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Tp53 R217H and R242H Mutant Zebrafish Display Dysfunctional p53 Hallmarks and Recapitulate LFS Phenotypes [WGBS]

ABSTRACT: Li-Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome associated with a highly penetrant and diverse tumor spectrum characterized by germline mutations in the TP53 tumor suppressor gene. To better understand how TP53 mutations predispose individuals with LFS to cancer development, we characterized tp53 R217H and R242H zebrafish lines as the first zebrafish p53 hotspot mutants (human R248 and R273H). These mutants have lost several key wildtype p53 functions and recapitulate many LFS phenotypes. Specifically, we have shown that the R217H and R242H alleles result in partial-to-no activation of key p53 target genes, are resistant to apoptosis in a dominant negative manner and exhibit a defective G1 cell-cycle checkpoint in vivo. The loss of these wildtype p53 functions predisposed the fish to develop spontaneous tumors as early as 6 months of age. Tumor histology resembles human sarcomas, a predominant LFS tumor type. tp53 R242H mutants developed tumors earlier with a higher lifetime incidence than tp53 null or R217H mutants, suggesting it is a more aggressive mutation, while the R217H allele may be hypomorphic. Additionally, we observed mutation-specific differences both in the tumor type and sex bias across tp53 null, R217H, and R242H genotypes with associated diverse transcriptomic and DNA methylome profiles, impacting metabolism, cell signalling, and biological macromolecule synthesis and degradation. These tp53 zebrafish mutants demonstrate fidelity to their human counterparts and provide new insights into underlying tumorigenesis mechanisms and kinetics, which may inform more tailored tumor surveillance approaches and novel therapeutic targets in LFS.

ORGANISM(S): Danio rerio

PROVIDER: GSE268283 | GEO | 2024/05/29

REPOSITORIES: GEO

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Project description:Li-Fraumeni syndrome (LFS) is a disorder due to inherited mutations in the TP53 gene resulting in an increased risk of developing several types of cancer. MicroRNA miR-34a has been implicated downstream of p53 on the basis of being a direct transcriptional target and, when over-expressed, having pro-apoptotic phenotypes in cell lines. Moreover, miR-34a has been shown to be a modifier gene in the context of LFS, since its epigenetic silencing increases the likelihood of tumour development in patients with mutant TP53. However, the in vivo consequences of miR-34 loss are still unclear. For example, mice lacking all three (a,b,c) miR-34 homologs show no detectable abnormalities in p53 responses. The relative expression of different miR-34 genes in zebrafish was studied using qRT-PCR with specific assays. The miR-34a, miR-34b and miR-34c display unique onset of developmental expression and expression levels, with miR-34a being the most abundant and constant in expression. All of the miR-34 genes also showed clear induction by p53 when DNA-damaging treatments are performed. Using CRISPR-Cas9 technology, we generated a zebrafish miR-34a deletion mutant to further investigate the roles of miR-34a on its own and its association with the p53 pathway. Predictably, a miR-34a deletion mutant demonstrated absence of miR-34a, though without miR-34b and miR-34c compensation beyond baseline expression levels. Mutants survive to adulthood, show no overt phenotypes and have normal apoptotic responses to DNA-damaging irradiation or camptothecin treatments. To further explore the effects of miR-34a, we performed gene expression profiling using RNA-seq of wild-type and miR-34a deletion mutant zebrafish embryos at 72 hpf. We wanted to explore how miR-34a loss affects differentiated organs in larval zebrafish. This resulted in 389 genes UP and 374 genes DOWN at 1.5-fold change and FDR < 0.05. There was no significant enrichment of predicted miR-34a target genes among the differentially regulated genes but some interesting biological trends were found and will be described in the paper associated with this dataset.

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Tp53 R217H and R242H Mutant Zebrafish Display Dysfunctional p53 Hallmarks and Recapitulate LFS Phenotypes [WGBS]

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