Specific Repression of Prolactin Gene Expression

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DESCRIPTION (provided by applicant): Our ultimate goal is to better understand how protein interactions in the cell nucleus control specific programs of gene expression, and how this is disrupted by disease. PRL is required to maintain normal reproductive processes, and its regulated expression is critical to the control of homeostasis. In the pituitary, transcription of the prolactin (PRL) gene is restricted to cells of the somatolactotroph lineage, making it an excellent model for defining the mechanisms that specify cell-selective gene transcription. The pituitary-specific homeodomain protein Pit-1, is required to establish the somatolactotroph cell lineage, and is also necessary, but not sufficient, for regulated transcription of the PRL gene. The role of Pit-1 in the genesis of pituitary cell-types is made clear by patients harboring mutations that affect the activity of Pit-1 who develop combined pituitary hormone deficiency (CPHD), a disease characterized by the lack of the hormones produced by these cells. The control of selective gene expression by Pit-1 occurs through balanced interactions with other transcription factors and co regulatory proteins. The association of Pit-1 with a co-activator complex includes the CBP that mediates hormone-stimulated PRL gene transcription. This activation is counter-balanced by the association of Pit 1 with the nuclear co-repressor proteins including N-CoR and SMRT. Recent work by ourselves and others has revealed that transcription factors and co-regulatory proteins localize to specific sites within the cell nucleus. We have developed innovative imaging approaches to directly visualize cooperative protein interactions in the living ceil nucleus. Our observations indicate that disruption of protein interaction surfaces formed by Pit-l, and its nuclear protein partners can critically affect where these protein complexes are assembled in the nucleus, and ultimately the regulation of PRL transcription. We showed the importance of these interaction surfaces by observation of a Pit-1 protein mutation associated with CPHD that dramatically affected cooperative protein interactions when viewed in living cells. Through the combination of in vitro analysis and live-cell imaging we will address the following questions: 1. How do protein interaction surfaces on Pit-1 function to guide the cooperative interactions with other transcription factors that activate PRL transcription? 2. How are the functional interactions of Pit-1 and co-repressor proteins regulated? 3. Is Pit-1 engaged in transcriptional complexes that directly control endogenous target genes in pituitary cells? If we are to understand disease processes and design therapeutic strategies, it is important to define how specific gene regulatory complexes are assembled within the nucleus and how their positioning influences the combinatorial code specifying the expression of particular genes. Our analysis will provide important organizational principals that underlie pituitary cell specific gene expression and form a basis for understanding the mechanisms involved in the control of eukaryotic gene expression in general.