Epigenetic modification cooperates with Zeb1 transcription factor to regulate Bmp4 to promote chicken PGCs formation

BMP4 is the critical gene of primordial germ cell formation in mammal, however, the mechanism of PGCs formation in chicken still unknown. In this research, we compared the evolution relationship of different species. Although the protein sequence is highly conservative between mouse, human and chicken, promotors vary among avian and mammal species. Therefore, it is easily to predict that there would be different regulation mechanism of Bmp4 expression in chicken. Here, we elucidate the function of chicken Bmp4 during PGCs formation. In vivo, Bmp4 can promote PGCs development and migration, and increase the expression of key genes (Cvh, c-kit, cxcr4, etc.). Whereas, the expression of these genes will decrease after knocking out Bmp4. After over-expression and knockout Bmp4 in vitro, we found that overexpression of Bmp4 could promote the formation of embryoid bodies (EB) and up-regulate the key genes of PGCs formation and migration, while knockout Bmp4 could inhibit the formation of embryoid bodies and decrease the expression of related genes. Flow and indirect immunofluorescence also indicated the same result. These all results proved that chicken Bmp4 could also promote the formation of PGCs. Furthermore, dual-luciferase activity detection showed that the promotor activity of Bmp4 was positively regulated by transcription factor Zeb1. Overexpression of Zeb1 can also increase the mRNA and protein expression of Bmp4. At the same time, DNA methylation inhibited Bmp4 transcription and histone methylation was able to promote its transcription. In conclusion, this study established that chicken Bmp4 can promote the formation of chicken PGCs. This gene is regulated by DNA, histone methylation and transcription factor Zeb1. These results lay a theoretical foundation for exploring the function and molecular mechanism of Bmp4 in the process of PGCs formation.