The Role And Mechanism Of TP63 Gene In The Pathogenesis Of Premature Ovarian Insufficiency

Background:Premature ovarian insufficiency（POI）is a reproductive dysfunctional disease characterized by the decline of ovarian activity before the age of 40 years that is hard to prevent and treat.The incidence of POI is about 3.7%,which is trending upward and seriously affects the physical and mental health of women.Most female patients have experienced irreversible ovarian failure when diagnosed clinically,but currently there are no effective therapeutic strategies to improve or restore ovarian function.Therefore,research on the etiology of POI has significance for its early warning,diagnosis and intervention.The etiology of POI is highly heterogeneous,including genetic defects,autoimmune abnormalities,infections,iatrogenic factors,and environmental factors.However,the etiology of about half of the patients with POI remains unknown.Therefore,it is crucial to identify the etiology of POI through genetic screening and explore the pathogenesis of POI to achieve early diagnosis and treatment.The transcription factor p63 gene（TP63 in humans）,a member of the p53 family,encodes the TAp63α isoform that is specifically expressed in oocytes of the primordial follicle.Under physiological conditions,TAp63α is maintained in an inactive dimeric state by the binding of its C-terminal transactivation inhibitory domain（TID）with the N-terminal transcriptional activation domain（TAD）.When DNA damage occurs,TAp63α is activated through sequential phosphorylation,and this triggers the transition from an inactive dimer to an open and active tetramer.Activated TAp63α induces apoptosis in oocytes and serves as a guardian of the female germline.In previous studies,only a few case reports have found that TP63 mutations may contribute to the occurrence of POI by affecting the function of TAp63α.However,due to the limitations of small sample size,large phenotypic variability,and lack of systematic functional studies,the genetic contribution of the gene variants to POI and the correlation between genotype and phenotype still lack clinical evidence and mechanistic studies in large samples.Objective:To elucidate the role and molecular mechanism of TP63 gene mutation in the pathogenesis of POI.Methods:Firstly,we performed mutation screening,validation,pathogenicity prediction,and mutation location analysis of the TP63 gene based on the 1030 cases of the POI-Whole exome sequencing（WES）database constructed in our laboratory previously;The wildtype and mutant TAp63α plasmids were overexpressed in SAOS-2 cells（Human osteosarcoma cell line without endogenous TP63 gene expression）,and the expression and aggregation states of wild-type and mutant TAp63α proteins were analyzed by Western blot and BN-PAGE,and to detect the binding variation of mutant TID to TAD by Co-IP assay to clarify the reason for the altered aggregation state of mutant TAp63αprotein;We further performed luciferase reporter assay,western blot detection of BAX apoptosis protein,and TUNEL assay to compare the transcriptional activity of wildtype and mutant TAp63α and the effect on apoptosis in SAOS-2 cells,and to clarify the effect of mutation on TAp63α activity and function.To further define the role and mechanism of the defective TP63 gene function in the occurrence of POI,we constructed TID deletion(p63^+/ΔTID)mice,and then performed fertility test and the phenotype analysis of the reproductive system in p63^+/ΔTID mice to determine the effect of TED deletion on female fertility.At the same time,immunofluorescence staining of the apoptotic marker Cleaved-PARP1 in p63^+/ΔTID mouse ovaries,western bolt of apoptosis protein BAX expression,and mRNA expression levels of the apoptosis-related target genes Puma and Noxa by qRT-PCR were performed to clarify whether the rapid depletion of TID-deficient mouse oocytes through the apoptosis pathway.Mouse wild-type p63 and mutant p63ΔTID plasmids were transfected in SAOS-2 cells,and the expression levels,aggregation status,and transcriptional effects on downstream apoptosis-related target genes of p63 and p63ΔTID protein were detected to further reveal the mechanism by which p63ΔTID functions.Finally,to clarify the pathogenicity of point mutation in the TP63 gene originating from clinical patients,we generated TID point mutant(p63^+/R647C)mice and performed fertility test and reproductive phenotype