The Role And Mechanism Of FAAP100 Gene In The Pathogenesis Of Premature Ovarian Insufficiency

Background Premature ovarian insufficiency(POI)is a common reproductive disorder that seriously affects the long-term physical and mental health of 6 millions Chinese women.The etiology of POI is highly heterogeneous and the causes for more than half of POI patients are uncertain.Therefore,promoting research on the aetiology and pathogenesis of POI will help to provide early screening,early diagnosis,subsequent fertility guidance and interventional treatment for POI patients.Among the known etiologies of POI,genetic factors,which account for 20-25%,are important pathogenic factors.Recent years,novel POI pathogenic genes have been identified through whole exome sequencing(WES)and more than half of these causative genes participate in DNA damage repair,suggesting that DNA damage repair pathways is crucial for the maintenance of female ovarian function.This points to a new direction for the etiological study of POI.The Fanconi anemia(FA)pathway is an essential DNA damage repair pathway and mainly involved in DNA inter-strand crosslink(ICL)repair.Recently,increasing evidences indicated that it also played an important role in replication stress response,especially in the resolution of transcription-replication conflicts(TRC).FA pathway includes 22 FA genes whose mutations cause Fanconi anaemia syndrome,which is often characterized with reproductive disorder.Up to now,18 FA gene deficient mice all have POI-like phenotypes and 5 FA gene,including FANCA、FANCD1/BRCA2、FANCL、FANCM、FANCU/XRCC2,have been identified as pathogenic genes of POI,indicating that FA genes participate in ovarian development and function maintenance.FAAP100 is a key member of FA pathway.It associates with FANCB and FANCL to form the E3 ubiquitin ligase catalytic module,ubiquitinating the FANCI-FANCD2 complex,and is essential for the activation of the FA pathway.However,the specific role of FAAP100 in ovarian development and its pathogenicity in human POI remain unclear.Objective In this study,Faap100 knockout mouse model was used to explore the role of FAAP100 in ovarian development and functional maintenance.Meanwhile,FAAP100 gene mutation screening in POI patients and functional exploration were conducted.We will reveal the role and mechanism of FAAP100 in the pathogenesis of POI.Methods The Faap100 knockout mice were constructed using CRISPR/Cas9 technology.The effect of Faap100 deletion on ovarian function was determined by observing fertility and estrous cycles,detecting serum sex hormone levels,analysing ovarian histomorphology.The influence of Faap100 knockout on germ cell development was confirmed by the embryonic gonads observation and germ cells count.PGC apoptosis,proliferative capacity,cell cycle,transcriptional levels,epigenetic reprogramming and FA pathway activity and DNA damage were assessed to confirm that Faap100 gene was involved in the genome stability maintainence of PGC.TRC frequency evaluated by proximity ligation assay and R-loop levels detected by S9.6 staining were detected to elucidate that FAAP100 participate in the resolution of TRC and R-loop.The mechanism of FAAP100 in safeguarding genomic stability was further investigated in mouse embryonic fibroblast(MEF).After using mitomycin(MMC)to induce DNA ICL damage as well as aphidicolin(APH)and hydrouria(HU)to induce replication stress,FA pathway activity,replication stress response and DNA damage markers were detected to to clarify the involvement of FAAP100 in replication stress response.Further,R-loop levels,TRC frequency,replication fork progression and stability were assessed to elucidate the mechanism by which FAAP100 resolve TRC and maintains PGC genome stability.FAAP100 gene mutations were screened in the WES database of POI and verified by sanger sequencing.Conservativeness analysis and pathogenicity prediction of mutations were performed.Further,wild-type and mutant FAAP100 proteins were overexpressed in HEK293 cells.Western blot and immunofluorescence were conducted to detect proteins expression and localization.After using APH to induce replication pressure,the ubiquitination of FANCD2 was assessed by Western blot to clarify the effect of FAAP100 mutations on FA pathway activation.Results 1.Faap100 gene knockout caused abnormal development of primordial germ cells in mice.The Faap100 knockout mice were verified to be constructed successfully from genotype identification and mRNA