The Study Of The Expression And Function Of Chicken Prdm1

PRDM1(positive regulatory domain1) is a transcriptional repressor that has been identified in various species and is crucial for cell growth, differentiation, and development. However, the expression pattern and role of PRDM1in development has not been sufficiently established in birds. We have therefore investigated the spatiotemporal expression of PRDM1in various tissues, especially in the germline, during chicken development, and explored the role of PRDM1in chicken development and the cell cycle of DF-1cell by forced expression of PRDM1.Our results showed that prdml mRNA was expressed in blastodermal cells (BCs) at stage X and in various tissues including the liver, skin, lung, kidney, eye, bursa of fabricius, spleen, proventriculus, gizzard, intestine, testis, ovary, tongue, feathers, and thymus but was not or was only sparcely present in the heart, brain, and skeletal muscle. The level of prdml mRNA was the highest in the BCs among all tissues tested and significantly changed during development in many tissues, such as the blastoderm, bursa of fabricius, spleen, feathers, and germline. Furthermore, the expression of the PRDM1protein generally paralleled the mRNA results, except for in the gizzard. Immunohistochemistry also revealed that PRDM1was localized in the smooth muscle. In addition, during germline development, PRDM1was found to be continuously expressed in the presumptive primordial germ cells (primordial germ cells, PGCs) at stage X, the circulating PGCs in blood, and the germ cells in the gonads from embryonic day6to adult in both males and females.The expression pattern of PRDM1in chicken thus suggests that this protein plays an important role during chicken development, such as in BCs differentiation, feather formation, and germ cell specification. Therefore, the lentiviral vector GV208-PRDM1was constructed, and injected into chicken embryos in subgerminal cavity or blood vessel. In the embryos injected with GV208, green fluorescent signals can be seen widely in various tissues. However, interestingly, green fluorescent signals were not observed in the embryos injected with GV208-PRDM1. The genomic DNA of15chicken embryos injected with GV208-PRDM1in blood vessel were analyzed by PCR, and8of the15chicken embryos were GFP positive. However, the exogenous prdml mRNA were not detected in all chicken embryos. These resulrts suggested the silence of exogenous prdml was resulted from the transcriptional regulation or post-transcriptional regulation.In addition, we explored the role of PRDM1in the cell cycle by forced expression of PRDM1in DF-1cell line. Our results showed that there was no endogenic PRDM1and exogenous PRDM1was specifically present in the nucleus of DF-1cell. PRDM1overexpression caused an increase in the cell population at G0/G1phase. Our data further demonstrated that the levels of p53, mdm2, Rb and e2f-1mRNA were significantly increased in DF-1cells transfected with pEGFP-N1-PRDM1. Interestingly, the mRNA expression levels of Cyclin C and Cyclin D2were significantly increased. The mRNA expression levels of the other cyclin genes were also increased, although the differences were not significant. Futhermore, PRDM1overexpression inhibited cell proliferation and changed cell morphology. These results showed that PRDM1overexpression induce GO/G1arrest through multiple parallel mechanisms including the p53and Rb pathway in DF-1cells.