Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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Item DIFFERENTIAL ABILITIES OF THE CHICKEN PIT1 ISOFORMS TO REGULATE THE CHICKEN GROWTH HORMONE PROMOTER(2011) Mukherjee, Malini; Porter, Tom E; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)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.Item Identification of a single nucleotide polymorphism associated with adiposity following transcriptional profiling of gene expression in the anterior pituitary gland(2006-07-23) muchow, michael; Porter, Tom E; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Although the anterior pituitary secretes three hormones that affect metabolism and body fat stores, a comprehensive analysis of pituitary gene expression associated with body fat has not been performed. This research used cDNA microarrays to investigate pituitary gene expression in two chicken lines that were selected for low and high body fat (Lean and Fat). RNA was extracted from pituitaries at 1, 3, 5, and 7 weeks of age. 386 genes that showed significant differences in expression levels by line or in the line-by-age interaction were analyzed further. Differentially expressed genes between lines are potential candidates as genetic markers for high and low potential for body fat accumulation. One such candidate, the lysophosphatidic acid (LPA) receptor-1 (LPAR1), was identified as a potential marker, being differentially expressed between the 2 lines at the early ages. Genomic DNA from the Fat and Lean F0 generation was sequenced upstream of the LPAR1 coding region. A SNP consisting of a T to C transversion that introduces a GATA-1 transcription factor binding site was identified in the Lean line (Fisher's Exact Test, p ≤ 0.001). The fattest and leanest animals of both sexes in the back-crossed F2 generation (n=48 each) were genotyped by allele-specific PCR, and an association was present between the genotype and phenotype (generalized linear model, p ≤ 0.05). Expression of GATA transcription factors in mice inhibits differentiation of preadipocytes into mature adipocytes. LPAR1 also inhibits differentiation of preadipocytes in mice, and LPAR1 knock-out mice become significantly fatter than wild-type mice. A SNP that introduces a GATA site in the promoter of LPAR1 could up-regulate its expression in the Lean line, and increased LPA signaling could then inhibit preadipocyte differentiation. Conversely, loss of the GATA binding site could explain decreased levels of LPAR1 expression and attenuated inhibition of adipocyte maturation in the Fat line.