levels.The birth ratio of Faap100-/-mice was consistent with Mendelian inheritance,and no significant alterations in growth and development were observed.Faap100-/-adult females showed POI-like phenotypes,such as infertility,disturbance of estrous cycle,elevated serum FSH levels and ovarian atrophy without follicles.The numbers of primordial follicles in ovaries of 3-day-old Faap100-/-females and germ cells in the testicular spermatogenic tubules of 3-day-old Faap100-/-males were significantly reduced,suggesting that the germ cell defects may arise during the embryonic stage.Further observation of the embryonic gonads revealed that the number of PGC in Faap100-/-embryos was comparable with that of Faap100+/+ embryos at embryonic day 8.5(E8.5),but it began to reduce from E9.5,and decreased significantly at E11.5 with a prolonged doubling time.The development of somatic cells in the gonads of Faap100-/-embryos was normal.However,there were no obvious development abnormalities of somatic cells in the gonads of Faap100-/-embryos.2.The role and mechanism of FAAP100 in the development of primordial germ cells in mice.Studies in vivo showed that E11.5 Faap100-/-PGC manifested with increased apoptosis,reduced proliferation and G2 arrest,while there were no obvious abnormalities in the processes of transcriptional upregulation and epigenetic reprogramming.Further,Faap100-/-PGC showed FA pathway inactivation and increased 53BP1 foci and p-p53 positive rates,suggesting that FA pathway inactivation led to increased DNA damage and p53 hyperactivation.Besides,the PGC loss in E11.5 Faap100-/-embryos was found to be partially rescued by p53 knockout,demonstrating that p53 was indeed involved in the loss of PGC in Faap100-/-embryos.Finally,elevated TRC and R-loop were observed in Faap100-/-PGC,indicating that impaired endogenous replication pressure resolution caused by FAAP100 deficiency led to PGC genomic instability and proliferation defect.Studies in vitro showed that Faap100-/-MEF manifested with FA pathway inactivation,increased DNA damage,p53 hyperactivation and elevated genomic instability under physiological situation,DNA ICL damage induced with MMC or replication stress induced with APH and HU.Further,increased R-loop and TRC levels,as well as the slowed replication rate were observed in Faap100-/-MEF.Whereas,after overexpressing RNase H1 to remove R-loop,the increased TRC,slowed replication rate and elevated DNA damage were partially rescued,suggesting that the accumulated R-loop in Faap100-/-MEF exacerbated TRC and lead to increased genomic instability.In addition,the nascent DNA strand was shorter after replication stress inducers APH and HU treatment in Faap100-/-MEF,indicating that Faap100 deletion lead to nascent DNA strands degradation by nucleases and increased replication fork instability under replication stress.Together,these data clarify that FA pathway counteracts TRC through R-loop resolution and replication forks protection to maintain the rapid proliferation of PGC and ensure the normal establishment of reproductive reserve.3.Screening and functional exploration of FAAP100 gene mutation in POI patients.Four carriers with FAAP100 heterozygotic mutations were identified in 1030 POI WES database,including c.1474G>A(p.V492M),c.1 742C>T(p.T581M),c.2335G>A(p.A779T),c.2519T>C(p.L840P).Mutation sites were highly conserved among different species.Multiple functional prediction software revealed that mutation sites were potentially pathogenic.Functional exploration showed that compared with wild-type FAAP100 protein,p.A779T and p.L840P mutant FAAP100 impaired the activation of FA pathway,suggesting that FAAP100 mutation may be the cause of POI.Conclusion 1.FA pathway inactivation,increased TRC and R-loop caused by Faap100 deletion lead to elevated DNA damage,p53 hyperactivation and proliferation defect of PGC,affecting the establishment of ovarian reserve and ultimately resulting in the premature depletion of follicular.This further confirms that underestablishment of reproductive reserve due to PGC developmental disorders is the pathogenesis of POI.2.FAAP100 participates in removing R-loop and protecting replication fork to facilitate TRC resolution.This clarifies the important role of FAAP100 in the maintenance of PGC genomic stability.3.FAAP100 gene mutation lead to the impaired activation of FA pathway and may be the cause of POI.This provides theoretical basis for genetic counseling and early intervention of POI patients.