Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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Item Role of Epigenetic Modifications and Dexras1 in glucocorticoid regulation of growth hormone expression(2011) Narayana, Jyoti; Porter, Tom E; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Glucocorticoid induction of growth hormone in anterior pituitary cells of embryonic chickens is an indirect effect. A genome-wide microarray screen conducted on embryonic day 11 chickens identified Dexras1 as a novel candidate gene involved in mediating the glucocorticoid effects on GH mRNA expression. The following studies were aimed at characterizing glucocorticoid regulation of chicken Dexras1 and identifying potential involvement of Dexras1 in mediating glucocorticoid effects on GH expression. We determined that glucocorticoid induction of Dexras1 requires glucocorticoid receptor and both MEK1/2 and Ras cell signalling pathways. Overexpression of Dexras1 vector had no effect on GH reporter activity. Transfection of 1ng or 10ng of Dexras1 expression plasmid stimulated basal GH mRNA levels, whereas transfection of 1000ng of Dexras1 expression plasmid significantly inhibited levels of glucocorticoid-induced GH mRNA. YFP-tagged Dexras1 protein was predominantly found in the cytoplasm and glucocorticoid treatment did not cause Dexras1 to translocate to the plasma membrane. Downstream targets of Dexras1 are not known. Additionally, glucocorticoid-regulated histone modifications within 2kb of the GH gene were investigated using chromatin immunoprecipitation assays. The effects of glucocorticoid (1.5 h or 6 h) on histone H3 modifications, RNA Polymerase II (Pol II) recruitment, and association of GR, Pit-1, and Ets1 with the chicken GH gene were examined. We found increased H3 acetylation and tri-methylation of lysine 4 at both Pit-1 sites and the transcription start site (TSS) in response to 1.5 h glucocorticoid treatment. Furthermore, 1.5 h glucocorticoid treatment significantly increased recruitment of Pit-1 and Pol II to the proximal Pit-1 site and the TSS, respectively. GR was recruited to the glucocorticoid-responsive region (GCRR) and the distal Pit-1 site in response to 1.5 h glucocorticoid treatment, while 6 h glucocorticoid treatment resulted in Ets1 dissociation from the GCRR. Collectively, these results indicate that glucocorticoid induces dynamic changes in histone modifications and transcription factor recruitment within the 5'-flanking region of the chicken GH gene. We conclude that glucocorticoid induction of GH gene expression in chickens is achieved by a combination of genomic and nongenomic pathways. Our studies provide novel mechanisms of GH regulation in chickens, some of which may be found in other vertebrate species as well.Item Ontogenic and glucocorticoid-regulated gene expression in the developing neuroendocrine system(2010) Ellestad, Laura Elizabeth; Porter, Tom E; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The neuroendocrine system is a critical regulator of vertebrate homeostasis that includes five hypothalamic-pituitary axes which develop during embryogenesis. Adrenal glucocorticoids play an important role in functional maturation of the anterior pituitary through initiation of growth hormone (GH) production. These studies were aimed at characterizing ontogenic and glucocorticoid-regulated changes in gene expression during neuroendocrine system development in the chick. First, to ascertain timing of initiation and establishment of each neuroendocrine axis, we measured mRNA levels of hypothalamic regulatory factors, their pituitary receptors, and pituitary hormones from embryonic day (e) 10 through post-hatch day (d) 7. We found that the adrenocorticotropic axis is the first to be established (e12), followed by establishment of the thyrotropic (e18), somatotropic (e20), lactotropic (d1), and gonadotropic (d5) axes. Next, we examined in detail mechanisms through which glucocorticoids initiate pituitary GH expression during embryogenesis. We determined that glucocorticoids elevate GH mRNA levels on e11 by increasing transcriptional activity of the GH gene rather than enhancing mRNA stability, and protein synthesis, histone deacetylase activity, ras signaling, and ERK1/2 signaling are required for this activation. Conversely, sustained activation of ERK1/2 and p38MAPK pathways reduced glucocorticoid stimulation of GH expression, indicating the requirement for ERK1/2 activity is transitory. Finally, we identified ras-dva as a novel Pit-1 and glucocorticoid-regulated gene in the chicken embryonic pituitary gland. Pituitary ras-dva mRNA levels increased between e10 and e18, decreased just prior to hatch, and remained low or undetectable post-hatch. Ras-dva expression was highly enriched within the pituitary gland on e18, and glucocorticoids rapidly induced ras-dva mRNA in cultured pituitary cells through a mechanism involving transcriptional activation. Potential regulatory elements within the 5'-flanking region of chicken ras-dva responsible for pituitary-specific expression were identified, as was a 2 kb fragment necessary for its glucocorticoid induction in embryonic pituitary cells. These results enhance our understanding of neuroendocrine system development and establishment during embryogenesis, reveal mechanisms underlying glucocorticoid initiation of GH expression in somatotrophs, and identify a new Pit-1 and glucocorticoid target gene that may play an important role in pituitary development.