly advance our current understanding of gene regulation by steroid hormones. Insights into the fundamental regulatory mechanisms (both proximal and distal) of transcriptional initiation, elongation, and pausing are critical since steroid hormone control of gene expression underpins a wide array of physiological and pathophysiological responses. Using an innovative reconstitution assay of the transcriptional complex, we recently revealed that DNA-bound progesterone receptor (PR) recruits the steroid receptor coactivator-2 (SRC-2) to the transcriptional complex to drive target gene expression. This in vitro finding has in vivo significance since we have previously demonstrated a pivotal role for SRC-2 in PR-dependent reproductive functions in the female mouse and more recently in PR-driven mammary epithelial expansion and morphogenesis. These in vitro and in vivo findings are the basis of the following hypothesis: The PR functions together with SRC-2 in a multi-subunit transcriptional complex to drive transcriptional programs that underpin normal and abnormal mammary epithelial expansion and morphogenesis. Implicit in this testable hypothesis is that deregulation of SRC-2 levels can derail normal PR mediated transcriptional output, which leads to abnormal hormone responsiveness of the mammary epithelium, resulting in hyperplasia and cancer. The above hypothesis will be tested by the following four specific aims: Specific Aim 1: To determine the 3D structure of DNA-bound PR/SRC-2/Co- CoReg protein complexes on a progesterone response element via cryo-electron microscopy (Cryo-EM); Specific Aim 2: To codify the regulatory involvement of PR and SRC-2 in Growth Regulation by Estrogen in Breast Cancer 1 (GREB 1) promoter/enhancer topology and transcriptional activity; Specific Aim 3: To dissect the selective contributions of luminal epithelial and basal/myoepithelial derived SRC-2 to normal progesterone- dependent mammary gland morphogenesis in vivo; and Specific Aim 4: To elucidate the role of SRC-2 in hormone-dependent and -independent aberrant mammary epithelial expansion and morphogenesis. For these innovative specific aims, forefront molecular and genetic mouse technologies?Cryo-EM, a cell-free reconstitution assay to study transcriptional complex assembly, cell-type specific cre-mediated gene ablation or induction in the mouse, and the integrative analysis of genome-wide datasets as a molecular phenotyping tool?will be used to advance the above provocative hypothesis. The outcomes of this research program are predicted not only to significantly expand our current understanding NR/CoReg control of gene expression but will address the underexplored role of CoRegs in normal progesterone-dependent mammary epithelial proliferation and morphogenesis as well as for abnormal hormone responses that can lead to aberrant development such as cancer?a high-priority health issue for the National Institutes of Health.