analysis to clarify the effect of TID point mutation on female fertility.Immunofluorescence staining of apoptotic markers Cleaved-PARP1 in the ovaries of p63^+/R647C mice was performed to clarify the depletion of mouse oocytes through the apoptotic pathway.Moreover,due to the milder reproductive phenotype observed in p63^+/R647C mice,the quality of survival oocytes was further assessed by observing the first polar body（PB1）emission rates,spindle morphology（immunofluorescence staining for a-tubulin）,and mitochondrial membrane potential（JC-1 staining）in p63^+/R647C mice oocytes.Results:1.TP63 gene mutation screening and in vitro functional studyNine TP63 gene heterozygous variants（p.S285N,p.T538A,p.T567I,p.Q568fs*3,p.R594*,p.R643Q,p.L646P,p.R647C,and p.R655Q）were identified from 11 POI patients and were confirmed by Sanger sequencing.Most of these mutations are located at the C-terminus of TAp63a,as truncated mutations prior to TID（p.Q568fs*3,p.R594*）or point mutations located within the core sequence of TID（p.R643Q,p.L646P,p.R647C,and p.R655Q）.These amino acid sites of all identified mutations were highly conserved across species,and these mutations were mostly predicted to be damaging.We overexpressed wild-type and mutant TAp63a plasmids in human SAOS-2 cells and found that the six mutant proteins affecting TID（p.Q568fs*3,p.R594*,p.R643Q,p.L646P,p,R647C and p.R655Q）were transformed from inactivated dimeric conformation to actived tetramer by BN-PAGE analysis.Among them,p.Q568fs*3 and p.R594*directly caused deletion of TID,and p.R643Q,p.L646P,p.R647C,and p.R655Q mutants impaired the binding of TID to the N-terminal TAD.Meanwhile,these six TID-related mutants showed significantly increased transcriptional activity toward the NOXA,PUMA,and BAX reporters and subsequent cell apoptosis.These results suggest that the mutations affecting TID function are pathogenic.2.Study of POI-like phenotype and related mechanisms in p63^+/ΔTID miceThe Adult p63^+/ΔTID femals were infertile and ovarian atrophy.Ovarian Gections with HE staining showed that follicles were significantly reduced at P1.While there were no significant differences in oocyte number between E18.5 p63^+/ΔTID and their littermate controls,the oocyte number of P1 p63^+/ΔTID mice were decreased to～40%of those in WT mice.Few oocytes in p63^+/ΔTID mice survived at P5,and they completely disappeared by P10.To further investigate the underlying molecular mechanism of rapid oocyte loss,we examined cell apoptosis in mouse ovaries.Immunofluorescence staining of PI ovarian sections showed that Cleaved-PARP1-positive oocytes were significantly increased in p63^+/ΔTID ovaries.As expected,increased expression of Bax protein was detected in P1 p63^+/ΔTID ovaries.The mRNA levels of two target genes of p63,Puma and Noxa,were also significantly increased.In addition,overexpression of the mouse p63ΔTID mutant in SAOS-2 cells was observed to form an activated tetramer,and also transcriptionally activated the PUMA,NOXA,and BAX genes.3.Study of POI-like phenotype and related mechanisms in p63^+/R647C miceAdult p63^+/R647C females exhibit decreased fertility and the ovary size was significantly reduced.HE staining of ovarian sections showed that the number of follicles was significantly reduced in P10 and 2M p63^+/R647C ovaries.The number of oocytes stained by DDX4 in P10 p63^+/R647C mice was reduced to approximately 50%of that in WT mice.Furthermore,Cleaved-PARP1-positive oocytes were significantly increased in P1 p63^+/R647C ovaries compared with WT ovaries.In addition to the loss of oocyte number observed in p63^+/R647C mice,the remaining survival oocytes showed decreased the first polar body（PB1）emission rates,increased abnormal spindle/chromosomes rate,and lower mitochondrial membrane potential,suggesting that the quality of the oocytes was impaired.Conclusion:In this study,based on mutation screening in a large sample of clinical cases and systematic in vitro and in vivo functional studies,we reveal the molecular mechanism by which the gain-of-function mutations in the TP63 gene lead to impaired function of TID at the C-terminus of TAp63α protein,causing abnormal activation of TAp63αprotein,thereby inducing pathological apoptosis of oocytes and ultimately progression to POI.It provides the theoretical basis and intervention targets for fertility protection in women,especially in tumor patients undergoing radiotherapy/chemotherapy.