This competitive renewal application addresses the following challenging question in current biology: What are the molecular mechanisms by which related transcription factor isoforms, displaying identical DNA sequence specificity and expressed in the same cell, actually mediate distinct transcriptional responses. The POU-homeodomain transcription factor, Pit-1, controls the development of somatotroph, lactotroph and thyrotroph pituitary cell- types and regulates the cell-specific expression of GH, PRL and TSHbeta genes. The single Pit-1 gene is expressed in the three pituitary cell types as two alternatively-spliced mRNAs, resulting in Pit-1 and Pit- 1beta proteins. Pit-1beta contains a unique 26 amino acid (AA) beta- domain inserted at AA 48 of Pit-1 precisely in the middle of the transcription activation domain (TAD). Initial studies describing Pit- 1beta revealed that the two Pit-1 isoforms bound to the same DNA site with the same relative affinity, yet they displayed promoter selectivity. Although the functional role of Pit-1beta has been largely overlooked by the field, we have discovered the beta-domain imparts unique positive and negative regulatory features to Pit-1beta with regards to PRL promoter activation. Specifically, Pit-1beta inhibited basal and Ras-stimulated rPRL promoter activity in GH4 pituitary cells, yet Pit-1beta reconstituted basal activity and enhanced the PKA response of the rPRL promoter in HeLa non-pituitary cells. We have shown that it the identify the amino acid sequence of the beta-domain, and not just the altered spacing of the TAD generated by the beta-domain insertion, that confers upon Pit-1beta these unique transcriptional properties. Thus, the Pit-1/Pit-1beta pair provides a prototypical model to study transcription factor isoform-specific functions. We hypothesize that Pit-1 isoform specific responses are due to selective protein partnerships dictated by an isoform-specific protein-protein interaction domain(s). Thus, the beta-domain functions as a beta isoform-specific interaction surface, resulting in protein-protein interactions specific to the Pit-1beta isoform. Corollary to this is that proteins that bind to the Pit-1 TAD in the region disrupted by the beta-domain insert site (AA48), may no longer bind to Pit-1beta. To address this hypothesis, we propose five Specific Aims: (1) To determine the precise amino acids of the beta-domain that dictate Pit-1beta specific transcriptional properties; (2) To elucidate whether beta-domain function is dependent upon protein context; (3) To determine the effects of DNA recognition sites upon Pit-1 and Pit-1beta functional relevance of beta-specific protein-protein interactions. Insights gained from these studies will not only provide a better understanding of Pit-1 isoform-specific function, but will also provide a conceptual and experimental framework to study other highly related transcription factors that bind to overlapping DNA sites.
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