The Role And Mechanism Of DNA Damage Repair Protein FANCD2 In Germ Cell Development

Germ cells undergo complex processes to develop into mature gametes,including the specification,migration,and proliferation of primordial germ cells(PGCs),sex differentiation,and meiosis,which are regulated by complicated mechanisms.The development of germ cells determines the establishment and depletion of the reproductive reserve,and studies into the regulatory mechanisms underlying germ cell development will help to elucidate the pathogenesis of infertility and genetic diseases.Fanconi anemia(FA)is a rare genetic syndrome characterized by early bone marrow failure,cancer predisposition,congenital malformation,and severe reproductive defects.To date,22 FA genes have been identified in a complex pathway that orchestrates DNA interstrand crosslink(ICL)repair and maintains genome stability.The ubiquitination of human FANCD2 at Lysine-561 is a key step in the activation of the FA pathway.In addition,it has been reported that FANCD2 also regulated genome stability when its ubiquitination is not occurred.Thus,FANCD2 maintains genome stability through both ubiquitination-dependent and ubiquitination-independent functions.Consistent with FA patients,FA mutant mice are also infertile or only rarely fertile.Our recent study showed that the FA pathway counteracted transcription-replication conflict(TRC)induced endogenous replication stress in PGCs,thus safeguarding genome stability and ensuring the establishment of reproductive reserve.Combined with the findings in FA mutant mice,the drastically reduction in germ cells due to inactived FA pathway could be mainly attributed to PGC deficiency during embryonic development.However,meiotic defects and epigenetic abnormalities on sex chromosomes during spermatogenesis have also been reported in some FA mutant mice.These findings emphasize the significant roles of the FA pathway in maintaining germ cell in different stages of development.Although the mutant mice without FA core complex components,such as Fanca and Fancb,had defects in ubiquitination of FANCD2 and fertility,Fancd2 knockout mice displayed more severe germline phenotypes,including a significant reduction of PGCs in the embryonic period,meiotic chromosome synapsis defects,epigenetic modification dysregulation on sex chromosomes during spermatogenesis,and infertility in both female and male mice.These results suggest that FANCD2 might also function in germ cell development independently of FA pathway activation.Therefore,the ubiquitination-dependent and ubiquitination-independent functions of FANCD2 during gametogenesis need to be further determined.ObjectiveIn this study,Fancd2 knockout(Fancd2-/-,KO)and the ubiquitination-deficient mutant(Fancd2K559R/K559R,MT)mice were used to explore the ubiquitination-dependent and ubiquitination-independent functions of FANCD2 during germ cell development.MethodsThe KO and MT mouse models were generated by the CRISPR/Cas9 technology.The functions of ubiquitinated and nonubiquitinated FANCD2 in PGC development were determined through the observation of embryonic gonads and PGC count.The frequency of the colocalized RNA polymerase Ⅱ(Pol Ⅱ)and proliferating cell nuclear antigen(PCNA),that is TRCs,was detected by the proximity ligation assay(PLA),and DNA damage was evaluated by immunofluorescence staining against DSB marker 53BP1 foci,so as to clarify the mechanism of ubiquitinated and nonubiquitinated FANCD2 in reducing TRCs and maintaining genome stability in PGCs during expansion.To determine the influence of ubiquitinated and nonubiquitinated FANCD2 dysfunction on germ cells development after birth,H&E staining of ovaries,oocyte count,H&E staining of testis and epididymis,seminiferous tubules analysis,and fertility test of adult male mice were performed.To determine the functions of ubiquitinated and nonubiquitinated FANCD2 in spermatogenesis,meiotic homologous chromosome synapsis,DNA double-strand break(DSB)formation and repair,homologous recombination(HR),crossover formation,and histone methylation on chromosomes were detected through the immunofluorescence staining on meiotic chromosome spreads.To explore the function of ubiquitinated and nonubiquitinated FANCD2 in somatic cell development,embryonic crown-lump length,Mendelian ratio,and body weight of postnatal mice were examined.Results1.The role and mechanism of FANCD2 in the development of PGC in mouse embryosThe KO and MT mice were verified to be constructed successfully by genotype identification and Sanger sequencing.The count of PGCs showed that the number of PGCs was reduced in both KO and MT embryos at embryonic day 9.5(E9.5).PGCs were dramatically decreased in MT embryos than in WT controls in the E11.5 genital ridges,and fewer PGCs were seen