While it is widely accepted that the risk of developing breast cancer is directly related to one's lifetime exposure to estrogen, the precise role of estrogen in the initiation and progression of breast cancer has yet to be determined. We reported that estrogens downregulate the expression of the transcription factor Hexamethylene-bis-acetamide-inducible protein 1 (HEXIM1), and HEXIM1, in turn, inhibits Estrogen Receptor (ERa) transcriptional activity. Our mechanistic studies suggest that HEXIM1 mediates a novel link between ERa and positive transcription elongation, and that this HEXIM1 function plays a role in mammary gland development/tumorigenesis. ERa interacts with cyclin T1, a component of a protein complex called the positive transcription elongation factor b (P-TEFb) that phosphorylates the carboxy terminal domain of RNA polymerase II (RNAP II. This action promotes productive phases of transcriptional elongation. HEXIM1 inhibits the co-recruitment of ERa and cyclin T1 to the promoter region of cyclin D1. As a result, HEXIM1 inhibits the phosphorylation of the carboxy terminal domain of RNAP II and thereby prevents ERa-mediated transcriptional elongation in breast epithelial cells. A mouse model created in my laboratory whereby HEXIM1 overexpression was targeted to the mammary gland produced results consistent with HEXIM1 inhibition of cyclin D1 and mammary epithelial cell proliferation via repression of P-TEFb activity. Studies using another mouse model created in my laboratory which expresses a mutant HEXIM11-312 protein that is able to inhibit P-TEFb, indicate that HEXIM1 has important physiological functions which are not dependent upon its ability to interact with and to inhibit P-TEFb. This mutant HEXIM1 protein is able to actively promote mammary tumorigenesis. Our studies indicate that HEXIM1 regulates mammary tumorigenesis by downregulating angiogenesis and the expression of Vascular Endothelial Growth Factor (VEGF), and this is independent of the ability of HEXIM1 to regulate P-TEFb activity. That regulation of VEGF by HEXIM1 is cell autonomous and occurs at the transcriptional level is also evident from these studies. Based upon our findings, we hypothesize that modulation by HEXIM1 of estrogen regulated mammary gland development/tumorigenesis involves inhibition of cyclin D1 and VEGF expression through P-TEFb dependent and independent mechanisms, respectively. The main specific aims of the proposed studies are to determine (1) What are the downstream transcriptional events influenced by HEXIM1- ERa-cyclin T1 functional interaction and their relevance in mammary epithelial cell proliferation? (2) What is the molecular basis for HEXIM1-regulated VEGF expression and its relevance in endothelial cell function? (3) What are the relative roles of Cyclin D1 and VEGF in HEXIM1 modulation of estrogen-regulated mammary tumorigenesis.