TP63 gain-of-function mutations cause premature ovarian insufficiency by inducing oocyte apoptosis
Chengzi Huang, … , Zi-Jiang Chen, Shidou Zhao
Chengzi Huang, … , Zi-Jiang Chen, Shidou Zhao
Published March 1, 2023
Citation Information: J Clin Invest. 2023;133(5):e162315. https://doi.org/10.1172/JCI162315.
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Research Article Genetics Reproductive biology

TP63 gain-of-function mutations cause premature ovarian insufficiency by inducing oocyte apoptosis

Abstract

The transcription factor p63 guards genome integrity in the female germline, and its mutations have been reported in patients with premature ovarian insufficiency (POI). However, the precise contribution of the TP63 gene to the pathogenesis of POI needs to be further determined. Here, in 1,030 Chinese patients with POI, we identified 6 heterozygous mutations of the TP63 gene that impaired the C-terminal transactivation-inhibitory domain (TID) of the TAp63α protein and resulted in tetramer formation and constitutive activation of the mutant proteins. The mutant proteins induced cell apoptosis by increasing the expression of apoptosis-inducing factors in vitro. We next introduced a premature stop codon and selectively deleted the TID of TAp63α in mice and observed rapid depletion of the p63+/ΔTID mouse oocytes through apoptosis after birth. Finally, to further verify the pathogenicity of the mutation p.R647C in the TID that was present in 3 patients, we generated p63+/R647C mice and also found accelerated oocyte loss, but to a lesser degree than in the p63+/ΔTID mice. Together, these findings show that TID-related variants causing constitutive activation of TAp63α lead to POI by inducing oocyte apoptosis, which will facilitate the genetic diagnosis of POI in patients and provide a potential therapeutic target for extending female fertility.

Authors

Chengzi Huang, Simin Zhao, Yajuan Yang, Ting Guo, Hanni Ke, Xin Mi, Yingying Qin, Zi-Jiang Chen, Shidou Zhao

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Figure 1

Analysis of human TAp63α-mutant pathogenicity.

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Analysis of human TAp63α-mutant pathogenicity. (A) Schematic diagram sho...
(A) Schematic diagram showing the 5 key domains of TAp63α: the TAD, the DBD, the oligomerization domain (OD), the SAM, and the TID. The positions of the variants identified in this study and reported in the literature are indicated in red and blue, respectively. Circles signify isolated POI; squares signify syndromic POI. The amino acid sequence of the EAO (504~550) is indicated. (B) After transfection with WT and mutant TAp63α plasmids in SAOS-2 cells, the intracellular protein level of TAp63α was detected by Western blotting. β-Actin was used as the loading control. (C) Oligomeric state analysis of WT and mutant TAp63α by BN-PAGE. The oligomeric conformation is indicated by T (tetramer), D (dimer), and M (monomer). In the protein samples of mutant TAp63α, no WT protein was present. (D) TAp63αΔTID and WT or mutant GFP-TID plasmids were cotransfected into HEK293 cells. Cells were harvested for co-IP assays and were immunoprecipitated with anti-p63 antibody, and then WT and mutant GFP-TID protein were detected by GFP antibody by Western blotting. IgG was used as the negative control. (E) Quantitative analysis of the amount of co-IP between TAp63αΔTID and WT or mutant GFP-TID. The immunoprecipitated GFP-TID was compared with the input. Data are presented as the mean ± SD of 3 independent experiments. **P < 0.01 and ***P < 0.001, by 1-way ANOVA followed by Dunnett’s test.