title: Modification of Tet1 and histone methylation dynamics in dairy goat male germline stem cells.
abstract: OBJECTIVE
  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-renewal of male germline stem cells (mGSCs) and spermatogenesis. mGSCs are the only type of adult stem cells 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 dynamics make establishment of stable livestock mGSC cell lines difficult. The present study was conducted to detect effects of DNA methylation and histone methylation dynamics in dairy goat mGSCs self-renewal and proliferation, through overexpression of Tet1.
  
  
  METHODS
  An immortalized dairy goat mGSC cell line bearing mouse Tet1 (mTet1) gene was screened and characteristics of the cells were assayed by quantitative real-time PCR (qRT-PCR), immunofluorescence assay, western blotting, fluorescence activated cell sorting (FACS) and use of the cell counting kit (CCK8) assay.
  
  
  RESULTS
  The screened immortalized dairy goat mGSC cell line bearing mTet1, called mGSC-mTet1 cells was treated with optimal doxycycline (Dox) concentration to maintain Tet1 gene expression. mGSC-mTet1 cells proliferated at a significantly greater rate than wild-type mGSCs, and mGSCs-specific markers such as proliferating cell nuclear antigen (PCNA), cyclinD1 (CCND1), GDNF family receptor alpha 1 (Gfra1) and endogenic Tet1, Tet2 were upregulated. The cells exhibited not only reduction in level of histone methylation but also changes in 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 nuclei of mGSC-mTet1 cells, while it was uniformly distributed throughout nuclei of mGSCs. Our results conclusively demonstrate that modification of mGSCs with mTet1 affected mGSC maintenance and seemed to promote establishment of stable goat mGSC cell lines.
  
  
  CONCLUSIONS
  Taken together, our data suggest that Tet1 had novel and dynamic roles for regulating maintenance of pluripotency and proliferation of mGSCs by forming complexes with PCNA and histone methylation dynamics. This may provide new solutions for mGSCs stability and livestock mGSC cell line establishment.
authors.affiliation: College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China.
authors.forename: Liming
authors.initials: L
authors.lastname: Zheng
authors.affiliation: College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China.
authors.forename: Yuanxin
authors.initials: Y
authors.lastname: Zhai
authors.affiliation: College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China.
authors.forename: Na
authors.initials: N
authors.lastname: Li
authors.affiliation: College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China.
authors.forename: Chongyang
authors.initials: C
authors.lastname: Wu
authors.affiliation: College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China.|College of Life Science, Yulin University, Yulin, Shaanxi, 719000, China.
authors.forename: Haijing
authors.initials: H
authors.lastname: Zhu
authors.affiliation: Key Laboratory for Mammalian Reproductive Biology and Biotechnology, Ministry of Education, Inner Mongolia University, Hohhot, 010021, China.
authors.forename: Zhuying
authors.initials: Z
authors.lastname: Wei
authors.affiliation: Key Laboratory for Mammalian Reproductive Biology and Biotechnology, Ministry of Education, Inner Mongolia University, Hohhot, 010021, China.
authors.forename: Chunling
authors.initials: C
authors.lastname: Bai
authors.affiliation: Key Laboratory for Mammalian Reproductive Biology and Biotechnology, Ministry of Education, Inner Mongolia University, Hohhot, 010021, China.
authors.forename: Guangpeng
authors.initials: G
authors.lastname: Li
authors.affiliation: College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China.
authors.forename: Jinlian
authors.initials: J
authors.lastname: Hua
mesh_terms: D053687:Adult Stem Cells / Q000378:metabolism*; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D019938:Cyclin D1 / Q000378:metabolism; D019175:DNA Methylation / Q000502:physiology*; D004318:Doxycycline / Q000494:pharmacology; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D005854:Germ Cells / Q000378:metabolism; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors / Q000378:metabolism; D006041:Goats / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D008297:Male; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D013091:Spermatogenesis / Q000502:physiology
chemical_list: D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D006657:Histones; D018809:Proliferating Cell Nuclear Antigen; D011518:Proto-Oncogene Proteins; D019938:Cyclin D1; D006899:Mixed Function Oxygenases; D004318:Doxycycline