Abstract
Epigenetic modification plays key roles in spermatogenesis, especially DNA methylation dynamic is important in sustaining normal spermatogenesis. Ten-eleven translocation 1 (Tet1) is not only a key demethylase, which works in specific gene regions, but also crosstalks with partners to regulate epigenetic progress as protein complexes. Dairy goat is an important livestock in China, while the unstable culture system in vitro inhibits optimization of new dairy goat species. The study of epigenetic modification in male germline stem cells (mGSCs) is beneficial to the optimization of adult stem cell culture system in vitro, and the improvement of sperm quality and breeding of selected livestock. In our study, we not only analyzed the morphology, gene expression, DNA methylation and histone methylation dynamic in mouse Tet1 (mTet1) modified mGSCs, we also analyzed the stemness ability by in vivo transplantation and explored the functional mechanism of Tet1 in dairy goat mGSCs. The results showed mTet1 modified mGSCs had better self-renewal and proliferation ability than wild-type mGSCs, mTet1 could also up-regulate JMJD3 to decrease H3K27me3, which also showed to suppress the MEK-ERK pathway. Furthermore, Co-IP analysis demonstrated that TET1 interact with PCNA and HDAC1 by forming protein complexes to comprehensively regulate dairy goat mGSCs and spermatogenesis.
Highlights
Epigenetic modification on DNA and histone level plays important roles in the development of embryonic and male germline stem cells (mGSCs) through gene regulation, genomic imprinting, X-chromosome inactivation and carcinogenesis[1,2]
The positive clones were identified by green fluorescent protein (GFP) expression and we screened 5 clones to analyze (Fig. 1A) and the clone purity was higher than 93% (Fig. 1B)
We choose Pcna as a predicted target gene to get overexpression in mGSCs transfected with pIRES2-Pcna, and western blot showed proliferating cell nuclear antigen (PCNA) was up-regulated in mGSCs (P < 0.001), which had almost the same tendency as mGSC-pCDH-mTet[1] cells compared with mGSC-pCDH cells (P < 0.05) (Fig. 5C). These results suggested TET1 interact with PCNA is a positive regulator to PCNA, the Co-IP assay showed TET1 form protein complex with PCNA and modify mGSC proliferation in protein level (Fig. 5D)
Summary
Epigenetic modification on DNA and histone level plays important roles in the development of embryonic and mGSCs through gene regulation, genomic imprinting, X-chromosome inactivation and carcinogenesis[1,2]. Sin3A, a member of the Sin[3] family, which is linked to tumorigenesis and regulate gene expression through their role as histone deacetylases (HDACs)[32], it was distinctly required in differentiating spermatogonia and cell cycle progression[33,34]. Another histone deacetylase we noted is Hdac[1], which could cooperate with Sin3A as SIN3A-HDAC1 complex, Hdac[1] could protect the testicular damage[35,36]. In order to maintain pluripotency and reprogramming efficiency of dairy goat mGSCs, we investigate the mGSC biology through Tet[1] overexpression, verified the self-renewal and proliferation ability in vivo and in vitro to explore the role of Tet[1] in mGSC characters and development potentiality
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