The Role And Mechanism Of UBE2T In The Pathogenesis Of Premature Ovarian Insufficiency

BacgroundPremature ovarian insuficiency(POI)is a common ovarian dysfunction disease,affecting approximately 6 million women of childbearing age in China.The etiology of POI is highly heterogeneous,with about 30-35%of patients attributed to genetic factors.In recent years,many pathogenic genes for POI have been identified using the whole-exome sequencing(WES)technique,with a significant portion of them enriched in the DNA damage repair pathway,suggesting that the DNA damage repair pathway plays an important role in the pathogenesis of POI.It is noteworthy that currently about 50%of POI patients have unknown causes,therefore,discovering new genes and deciphering their pathogenic mechanisms is crucial for early diagnosis and treatment of POI.Fanconi anemia(FA)pathway is an important DNA damage repair pathway,whose classical function is to resolve inter-strand crosslink(ICL)damage.In addition,the FA pathway is also involved in replication stress response,especially in responding to transcription-replication conflicts(TRC)and replication stress from common fragile sites(CFS),and plays an essential role.Most FA gene knockout mice exhibit reproductive barrier phenotypes,and five FA pathway genes,FANCM、FANCU/XRCC2、FANCD1/BRCA2、FANCL、and FANCA,have been shown to be associated with the pathogenesis of POI,indicating that the FA pathway plays an indispensable role in ovarian development and function maintenance.UBE2T is a type of E2 ubiquitin-conjugating enzyme,whose main function is to synergize with E3 ubiquitin ligase FANCL to promote the ubiquitination of FANCI-FANCD2 dimer,thereby activating the FA pathway.However,the role of UBE2T in ovarian development and function maintenance,as well as its specific mechanisms in the pathogenesis of POI,remain to be elucidated.ObjectiveThis study aims to explore the role and mechanism of Ube2t in the development and function maintenance of the ovary by using Ube2t gene knockout mouse model.Furthermore,we conducted UBE2T gene mutation screening in patients with POI and studied the functional significance of the mutation sites to elucidate the contribution of UBE2T gene mutations in POI pathogenesis.MethodsWe used CRISPR/Cas9 technology to construct Ube2t gene knockout mice and determined the impact of Ube2t gene knockout on ovarian function by measuring serum hormone levels,observing estrous cycles,and studying ovarian morphology.Furthermore,we investigated the effect of Ube2t gene on the development of early germ cells by observing embryonic gonads and germ cell counts.PGC epigenetic modifications,transcription levels,apoptosis,cell cycle,DNA damage and FA pathway activation were assessed to confirm the role of Ube2t gene in maintaining PGC genome stability.Using proximity ligation assay to access TRC frequency,S9.6 immunofluorescence staining to detect the accumulation of R-loops in PGCs,and detecting 53BP1 nuclear bodies in G1-phase PGCs to confirme the role of Ube2t in responding to replication stress caused by TRCs and CFS in PGCs.Furthermore,shRNA adenovirus was used to knock down Ube2t in P19 cells,and Ube2t gene knockout mouse embryonic fibroblasts(MEFs)were used to explore the role of UBE2T in maintaining genome stability in vitro.Aphidicolin(APH)or hydroxyurea(HU)to induce replication stress,mitomycin(MMC)was used to induce ICL damage.FA pathway activation,cell cycle,DNA damage,R-loop accumulation,TRC frequency,replication fork stability,CFS-related damage,ultra-fine anaphase bridges,mitotic DNA synthesis(MiDAS)and R-loop on large genes in CFS regions were detected in physiological P19 cells and/or drug-treated MEFs to elucidate the mechanism by which UBE2T participates in responding to replication stress caused by TRCs and CFS to reduce DNA damage and ultimately maintain PGC genome stability.We screened for UBE2T gene mutations in 1030 POI patients’ WES databases and validated mutation sites through Sanger sequencing.We overexpressed wild-type or mutant UBE2T in MEFs to examined UBE2T protein expression,intracellular localization,FA pathway activation and replication stress response function,and tested for haploinsufficiency and dominant negative effects in MEF cells to clarify the role of UBE2T gene mutations in POI pathogenesis.Results1.Ube2t gene knockout causes abnormal development of mouse primordial germ cellsUsing CRISPR/Cas9 technology,Ube2t knockout mice were constructed,and PCR identification and protein level verification results indicated that the knockout mice were successfully constructed.The genotype ratio of Ube2t-/-mice at birth conformed to Mendelian inheritance,and the appearance and weight of adult Ube2t-/-mice showed no significant difference compared to wild-type mice.However,Ube2t-/-female mice exhibited POI-like phenotypes,such as ovarian atrophy,depletion of follicles,elevated serum FSH levels and disappearance of estrous cycles.In