The role of DNA methylation in Glucocorticoid Receptor-mediated repression of Corticotropin releasing hormone (CRH) gene.

Glucocorticoid receptor(GR)-mediated repression of corticotropin releasing hormone gene(crh) is an important component of a negative feedback which controls hypothalamic pituitary adrenal(HPA) axis. Failure of this negative feedback is often the cause of HPA axis dysregulation. The molecular mechanisms that lead to this failure are still unclear. The dysregulation of HPA axis is associated with many neuropsychiatric disorders such as depression, cognitive impairment, neuro-degeneration and mood disorders. Here we investigate the molecular mechanism by which GR maintains the repressed levels of crh. DNA methylation of crh promoter plays an important role in gene regulation. Sharma et al. have shown that, Dexamethasone(Dex)- a GR ligand treatment increases the methylation of CpG dinucleotides present in the crh promoter in IVB rat hypothalamic cells. Also,according to same study, Dex increases the recruitment of methyl CpG binding protein 2(MeCP2) to the crh proximal promoter region. Here we tested the hypothesis that whether DNA methyalation and MeCP2 are required for maintaining the GR mediated repressed levels of crh. We found that, in IVB cells inhibition of DNA methylation by 5-Aza-2-deoxycytidine (5-AzaDC) increased the crh expression in dose dependent manner. We also found that 5-AzaDC at 0.5uM concentration significantly inhibited the crh promoter methylation. Further we observed that Dex fails to maintain the repressed levels of crh in cells pretreated with 5-AzaDC. We used RT-qPCR for gene expression analysis. We also analyzed the role of MeCP2 in GR mediated repression of crh. Our data indicated that siRNA mediated knock down of MeCP2 protein leads to increased basal levels of crh expression. Also, Dex fails to repress the crh in absence of MeCP2 protein. Taken together our results indicate that DNA methylation and MeCP2 are required for maintaining the GR mediated repressed levels of crh.