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 5

The oocytes in p63+/ΔTID mouse ovaries died by apoptosis.

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The oocytes in p63+/ΔTID mouse ovaries died by apoptosis. (A) IF stainin...
(A) IF staining for DDX4 (green) and cleaved-PARP1 (red) in ovary sections from P1 WT and p63+/ΔTID mice. Cell nuclei were counterstained with DAPI (blue). Scale bars: 50 μM. (B) Quantitative analysis of cleaved-PARP1–positive oocytes in each group (n = 5). (C) Western blot analysis of BAX expression in P1 WT and p63+/ΔTID ovaries. β-Actin was used as the loading control. (D) Quantitative RT-PCR analysis of Puma and Noxa gene expression in P1 ovaries of WT and p63+/ΔTID mice. Gapdh was used as the internal control. Three mice for each genotype were used for each independent experiment, and 3 independent experiments were conducted. (E) The p63 and p63ΔTID plasmids were transfected into SAOS-2 cells, and protein levels were detected by Western blotting. β-Actin was used as the loading control. (F) Oligomeric state analysis of p63 and p63ΔTID using BN-PAGE. The oligomeric state is indicated by T, D, and M. (G) Transcriptional activity of p63ΔTID in SAOS-2 cells on the NOXA, PUMA, and BAX promoters. The activity of the p63 group was set to 1, and 3 independent experiments were conducted. In B, D and G, data are shown as the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired, 2-tailed Student’s t test.