DEXAMETHASONE INDUCES FORMATION OF A PUTATIVE REPRESSOR COMPLEX AND ASSOCIATED CHROMATIN MODIFICATIONS IN THE REGION OF THE CORTICOTROPIN-RELEASING HORMONE GENE PROMOTER

Purpose: Glucocorticoids down-regulate expression of hypothalamic CRH; however, mechanisms by which they do so are not fully understood. The proximal promoter cAMP response element (CRE), negative GRE (nGRE), and methylated CpG islands all play a role in crh down-regulation. Here we are reporting the involvement of DNA and Histone methyltion in the regulation of crh gene expression. Methods: Real time PCR to measure the levels of mRNA. Immunocytochemistry to localize immunoreactive proteins. Western Blotting to check the expression of Proteins. Coimmunoprecipitation to look for the interaction of various proteins involved. Chromatin immunoprecipitation to analyze the recruitmentment of various regulatory molecules at the promoter. Promoter methylation analysis by sodium bisulphite sequencing Results: Previously, we showed that Dex-repressed crh expression is associated with GR and histone deacetylase 1 (HDAC1) recruitment to the crh promoter region. Given that HDAC1 may be present in methylated CpG binding protein 2 (MeCP2) complexes, and that MeCP2 is known to play a role in regulating crh expression, we sought to determine whether or not HDAC and/or MeCP2 could interact with the GR. Dex enhanced GR interactions with both proteins.Glucocorticoid regulation of crh has also been associated with CpG methylation, thus we assessed whether GR could interact with a DNA Methyltransferase (DnMT). Indeed, the GR interacted with DnMT3b but not DnMT3a. In addition, Dex also induced occupancy of crh by, HDAC1, MeCP2, and DnMT3b in the promoter region and consequently it led to an increase in the level of promoter methylation that appeared to be CpG site specific. Lastly, to extend previous assessment of chromatin modifications in this promoter region, the degree of histone methylation was measured. Dex increased tri-methylation of histone 3 lysine 9 (H3K9), a marker of gene suppression, however levels of di and tri methylated H3K4 were not significantly changed. Conclusions: The data indicate that a GR:HDAC1:MeCP2 repressor complex participates in Dex mediated crh down regulation. Furthermore, recruitment of this complex occurs in parallel with GR interaction with DnMT3b and DnMT3b recruitment to the crh proximal promoter and simultaneous increase in site specific promoter methylation. Lastly, specific histone methylation appears to play a part in crh downregulation, as well, in that Dex treatment leads to a co-temporaneous increase in tri- methylated H3-K9.