Role of Epigenetic Modifications and Dexras1 in glucocorticoid regulation of growth hormone expression

Abstract

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.