The Modification of Tet1 and Histone Methylation Dynamics in Dairy Goat Male Germline Stem Cells

摘要

Tet (ten-eleven translocation) protein 1 is a key enzyme for DNA demethylation,which modulates DNA methylation and gene transcription.DNA methylation and histone methylation are critical elements in self-renew of male germline stem cells (mGSCs) and spermatogenesis.The mGSCs are the only type of adult stem cells that are able to achieve intergenerational transfer of genetic information,which is accomplished through differentiated sperm cells.However,numerous epigenetic obstacles including incomplete DNA methylation and histone methylation dynamic make the establishment of stable livestock mGSC cell lines difficult.The present study was conducted to detect the effect of DNA methylation and histone methylation dynamic to dairy goat mGSCs self-renew and proliferation through overexpression of Tet1.Materials and methods:An immortalized dairy goat mGSC cell line bearing mouse Tet1 (mTet1) gene was screened; the characteristics of cells were assayed by quantitative real-time PCR (qRT-PCR) assay,immunofluorescence assay,western blot assay,fluorescence activated cell sorter (FACS) and cell counting kit (CCK8) assay.Results: The screened immortalized dairy goat mGSC cell line bearing mTet1,which was named mGSC-mTet1 and treated with an optimal doxycycline (Dox) concentration to maintain Tet1 gene expression.The mGSC-mTet1 cell line proliferated at a significantly greater rate than wild mGSCs and mGSCs-specific gene markers such as Proliferating Cell Nuclear Antigen (PCNA),cyclinD1 (CCND1),GDNF family receptor alpha 1 (Gfra1) and endogenic Tet1,Tet2 were upregulated.The mGSC-mTet1 cells exhibited not only a decrease in the level of histone methylation but also a change in the nuclear location of that methylation marker.While H3K9me3 was uniformly distributed throughout the nucleus of mGSC-mTet1 cells,it was present in only particular locations in mGSCs.H3K27me3 was distributed surrounding the edges of the nuclei of mGSC-mTet1 cells,while it was uniformly distributed throughout the nuclei of mGSCs.Our results conclusively demonstrate that modification of mGSCs with mTet1 affects mGSC maintenance and may promote the establishment of stable goat mGSC cell lines.Conclusions: Taken together,our data suggest that Tet1 has the novel and dynamic roles in regulating the maintenance of pluripotency and proliferation in mGSCs by forming complex with PCNA and histone methylation dynamic,which may provide new solutions for mGSCs stability and livestock mGSC cell lines establishment.