addition,a significant loss of germ cells was observed in the testes of 3-day-old Ube2t-/-male mice,suggesting abnormal development of embryonic germ cells.Further observation of embryonic gonadal tissue showed that the number of Ube2t-/-PGCs decreased from E9.5 days and significantly decreased by E11.5 days.2.The role and mechanism of UBE2T in the developmemt of mouse primordial germ cellsE11.5 Ube2t-/-embryonic PGCs exhibited increased apoptosis,defective proliferation,and G2 phase arrest,but no significant abnormalities in transcriptional activation or epigenetic reprogramming were observed.Disappearance of FANCD2 foci,increased 53BP1 foci and p-p53 positive PGCs in Ube2t-/-embryos suggested that Ube2t gene knockout impaired FA pathway activation in PGCs,leading to increased DNA damage and excessive p53 activation.Further findings revealed that partial rescue of Ube2t-/-embryonic PGC loss can be achieved by p53 gene knockout,indicating the involvement of excessive p53 activation in PGC loss.Subsequently,increased PLA foci for RNA pol Ⅱ and PCNA,elevated R-loop nuclear average fluorescence intensity,and increased G1 phase 53BP1 nuclear body positivity in Ube2t-/-embryonic PGCs suggested that Ube2t knockout causes impaired intracellular replication pressure response from both TRC and CFS sources,resulting in increased DNA damage and proliferation defects in PGCs.We then explored the mechanisms by which UBE2T maintains genome stability in mouse P19 cells and MEFs.It was discovered that Ube2t knockdown in P19 cells under physiological conditions and/or Ube2t-/-MEFs after induction of ICL or replication stress led to inactivation of FA pathway,resulting in proliferation defects,G2-phase arrest,increased DNA damage,and excessive p53 activation.Furthermore,R-loop accumulation,increased TRC frequency,and slower replication speed were observed in Ube2t-/-MEFs,while overexpression of RNH1 could relieve the accumulation of R-loops,reduce TRC frequency,and restore damaged replication speed.R-loop CUT&Tag results showed that R-loop signals in the Ube2t knockdown group of P19 cells increased genome-wide,indicating that UBE2T deficiency caused R-loop accumulation,leading to TRC resolving dysfuction.In addition,after replication stress drugs caused replication fork stalling,the length of newly synthesized DNA strands in Ube2t-/-MEFs and knockdown P19 cells became shorter.The application of mirin,which was the MRE11 nuclease inhibitor,could restore the length of newly synthesized DNA strands,suggesting that UBE2T deficiency caused increased instability of replication forks and that newly synthesized DNA strands were cleaved by the MRE11 nuclease.Subsequently,it was found that CFS-related damage increased in Ube2t knockdown P19 cells and/or Ube2t-/-MEFs after induction of replication stress,the rate of cells with ultra-fine anaphase bridge increased,MiDAS frequency decreased,and the recruitment of MUS81 to chromatin decreased in premetaphase cells,and chromosome breaks or gaps in the metaphase cells decreased.These findings indicate that UBE2T deficiency inhibits the recruitment of MUS81 to chromatin,restricts MiDAS,and leads to increased CFS-related damage.Finally,it was found that R-loop signals were enriched in the promoter region of large genes nested in mouse CFSs,demonstrating that UBE2T deficiency caused R-loop accumulation in these regions,thereby hindering CFS replication in the S phase.3.Study on the pathogenic mechanism of UBE2T gene mutations in patients with primary ovarian insufficiencyTwo UBE2T heterozygous truncating mutations,c.205C>T:p.R69X and c.421 A>T:p.K141X,were identified in 2 out of 1030 patients with POI.Functional experiments revealed that UBE2T gene mutations caused abnormal intracellular localization or no expression of UBE2T protein,inactivation of the FA pathway,and compromised DNA damage repair capacity.It was also found that UBE2T gene mutations caused FA pathway inactivation and DNA repair capacity impairment through haploinsufficiency.These findings suggest that UBE2T gene mutations may be a cause of POI.Conclusion1.Ube2t gene knockout leads to FA pathway inactivation,increased DNA damage from TRCs and CFSs,and excessive p53 activation in PGCs,which hinders rapid proliferation of PGCs and insufficient establishment of reproductive reserves,confirming the important role of UBE2T in the development and functional maintenance of ovary.2.UBE2T counteracts TRCs by resolving R-loops and stabilizing stalled replication forks,and protects CFSs by promoting MiDAS and resolving R-loops located in the promoters of the large genes associated with CFSs,demonstrating the crucial role of UBE2T in maintaining genomic stability in PGCs.3.UBE2T gene mutations causing haploinsufficiency lead to FA pathway inactivation and compromised replication stress response capacity,which may be an etiology of POI,providing new evidence for the genetic counseling and reproductive guidance of POI.