Related Experiment Videos
Germline TP53 mutations result into a constitutive defect of p53 DNA binding and transcriptional response to DNA
Yasmine Zerdoumi1, Raphaël Lanos1, Sabine Raad1
1Normandie University, UNIROUEN, Inserm U1245 and Rouen University Hospital, Department of Genetics, F 76000, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.
Abstract:
Li-Fraumeni Syndrome (LFS) results from heterozygous germline mutations of TP53, encoding a key transcriptional factor activated in response to DNA damage. We have recently shown, from a large LFS series, that dominant-negative missense mutations are the most clinically severe and, thanks to a new p53 functional assay in lymphocytes, that they alter the p53 transcriptional response to DNA damage more drastically than null mutations. In this study, we first confirmed this observation by performing the p53 functional assay in lymphocytes from 56 TP53 mutation carriers harbouring 35 distinct alterations. Then, to compare the impact of the different types of germline TP53 mutations on DNA binding, we performed chromatin immunoprecipitation-sequencing (ChIP-Seq) in lymphocytes exposed to doxorubicin. ChIP-Seq performed in wild-type TP53 control lymphocytes accurately mapped 1287 p53-binding sites. New p53-binding sites were validated using a functional assay in yeast. ChIP-Seq analysis of LFS lymphocytes carrying TP53 null mutations (p.P152Rfs*18 or complete deletion) or the low penetrant 'Brazilian' p.R337H mutation revealed a moderate decrease of p53-binding sites (949, 580 and 620, respectively) and of ChIP-Seq peak depths. In contrast, analysis of LFS lymphocytes with TP53 dominant-negative missense mutations p.R273H or p.R248W revealed only 310 and 143 p53-binding sites, respectively, and the depths of the corresponding peaks were drastically reduced. Altogether, our results show that TP53 mutation carriers exhibit a constitutive defect of the transcriptional response to DNA damage and that the clinical severity of TP53 dominant-negative missense mutations is explained by a massive and global alteration of p53 DNA binding.
Insights
Li-Fraumeni Syndrome (LFS) arises from TP53 mutations. Dominant-negative TP53 mutations severely impact p53 DNA binding and transcriptional response to DNA damage, explaining LFS clinical severity.
Area of Science:
- Genetics
- Molecular Biology
- Cancer Genomics
Background:
- Li-Fraumeni Syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in the TP53 tumor suppressor gene.
- TP53 mutations can be missense (dominant-negative) or null (loss-of-function), with varying clinical severity.
- Dominant-negative TP53 mutations are associated with more severe LFS phenotypes, but the underlying molecular mechanisms are not fully elucidated.
Purpose of the Study:
- To confirm that dominant-negative TP53 mutations cause a more drastic alteration of the p53 transcriptional response to DNA damage compared to null mutations.
- To investigate the impact of different types of germline TP53 mutations on p53 DNA-binding activity using chromatin immunoprecipitation-sequencing (ChIP-Seq).
- To correlate the observed alterations in p53 DNA binding with the clinical severity of Li-Fraumeni Syndrome.
Main Methods:
- p53 functional assays in lymphocytes from 56 TP53 mutation carriers with 35 distinct alterations.
- Chromatin immunoprecipitation-sequencing (ChIP-Seq) in doxorubicin-exposed lymphocytes from LFS patients and wild-type controls.
- Validation of novel p53-binding sites using a functional assay in yeast.
Main Results:
- p53 functional assays confirmed a more severe defect in transcriptional response for dominant-negative TP53 mutations.
- ChIP-Seq analysis revealed a significant reduction in the number and depth of p53-binding sites for all TP53 mutation types compared to wild-type.
- Dominant-negative TP53 mutations (p.R273H, p.R248W) resulted in a drastic global decrease in p53 DNA binding (310 and 143 sites, respectively), far more pronounced than null mutations (949-620 sites).
Conclusions:
- TP53 mutation carriers exhibit a constitutive defect in the transcriptional response to DNA damage.
- The severe clinical presentation of LFS associated with dominant-negative TP53 missense mutations is directly explained by a massive and global impairment of p53 DNA binding.
- These findings highlight the critical role of p53 DNA-binding integrity in LFS pathogenesis and clinical outcome.