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

[摘要] 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.

[学科分类] 内分泌与代谢学