The ovarian steroid hormone progesterone fulfills a critical role in the growth and development of tissues of the female reproductive tract and the mammary gland, as well as growth and metastatic potentials of malignancies arising from these tissues. The progesterone receptor (PR), a hormone-dependent transcriptional activator, mediates the effects of progesterone by binding to specific regulatory elements of target genes to alter transcription of specific gene networks. Our overall goal is to uncover the molecular mechanisms that lead to the activation of the steroid receptor as a regular of gene expression. As such, an improved understanding of progestin action could lead to design of new therapeutic approaches to infertility and breast cancer in women. Because of the central importance of receptor binding to DNA, past research efforts have addressed mechanisms that regulate receptor recognition of specific DNA sequences. Within this framework, the present application focuses on the role of nuclear accessory proteins and protein manipulation of DNA structure. Our previous studies have identified the DNA bending protein HMG-1 as an accessory factor that facilitates PR binding.
In AIM #1 molecular and biochemical studies are proposed to investigate the mechanism by which HMG-1 facilitates binding of PR to its target DNA. Because we have shown that PR induces DNA flexure, many of these studies focus on the role of DNA conformation or bending in receptor-HMG-1-DNA interactions.
AIM #2 addresses the generality of the role of HMG-1 in steroid hormone action by examining whether it can serve as an accessory factor to other steroid receptors. While we have shown that a number of proteins, including related """"""""HMG box""""""""-containing proteins, do not functionally substitute for HMG-1, we have data that proteins in addition to HMG-1 can enhance PR-DNA binding in vitro.
AIM #3 therefore will assess the quantitative contribution of HMG-1 and other proteins to this activity and we will seek to purify and identify the other proteins involved.
In AIM #4 we seek to make the connection between in vitro binding results and the possible role of HMG-1 in receptor function in vivo and the receptor-mediated transcriptional enhancement. Three different strategies will be employed, including the manipulation of HMG- 1 levels in mammalian cells by anti-sense technology, in yeast by depletion of HMG-1 analogs and in vitro in cell-free receptor-dependent transcription assays. This multi-pronged attach will permit a powerful approach to linking in vitro binding results with an influence of HMG-1 or PR function in vivo.
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