Epigenetic regulation of the corticotropin releasing hormone (CRH) gene in human trophoblasts

Corticotropin Releasing hormone (CRH) is crucial in the central nervous system as well as in the female reproductive system. Placental CRH, synthesized and secreted in trophoblastic syncytium, is the main source of CRH in maternal plasma. The level of CRH increases as gestation advances, peaking at the time of delivery. The rise is strongly linked to gestational length. In women who are going into spontaneous preterm birth, the increase is in advance while the level is higher. In postterm pregnancy, the increase of maternal plasma CRH is obviously retarded.CRH gene expression shows a tissue-specific pattern. It is repressed in cytotrophoblasts, but greatly induced after syncytialization,. It has been reported that the change of expression is related to cAMP pathway. cAMP is a strong inducer of syncytial differentiation and CRH gene expression. However, the mechanism underlying the interactions between cAMP and its binding sites remain unclear. Epigenetics may be involved in the tissue-specific regulation. Epigenetics is defined as change of gene expression without any change of DNA sequence. The most well studied epigenetic regulations mainly include DNA methylation (literature review published and cited in Chapter 1) and histone modifications. There are nine CpG sites in the proximal CRH promoter, which could be the targets of DNA methylation. The most interesting CpG site is the one located in the cAMP response element (CRE). There have been several reports showing that epigenetic regulations are crucial in the CRH gene expression in central nervous system. Still, there was no research performed in human placenta. We propose that the change of CpG methylation as well as histone modifications in the proximal CRH promoter is involved in the dynamic change of transcription factor binding, participating in CRH gene regulation in human placenta. Bisulfite sequencing was used in BeWo cell line, a well-characterized trophoblastic cell line, to determine the methylation profile of the nine CpG sites before and after the induction of CRH gene expression by 8-Br-cAMP, while 5-aza-2 deoxycytidine (5-AZA-dC) was used to diminish DNA methylation and clarify the effects on CRH gene expression. We have detected that CRH promoter is dynamically and intermediately methylated in BeWo cells. CRH mRNA expression and the methylation of a subset of CpGs (including CpG2 in the CRE) increased spontaneously during culture. 8-Br-cAMP stimulated CRH expression without affecting the increase in methylation (published and cited as Chapter 3). We further employed primary trophoblast culture to determine the DNA methylation patterns of proximal CRH promoter and its interactions with histone acetylation, methylation and transcription factor binding. Cytotrophoblast cells were isolated and cultured with 8-Br-cAMP induction or through spontaneous syncytialization. Bisulfite sequencing was also performed to detect the specific CpG methylation before and after the CRH gene activation. Chromatin immunoprecipitation(ChIP) was adapted to determine histone modifications and specific transcription factor binding, such as pCREB, Pol II, and TBP. Notably, we used a novel method by bisulfite sequencing of ChIP-selected DNA to evaluate the CpG methylation patterns associated with particular histone modifications and transcription factors. First, DNA fragments subject to histone modifications or with transcription factors were picked up by specific antibodies. Bisulfite sequencing was then adapted to determine the CpG methylation patterns on these alleles. We showed that CRH gene expression was accompanied by increased activating and decreased repressive histone modifications at the promoter. No significant DNA methylation changes were detected. The nine CpGs in the CRH proximal promoter were partially and allele-independently methylated. Different transcription factors selectively associated with specific epialleles. Histone modifications also showed different epiallele preference. We propose that conditions impacting on epiallele distribution influence the number of transcriptionally active CRH gene copies in the trophoblast cell population determining the gestational trajectory of placental CRH production in normal and pathological pregnancies (published and cited as Chapter 4). In conclusion, these studies revealed the role of epigenetic regulation in placental CRH gene expression, which could provide molecular fundamentals for early treatment of abnormal pregnant outcomes. Hopefully, women with high levels of placental CRH, at the same time with high riskof preterm delivery, will greatly benefit from these studies.