The cytoplasmic components and nuclear effectors mediating growth factor/Ras-induced gene expression are common to all cell types. Thus, defining the precise molecular mechanisms by which ubiquitous components of growth factor/Ras signaling pathways result in cell-specific responses remains a central problem in biology today. Previously, we discovered an elegant and precise tri-paritite molecular code, involving the proto-oncoprotein c-Ets-1, the POUhomeodomain protein Pit-l, and a composite DNA element containing an Ets binding site (EBS) adjacent to a Pit-1 site (FPIV). Pit-l, Ets-1 and this composite site were shown to be required not only for the Ras response of the rat (r) prolactin (PRL) promoter, but also to reconstitute lactotroph-specific rPRL promoter activity. This was the first and remains the only example of a tissue-specific Ras response element (RRE). During the past funding cycle, we used NMR and GST pull-down methods to map the Ets-1/Pit-1 interaction surfaces to exact residues on the Pit-1 POUhomeodomain and to the Ets-1 RIII TAD. We reported that Ets-1 preferentially binds to the EBS in the RRE, whereas GABP preferentially binds to the BTE/EBS centered at -95. Finally, using dominant-negative and siRNA approaches, we verified that Ets factors, in particular GABP, are critical for basal and growth factor-induced rPRL promoter activity. These studies revealed that Ets-1 and GABP are critical for lactotroph-specific expression of the endogenous rPRL gene. While our previous studies have mostly used reporter gene assays, the broad mandate of this proposal is to use molecular, biochemical and transgenic approaches to dissect the physiological role of select Ets factors and Ets factor binding sites in goveming basal and Ras-regulated lactotroph-specific expression of the endogenous rPRL gene in GH4 cells and in mice. Unifying Hypothesis: We hypothesize that basal and Ras-regulated expression of the lactotroph-specific rPRL gene is governed by specific Ets factors, some acting in concert with Pit-l, whose binding to Ets binding sites on the proximal rPRL promoter is regulated by the physiological state of the cell.
Specific Aims : (1) To identify Ets factor occupancy on the endogenous, proximal rPRL promoter in GH4 rat pituitary cells q oncogenic Ras. (2) To identify the oncogenie Ras-dependent protein components of Ets-1/Pit-1 transcription factor complexes. (3) To determine the functional role of basal and oncogenic Ras-regulated transcription co-regulatory molecules (4) To elucidate the physiological role of rPRL Ets binding sites and the Ets factor GABP in PRL gene expression in transgenic mice. Information gained from these studies will provide critical insights into the mechanisms by which combinatorial Ets/Pit-1 protein interactions specify cell-restricted PRL gene expression, and also provide a novel insights into the mechanisms by which Ras/MAPK regulates gene expression.
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