Studies on Transcriptional and Epigenetic Mechanisms of NANOG gene in Chicken Primordial Germ Cells

Abstract

Primordial germ cells (PGCs), the precursors of sperm and oocytes, are the only cell type which can transfer genetic information to next generation. These distinct characteristics are largely dependent on germ cell- specific gene expression. They are controlled by coordinated actions of many key regulators such as transcription factors (TFs), RNA Binding proteins (RBPs) and specialized epigenetic modification. Nanog, the mostly conserved protein between species, are well- known to be a core transcriptional factor in the early formation of embryonic development. Nanog is also expressed in cells from pluripotent cells to gonads through the developmental status of PGCs, which means that the role of Nanog is to perpetually maintain germ cell characteristics containing stemness and germness in general germ cell development. Indeed, Nanog regulates PGCs by signaling pathways with other key transcription factors. Furthermore, induction of mouse PGC-like cells(PGCLCs) from epiblast-like cells(ESCs) is available with Nanog alone. In chicken, Nanog expression is observed from early development. And then, the pattern of the gene is restricted to PGCs after HH3. These results suggest that Nanog in chicken is a key factor of regulation of PGC characteristics. Recent studies find that epigenetic regulations, such as loss of 5mC in whole genome, DNA methylation, histone and chromatin modifications have a crucial role in PGCs. However, studies of histone and chromatin modifications and also the control of epigenetic patterns in chicken are highly limited. In the previous studies, methylation and acetylation during germ cell specification to differentiation activate germ cell specific genes and repress somatic cell genes in mouse. And also, in chicken, germ cells are also epigenetically regulated. In contrast to mammals, the H3K27me3 global level is reduced, whereas the H3K9me3 level is increased in chicken with still acetylation level in PGCs yet unknown. In this study, we investigated the elaborate regulatory mechanisms that govern epigenetic and transcriptional programs of Nanog. Histone deacetylase (HDAC) regulates NANOG in PGCs. And also, methylation patterns of CpG islands in cNanog upstream, the regions which the transcription factors bind to, are hypomethylated in PGCs. The result of this study using a series of experiment for functionality testing, including siRNA mediated knockdown, overexpression, immunocytochemistry, luciferase reporter assay, TFs motif analysis, and western blotting demonstrates that chicken has specific epigenetic regulation during PGC development. Intriguingly, we showed that transcriptional program of NANOG was strictly regulated by specific isotype of HDACs (HDAC1 and HDAC2) and the REST repressor complex, suggesting that avian PGCs have the different molecular regulatory mechanism from that of mammals. In conclusion, chicken PGCs display the unique epigenetic and transcriptional program of Nanog. Our findings first time provide valuable insight on chicken PGCs to unravel important regulatory components as well as biological roles regarding the genetic and epigenetic regulation for stemness and germness of Nanog and better understanding of germ cell fate. The study of avian species can be adapted to important vertebrate model for the research of developmental biology and speciation.