UMD Theses and Dissertations
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Item MEDIATION OF CORTICOSTERONE-INDUCED GROWTH HORMONE GENE EXPRESSION IN CHICKEN EMBRYONIC PITUITARY CELLS: IDENTIFICATION OF TRANS-ACTING FACTORS AND A NOVEL PITUITARY CELL TYPE(2024) Liu, Kuan Ling; Porter, Tom E.; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Growth hormone (GH) is responsible for up to 30% of growth in broiler chickens. Somatotrophs, or GH secreting cells, begin to differentiate around embryonic day (e)14, in conjunction with an increase in the primary plasma glucocorticoid (GC) corticosterone (CORT). CORT treatment of e11 chicken embryonic pituitary (CEP) cells induces premature GH secretion. This GC-induced process involves trans-acting factors because the GH gene lacks a canonical GC response element (GRE). In addition to the binding of ETS1 and the GC receptor (GR) to the GC-responsive region (GCRR; 1045/ 964), we hypothesize that there are other regulatory factors necessary for CORT responsiveness. By modifying the pGL3_-1742/+25 GH-luciferase reporter, we have constructed various other GH-luciferase reporters and analyzed them for promoter activity in response to CORT treatment. We identified a putative distal (d) ETS-Like 1 (ELK1) binding site that is necessary. The proximal (p)PIT1 site and pTATA box were also identified to be critical for CORT induction of the GH gene. Interestingly, cloning multiple copies of the extended GCRR (eGCRR; -1067/-900) further increased promoter activity in an additive manner under both basal and CORT treated conditions. Through single-cell RNA sequencing (scRNAseq), 8 members of the ETS family of transcription factors were identified in > 5% of the somatotroph population. Commercial antibodies were validated, and human (h)ETV1, hELF2, hELK3, and hETV6 antibodies were confirmed to recognize their recombinant chicken ortholog and to identify their corresponding protein in e11 CEP cells. Results from chromatin immunoprecipitation quantitative PCR suggest that multiple ETS members are involved in CORT induction of the GH gene with more evidence pointing towards ELF2 and ELK3. Identifying trans-acting factors for the GH gene inducible by CORT allows for better understanding of endogenous GH regulation in chickens. Further analysis of the scRNAseq data from e11 CEP cells revealed a cluster of cells expressing genes for more than one hormone-producing cell type (“premature nebulous” cluster). Within the premature nebulous cluster, a large population (~30%) was co-expressing proopiomelanocortin (POMC) and growth hormone (GH). We named this novel cell population the cortico-somatotrophs. Through RNA fluorescent in-situ hybridization (RNA-FISH) and dual label immunofluorescence, we verified the existence of the cortico-somatotrophs at both the mRNA and protein level, respectively. Cortico-somatotrophs were also shown to share genes for receptors normally specific to both corticotrophs (CRH-R1) and somatotrophs (GHRHR). Additionally, in response to CORT treatment, the cortico somatotrophs showed an increase in GH as well as a decrease in POMC mRNA levels. The discovery of the cortico-somatotrophs suggests a modification to the current dogma on pituitary cell lineages, where corticotrophs and somatotrophs may have overlapping developmental pathways. In conclusion, our discovery of the cortico somatotrophs has furthered our understanding of CEP development and opened the door for further exploration of the cell lineages during pituitary development.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.Item IDENTIFICATION OF A NON-CLASSICAL GLUCOCORTICOID-RESPONSIVE ELEMENT IN THE 5'-FLANKING REGION OF THE CHICKEN GROWTH HORMONE GENE(2010) Knubel, Kristina Heuck; Porter, Tom E; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Growth hormone (GH) effects growth and contributes to a lean phenotype in broiler chickens. GH secretion by the anterior pituitary begins on embryonic day (e) 14, concomitantly with a rise in adrenal glucocorticoids (GC) or corticosterone (CORT) secretion. CORT treatment of chicken embryonic pituitary (CEP) cells induces GH secretion prematurely. GC induction of the GH gene requires on-going protein synthesis or an intermediary protein, but the gene lacks a classical GC-response element. We hypothesized that a GC-responsive intermediary protein is necessary for the CORT induced increase in GH. Characterization of the upstream region of the gene may help identify such a protein. To this end, a fragment of the GH gene (-1727/+48) was cloned into a luciferase reporter and characterized in e11 CEP cells. CORT treatment increased luciferase activity and mRNA. Inclusion of CHX blocked CORT induction of luciferase mRNA. Through deletion analysis, we found that a GC-responsive region (GCRR) is located at -1045 to -954. By defining the GC-responsive region and cis-acting elements located within, trans-acting proteins involved in GC induction of the GH gene may be identified. The GCRR is CORT-responsive in either orientation, but it is context-dependent. Potential transcription factor motifs in the GCRR include ETS-1 and a degenerate GRE (GREF). Nuclear proteins bound to a GCRR probe in a CORT-regulated manner and unlabeled competitor DNA competed off binding. Mutation of the central portion of the DNA probe resulted in a significant decrease in protein binding. Mutation of the ETS-1 site or GREF site in the -1045/+48 GH construct resulted in ablation of luciferase activity. ETS-1 and GR are associated with the endogenous gene under basal and 1.5 h CORT-treated conditions, while GR recruitment increased after CORT treatment. GC regulation of the GH gene during chicken embryonic development requires cis-acting elements located 1 kb upstream from the transcription start site and includes recruitment of ETS-1 and GR. This is the first study to demonstrate involvement of ETS-1 in GC regulation of the GH gene during embryonic development. Characterization of GC regulation of the GH gene during embryonic development enhances our understanding of growth regulation in vertebrates.