Studies On Mechanism Of GGNBP2 Regulating DNA Double-strain Break Repair And Histone Ubiquitination In Mouse Spermatocytes

Spermatogenesis is an extremely complex process,which mainly includes three stages:proliferation and differentiation of spermatogonial stem cells,spermatocytes meiosis and spermiogenesis.In this complex process,a variety of gene regulation and protein expression modification changes are involved.In addition to studying the protein function of gene expression at the transcriptional level,it is also important to study protein post-translational modification at the protein level.Histone post-translational modifications include histoneacetylation,phosphorylation,methylation,ubiquitination and glycosylation.These post-translational modifications play an important role in chromatin remodeling,gene transcription regulation,as well as DNA damage repair.Gametogenetin binding protein 2?GGNBP2?,also called zinc finger protein 403,the binding protein of gametogenetin?GGN?,is highly expressed in testicular tissues.Yeast two-hybrid test confirmed that GGNBP2 could interact with GGN1,GGNBP1 and OAZ3,and play a decisive role in spermatogenesis.Our previous workhave found that Ggnbp2 knockout cause azoospermia and defective spermatid differentiation.In addition,the integrity of seminiferous epithelial lumen was destroyed,and the acrosome and nucleolus were deformed.Based on these findings,we intend to investigate the role of GGNBP2 and GGN1?the largest isoform of GGN protein?in spermatocyte meiosis,DNA double strand break repair,and to explore the molecular mechanism of GGNBP2 in regulating histone post-translational modification.The results are as follows:1.Reciprocal immunoprecipitation?IP?with GGN1 and GGNBP2 antibodies demonstrated that GGN1 and GGNBP2 formed a protein complex inthe testis.Both GGNBP2 and GGN1 were observed in the nucleus and cytoplasm of spermatocyte and they colocalized very well in spermatocytes,spermatids and spermatozoa.Both mRNA and protein levels in the Ggnbp2KO testes were noticeably reduced compared to WT testes of 18 days or adult mice.Immunofluorescence staining showed thatintensity of GGN1 in Ggnbp2KO spermatocytes was remarkably reduced and GGN1 foci that associated with chromosomes in the nucleus were significantly lower than that of WT spermatocytes.Flow cytometry revealed Ggnbp2KO markedly decreased haploid?1C?cell fraction and increased tetraploid?4C?cell fraction compared to age-matched either 30-or 60-day-old WT littermates.2.Ggnbp2 and Ggn gene knock-out spermatocytes?GC-2spd?were successfully constructed.XTT and flow cytometry showed that Ggnbp2 and Ggn knock-out could inhibit the proliferation and differentiation in 32?incubation,but could inhibit the differentiation of spermatocytes into haploid cells at 37?incubation.Knockout of Ggnbp2 reduced Ggn mRNA and protein expression.Overexpression of Ggnbp2 could rescue the Ggn mRNA and protein levels of Ggnbp2KO cells,and partially restore differentiation,but had no effect on the GgnKO cellsdifferentiation.3.Immunoprecipitation showed that GGNBP2 could interact with DNA repair protein FANCL,RAD51,RAD18.Ggnbp2 knockout did not affect loss does not affect the formation and disassembly of the synaptonemal complexes,and staining pattern ofg-H2AX persisted not only in the XY body but also in several patches along autosomes in the pachytene and diplotene stages.In addition,there was no significant difference in RAD51 staining in XY body,but the foci in autosome significantly increased,the expression of RAD18 in other parts except XY in vitro,while BRCC36 foci increased significantly compared to WT spermatocyte.4.In vivo and in vitro experiments showed that Ggnbp2 knockout upregulated histone H2A_K119119 ubiquitination.Immunoprecipitation assay showed that GGNBP2 bond with ASXL1,but not with BAP1.GGNBP2 loss did not affect ASXL1and BAP1 protein expression,but could disrupt ASXL1 and BAP1 interactionand result in BAP1 gathering in the cytoplasm and not expressed in the nucleus.5.In vitro and in vivo experiments showed that Ggnbp2 knockout down-regulated H2B_K120ubiquitination.Immunoprecipitation showed that GGNBP2 interacted with ubiquitin binding enzyme E2 B?UBE2B?,but not with ubiquitin ligase E3 RNF40.GGNBP2 abscence could block the interaction between UBE2B and RNF40,and up-regulate the expression of UBE2B without affecting the expression of RNF40 protein.6.GGNBP2 protein did not interact with ubiquitin specific protease superfamily?USP?,but Ggnbp2 knockout down-regulated the expression of specific H2A_K119119 deubiquinase USP16,USP21 and USP3,USP22.The expression of H2B_K120120 deubiquinaseUBP10,USP7 was not affected.This study draws the following conclusions:1.As a GGN binding protein,GGNBP2,together with GGN,is located in the nucleus and cytoplasm of spermatocytes,as well as the acrosome and tail of sperm cells,and plays an important role in sperm differentiation and the spermatocytemeiosis.2.Ggnbp2 knockout induced spermatogenic dysfunction in mice,which might be related to the damage of the DNA double strand repair during the meiosis in which GGN1 was involved.3.Histone ubiquitination may play an important role in the DNA double strand damage repair.GGNBP2 may affect H2A_K119119 deubiquitination by regulating ASXL1/BAP1 binding,or ubiquitin specific protease USP expression.Meanwhile,GGNBP2 regulates H2B_K120ubiquitination by affecting the interaction between ubiquitin binding enzyme E2B and ubiquitin ligase RNF40.