Effect of dynamic DNA methylation and histone acetylation on cPouV expression in differentiation of chick embryonic germ cells.

As a crucial pluripotency-related factor, the epigenetic regulation of Oct4 has been studied intensively in mammalians. However, its dynamic changes of DNA methylation and histone modification in avians remain poorly understood. In the present study, we first described the alterations of DNA methylation and histone acetylation in the promoter of chicken PouV (cPouV; the homologue of Oct4 in avian) during chick embryonic germ (EG) cell differentiation. The epigenetic modification analysis showed that DNA methylation in the cPouV promoter increased obviously, while histone acetylation decreased dramatically detected by chromatin immunoprecipitation assay in the process of differentiation. Gene expression analysis detection indicated that the levels of DNA methyltransferase 3a (Dnmt 3a), Dnmt 3b, and histone deacetylase 3 (HDAC 3) transcripts were significantly high, whereas the relative abundance of Dnmt 1, histone acetyltransferase (HAT), and cPouV mRNA was significantly decreased during the conversion of EG to embryoid body-like structures (EBs), which was correlated with the increased level of methylation and reduced level of H3 acetylation. Moreover, in vitro methylation assay indicated that the reporter gene was remarkably inhibited by the methylated promoter of cPouV. To further understand the effect of epigenetic modifiers on cPouV expression, we performed an analysis of EB cells treated with trichostatin A (TSA), Aza-2'-deoxycytidine (Aza), or TSA plus Aza (TSA/Aza). We observed that the effect of TSA/Aza is more sensitive to the reactivation of cPouV compared with TSA or Aza, indicating that these epigenetic inhibitors can function synergistically to facilitate the reprogramming process. The present study provided evidences that a critical role for cPouV activation/repression by DNA methylation and/or histone modifications is involved in the pluripotency maintenance and differentiation process of chick EG.