Corticotropin Releasing Hormone (CRH), is expressed in many regions of the central nervous system and in some peripheral tissues, and plays an important role in determining gestational length. In placenta, a cAMP regulatory site (CRE) is crucial for CRH gene regulation. The promoter of CRH gene has 9 CpG sites, which should be the targets of epigenetic regulation by DNA methylation. The BeWo cell line, derived from human gestational choriocarcinoma, has been widely used as an in vitro model for the placenta. BeWo cells only produce CRH after exposure to cAMP. The DNA methyl transferase (DNMT) inhibitor 5-aza-cytidine stimulates CRH expression 5-fold in camp treated BeWo cells, indicating the CRH promoter as a target of DNMTs. To evaluate methylation differences of the 9 CpG sites in CRH gene promoter in BeWo cells after treatment with cAMP. Genomic DNA was extracted from BeWo cells treated or not with cAMP. Sodium bisulfite conversion was used to modify the genomic DNA. PCR was used to amplify the CRH promoter region with primers that did not contain CpG sites. The PCR products were cloned and sequenced. The CpG methylation status of each sample was obtained by comparing the sequencing results with the original sequence. In non-stimulated cells (control) CpG -4 was methylated in 50% of the clones and CpG -6 was methylated in 75% of the clones, but the other 7 sites were methylated in every clone. In the cAMP treated cells however there was 100% methylation at CpG sites 6 through 9, but only partial methylation at CpG-1 and 3 (60%), CpG-4 and 5 (40%). Most interestingly, there was no methylation found at CpG-2 in any of the clones from cAMP treated cells, indicating that specific CpG de-methylation around the CRE is required for CRH gene expression.
Epigenetic regulation of DAT gene promoter modulates the risk of Externalizing and Internalizing behaviors on a normative population: an explorative study
