DNA methylation associated with induction of CRH gene expression in trophoblast cells (#274)
Background: Corticotropin Releasing Hormone (CRH) produced by human syncytiotrophoblasts rises exponentially in maternal plasma with advancing gestation predicting the timing of birth. The mechanisms controlling this increase are unclear. There are nine CpG dinucleotides in the human CRH promoter, which are potential targets for DNA methylation. CpG-2 locates within a cAMP response element (CRE), which is critically involved in controlling CRH expression. Cytosine-methylation of CpG-2 renders the CRE non-functional suggesting that promoter methylation might have regulatory role in placental CRH expression.
Objective: To determine CRH expression and CRH promoter CpG methylation in BeWo human choriocarcinoma cells and human primary cytotrophoblasts (PHT) during cAMP-induced and spontaneous syncytialisation.
Methods: BeWo cells and Percoll-purified term human cytotrophoblasts were cultured with 8-Br-cAMP (a cell-permeable cAMP analog) or 5-Aza-2’-deoxycytidine (ADC, a DNA-demethylating agent) individually or in combination, or vehicle for 24, 48 and 72 hours. CRH mRNA levels were measured by quantitative, reverse transcription-coupled PCR, and cytosine-methylation at the nine CpG sites was determined by bisulfite-sequencing.
Results: 25-60% of CRH promotes was methylated on CpG-2 in both BeWo and PHT cells at 0h incubation. CpG-2 methylation increased markedly by 72h in BeWo cells, which was partially blocked by 8-Br-cAMP. 8-Br-cAMP dose and time-dependently increased CRH mRNA level in both BeWo and PHT cells, reaching 60- and 40-fold stimulation, respectively, after 72 hours. ADC alone did not stimulate CRH mRNA expression, but augmented CRH gene induction by 8-Br-cAMP in BeWo approximately 5-fold.
Conclusions: These preliminary results suggest that the CRE is inactivated by methylation in a large proportion of CRH promoters in trophoblast populations, rendering them potentially unresponsive to cAMP-stimulation. Nevertheless, cAMP is a powerful inducer of the CRH gene presumably acting on promoters unmethylated on CpG-2. Active or passive demethylation of CpG-2 may play a major role increasing CRH expression in trophoblasts during gestation.