Studies Of The Function And Mechanism Of OVOL1 And OVOL2 In DNA Damage Repair And Male Meiosis

Background: OVO genes are essential for cell specific differentiation.OVO family play vital roles in several signaling pathways which are involved in early and late embryonic development.Besides,OVO genes also act as transcription factors in regulating genes expression during tissue development.Researches on OVO genes in mammalians mainly focus on OVOL1 and OVOL2.However,the functions of OVOL1 and OVOL2 on DNA damage repair and male meiosis are still unknown.Objective: This study aims to explore the role of OVOL1 and OVOL2 in DNA damage repair and spermatogenesis as well as the regulatory mechanism.Research methods: In this study,the sequences and structure of OVOL1 and OVOL2 proteins have been analyzed by bioinformatics.Then,the embryonic stem cells with OVOL1 and OVOL2 deletion have been constructed.The function of OVOL1 and OVOL2 in DNA damage repair were studied by biochemistry and molecular biology technologies,such as fluorescence quantitative PCR,tandem affinity purification,western blot,immunofluorescence,flow cytometry,gene mutation.Meanwhile,Stra8-GFPCre mice were used to establish OVOL1 and OVOL2 conditional knockout germ cells for studying the roles of OVOL1 and OVOL2 in spermatogenesis.Results:1.Sequences analysis showed that OVOL1 and OVOL2 in human had high similarity to OVOL1 and OVOL2 in mouse.The similarity of OVOL1 was 96% and the similarity of OVOL2 was 88%.All of the four proteins have four zinc-finger domains which have the function of binding DNA and regulating transcription.2.The signal or double OVOL1 and OVOL2 knockout mice model were successfully established and embryonic stem cells were acquired.Besides,the Ovol1 and Ovol2 were specifically knocked out in germ cells with the help of Stra8-GFPCre tool mice.3.After DNA damage was induced by laser in Hela cells,both of OVOL1 and OVOL2 were recruited to DNA damage sites.The results of tandem affinity purification showed that OVOL1 and OVOL2 could interact with single-strand DNA binding protein complex(RPA).Using Co-IP to examine the variants created by site directed mutagenesis showed that OVOL1 and OVOL2 could bind RPA complex through the three zinc-finger domains close to C terminal.4.Western blot and immunofluorescence showed that loss of OVOL1 or OVOL2 in embryonic stem cells did not affect the binding of RPA complex to chromosome and the recruitment of RPA complex to DNA damage sites.Western blot examination on phosphorylation of CHK1 showed that the phosphorylation process was affected by the OVOL1 or OVOL2 deficiency.Furthermore,FACS examination on OVOL1 or OVOL2 deficiency cells showed that G2/M cell cycle checkpoint could not be activated efficiently and DNA damage response was delayed which leads to the chromosomal abnormalities eventually.5.Animal model showed that Ovol1 or Ovol2 knockout male mice were fertile and matured sperms can be observed in their epididymides.There was no significant difference in testis size and weight between mutants with control.The meiosis was also normal in Ovol1 or Ovol2 knockout male mice.Conclusion: Deletion of Ovol1 or Ovol2 leads to DNA damage repair defects in mouse embryonic stem cells.OVOL1 and OVOL2 participate in DNA damage repair pathways upon their interaction with RPA complex,which is important to maintain the genomic stability in mouse embryonic stem cells.Inactivation of OVOL1 or OVOL2 did not affect the spermatogenesis,including male meiosis.