in KO embryos than those in MT embryos,suggesting that both ubiquitinated and nonubiquitinated FANCD2 are significant for PGCs development.Consistent with this,germ cells in both KO and MT gonads were dramatically reduced at E13.5 and E18.5.To explore the mechanisms of ubiquitinated and nonubiquitinated FANCD2 in maintaining PGC development,we examined the frequency of TRCs and the DNA damage in KO and MT embryo PGCs.The results showed that the frequency of TRCs and the proportion of 53BP1 positive PGCs were increased in both MT and KO PGCs,and a greater increase of TRCs and DNA damage were detected in KO PGCs than MT PGCs.Those results implied that TRCs resolution and genome stability maintenance in PGCs require both ubiquitinated and nonubiquitinated FANCD2.2.The role and mechanism of FANCD2 in the development of germ cells in postnatal miceTo clarify the influence of ubiquitinated and nonubiquitinated FANCD2 dysfunction in germ cell development in postnatal mice,we observed the ovary and testis of postnatal mice.Compared with WT females,follicles in postnatal day(PD)3 ovaries from KO and MT females were significantly reduced.Although more follicles were found in PD3 MT ovaries compared to KO ovaries,follicles were completely exhausted in 2-month KO and MT ovaries.The testes of KO and MT adult male mice were smaller than those of their WT siblings,with spermatogenic cells in testes and sperm in epididymis reduced significantly.Compared with MT mice,the reduction of germ cells in testis and epididymis was more obvious in KO mice.Consistent with this,fertility tests showed that 4 of 6 MT male mice were fertile,while only 1 of 6 KO male mice was fertile.These results indicate that there are more germ cells in MT male mice than KO male mice.Except for the difference in the number of PGC in embryonic stage,this difference may also be related to the different functions of ubiquitinated and nonubiquitinated FANCD2 in spermatogenesis.To elucidate the mechanism of ubiquitinated and nonubiquitinated FANCD2 in regulating spermatogenesis,immunofluorescence staining on meiotic chromosome slices was performed.The results showed that the number of spermatocytes with abnormal homologous chromosome synapsis was increased in KO mice than WT and MT mice,while there was no difference between WT and MT mice,suggesting that nonubiquitinated FANCD2 promote meiotic homologous chromosome synapsis.The number of spermatocytes with unrepaired DSBs was increased in MT mice and was further increased in KO mice.Consistently,the number of RAD51 foci,homologous recombination protein,in pachytene spermatocytes was increased in MT mice and was further increased in KO mice,suggesting that both ubiquitinated and nonubiquitinated FANCD2 promote meiotic DSB repair.The number of crossover sites indicated by MLH1 was increased in KO,but there was no difference between WT and MT spermatocytes,indicating that nonubiquitinated FANCD2 regulates the crossover formation in male meiosis.The detection of the histone methylation of meiosis chromosomes showed that the accumulation of H3K4me2 on diplotene XY chromosomes was decreased in KO spermatocytes,suggesting that the accumulation of H3K4me2 on sex chromosomes requires nonubiquitinated FANCD2.In addition,the intensity of H3K9me2 on diplotene XY chromosomes was reduced in both KO and MT spermatocytes,while the intensity of H3K9me3 was increased in both KO and MT spermatocytes,suggesting that ubiquitinated FANCD2 maintains H3K9me2 and inhibits H3K9me3 on XY chromosomes.3.The role of FANCD2 in somatic cells development in miceFinally,we observed the function of ubiquitinated and nonubiquitinated FANCD2 in somatic cells development in mice.Fetal crown-rump lengths of E11.5-E18.5 embryos were decreased in KO embryos than WT and MT embryos.Mendelian ratio analysis showed that the percentage of surviving KO pups was below the expected Mendelian ratio,whereas MT pups showed a close to normal Mendelian distribution.KO pups had reduced body sizes compared with WT and MT pups.The differences in body weight between KO,MT,and WT adult mice both in female and male disappeared.Collectively,we conclude that nonubiquitinated FANCD2 maintains the development of somatic cell,while ubiquitinated FANCD2 has less function in this process.ConclusionBoth ubiquitinated and nonubiquitinated FANCD2 have significant role in germ cell development.They ensure normal PGC development through reducing TRCs and maintaining genome stability;both the ubiquitinated and nonubiquitinated FANCD2 regulate meiotic DSB repair,ubiquitinated FANCD2 maintains H3K9me2 and inhibits H3K9me3 on XY chromosomes,nonubiquitinated FANCD2 promotes chromosomes synapsis,regulates crossover formation,and maintains the H3K4me2 on XY chromosomes,thus maintaining spermatogenesis.