The broad mandate of this proposal is to use transgenic, molecular and biochemical approaches to determine the in vivo biological effects specific to the Pit-IB isoform with regards to Pit-1-dependent pituitary cell ontogeny and gene expression, and to define the mechanism that mediates B-isoform-specific transcriptional responses in pituitary cells. 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 TSHB genes. The single Pit-1 gene is expressed in the Pit-1 lineage as two alternatively-spliced mRNAs, resulting in Pit-1 and Pit-IB proteins. Pit-IB contains a unique 26 amino-acid (AA) S-domain inserted at AA 48 of Pit-1, precisely in the middle of the transcription activation domain (TAD). However, Pit-1and Pit-IB share identical structures otherwise. Naturally occurring Pit-1mutations in mice and humans resulting in heritable dwarfism map to regions common to both Pit-1 and Pit-IB. However, the precise biological contributions of Pit-IB to pituitary cell-specific ontogeny and gene expression remain unknown. AlthoughPit- 1B has been overlooked after its initial description, during the previous funding period we made important, novel and unique contributions to our understanding of the precise molecular mechanisms by which the Pit-1 B-domain functions as a transcription switch motif resulting in B-isoform-specific transcriptional responses. We found that Pit-IB consistently inhibits PRL, GH and TSHB promoter activities in all pituitary cell lines tested, whereas Pit-IB consistently activates these same promoters in all nonpituitary cell lines tested. Moreover, Pit-IB inhibited basal, cAMP- and Ras- stimulated rPRL promoter activity in GH4 pituitary cells. We elucidated mechanism, demonstrating that the B-domain functions as a pituitary-specific represser motif acting via five hydrophobic amino acids that recruit HDAC activity to alter the histone acetylation state of the proximal rPRL promoter. Importantly, the B-domain can function as an autonomous, modular, active and HDAC-dependent represser motif when fused to the Gal4 DBD. Finally, adenoviral encoded HA-Pit-1B inhibits endogenous PRL and cyclinDI, but activates RB gene expression;and inhibits GH4 cell proliferation and tumor growth in nude mice, providing evidence of its biological effects. Thus, the Pit-1/Pit-IB pair provides a prototypical model to study transcription factor isoform-specific functions. We hypothesize that a pituitary- restricted represser complex that associates with the B-domain dictates Pit-IB isoform-specific transcriptional responses. A corollary hypothesis is that pituitary cells expressing increased Pit-IB- will have a distinct phenotype. To address this hypothesis, we propose four Specific Aims: (1) To determine whether HA-Pit-1B- transgene expression governs the ontogeny and/or expansion of the Pit-1 lineage in transgenic mice. (2) To determine the biological responses specifically induced by Pit-IB in GH4 cells. (3) To use RNAi to determine the biological role of Pit-IB and defined co-repressors in mediating B-specific responses. (4) To identify and functionally validate the represser complex associated with the B-domain. Insights gained from these studies will not only provide a better understanding of Pit-IB isoform-specific functions, but will also provide a conceptual and experimental framework to study other highly-related transcription factors that bind to overlapping DMA sites.
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