Methylation of the Corticotropin Releasing Hormone Gene Promoter in BeWo Cells: Relationship to Gene Activity

Abstract

Corticotropin releasing hormone (CRH) production by the human placenta increases exponentially as pregnancy advances, and the rate of increase predicts gestational length.CRHgene expression is regulated by cAMP in trophoblasts through a cyclic AMP-response element (CRE), which changes its transcription factor binding properties upon methylation. Here we determined whether methylation of theCRHproximal promoter controls basal and cAMP-stimulatedCRHexpression in BeWo cells, a well-characterized trophoblastic cell line. We treated the cells with 8-Br-cAMP and the DNA methyltransferase inhibitor 5-aza-2′ deoxycytidine (5-AZA-dC) and determined the effects onCRHmRNA level and promoter methylation. Clonal bisulfite sequencing showed partial and allele independent methylation of CpGs in theCRHpromoter.CRHmRNA expression and the methylation of a subset of CpGs (including CpG2 in the CRE) increased spontaneously during culture. 8-Br-cAMP stimulatedCRHexpression without affecting the increase in methylation. 5-AZA-dC decreased methylation and augmented 8-Br-cAMP-stimulatedCRHexpression, but it blocked the spontaneous increase ofCRHmRNA level. We conclude that theCRHpromoter is a dynamically and intermediately methylated genomic region in BeWo cells. Promoter methylation did not inhibitCRHgene expression under the conditions employed; rather it determined the contribution of alternative cAMP-independent pathways and cAMP-independent mechanisms toCRHexpression control.