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Authors: Mukherjee, Malini
Advisors: Porter, Tom E
Department/Program: Molecular and Cell Biology
Type: Dissertation
Sponsors: Digital Repository at the University of Maryland
University of Maryland (College Park, Md.)
Subjects: Molecular biology
Keywords: chicken
gene expression
transcription factor
Issue Date: 2011
Abstract: Pit1, a pituitary-specific transcription factor, regulates differentiation of cells of the PIT1 lineage in the anterior pituitary. PIT1 also regulates the synthesis of peptide hormones from these cell types, including growth hormone (GH). A founding member of the POU-homeodomain family of transcription factors, PIT1 is characterized by a serine-threonine rich N-terminal transactivation domain and a C-terminal POU-domain. Alternative forms of PIT1, differing from each other in the N-terminal domain have been reported in several species, but the functional implication of having multiple isoforms is not known. Several Pit1 isoform mRNAs exist in chickens which have not been characterized. The main aim of this study was to determine which, if any, of the chicken PIT1 isoforms regulated the chicken Gh (cGh) promoter. PIT1β2, a novel isoform of chicken PIT1 was discovered, and known and novel isoforms (PIT1α, PIT1β1, PIT1β2 and PIT1γ) were characterized. A luciferase reporter construct containing 1775bp of the cGh promoter driving expression of firefly luciferase was used to determine the ability of the isoforms to regulate the target gene promoter activity in chicken LMH cells. We showed that three of the isoforms, PIT1α, PIT1β1 and PIT1β2, expressed from recombinant plasmids, regulated the cGh promoter, while PIT1γ did not. All the isoforms localized to the nucleus in both non-pituitary and pituitary cells. Results from gel-shift assays show that PIT1γ did not bind the proximal PIT1-binding site of the cGh promoter as well as the other isoforms, suggesting a possible mechanism behind the inactivity. Our result did not suggest a negative regulatory role for this isoform. In contrast, we found a functional advantage for having multiple isoforms. PIT1β1, the isoform that activated the promoter most strongly, when co-transfected with other activating isoforms, such as PIT1α and PIT1β2, induced significantly higher level of activation than one isoform alone. Whether this increased activation required, or was facilitated by, heterodimerization of two isoforms is not known. Nevertheless, identification of isoforms with specific functions will facilitate identification of their respective interacting partners, which are essential for GH gene expression.
Appears in Collections:Cell Biology & Molecular Genetics Theses and Dissertations
UMD Theses and Dissertations

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