Allele-specific silencing of EEC p63 mutant R304W restores p63 transcriptional activity

Abstract

EEC (ectrodactily-ectodermal dysplasia and cleft lip/palate) syndrome is a rare genetic disease, autosomal dominant inherited. It is part of the ectodermal dysplasia disorders caused by heterozygous mutations in TP63 gene. EEC patients present limb malformations, orofacial clefting, skin and skin's appendages defects, ocular abnormalities. The transcription factor p63, encoded by TP63, is a master gene for the commitment of ectodermal-derived tissues, being expressed in the apical ectodermal ridge is critical for vertebrate limb formation and, at a later stage, for skin and skin's appendages development. The ΔNp63α isoform is predominantly expressed in epithelial cells and it is indispensable for preserving the self-renewal capacity of adult stem cells and to engage specific epithelial differentiation programs. Small interfering RNA (siRNA) offers a potential therapy approach for EEC patients by selectively silencing the mutant allele. Here, using a systemic screening based on a dual-luciferase reported gene assay, we have successfully identified specific siRNAs for repressing the EEC-causing p63 mutant, R304W. Upon siRNA treatment, we were able to restore ΔNp63-WT allele transcriptional function in induced pluripotent stem cells that were derived from EEC patient biopsy. This study demonstrates that siRNAs approach is promising and, may pave the way for curing/delaying major symptoms, such as cornea degeneration and skin erosions in young EEC patients.

Integrin-β4 is a novel transcriptional target of TAp73

“…Recently, mutant p53 enhanced ovarian cancer-mesothelial cell adhesion has been associated with the integrin b4 and Akt signalling [63,64]. In addition DNp63, which plays a critical role in stratified epithelia development [65][66][67][68][69][70], was characterized as a critical regulator in cellular adhesion and survival of epithelial cells by directly regulating cell adhesion-associated genes including ITGB4 [71]. Hence, our findings provide further support for the role of p53 family in integrin regulation, especially integrin b4.…”

Mutant p63 affects epidermal cell identity through rewiring the enhancer landscape

“…This indicates that the mutant p63 protein has a dominant effect on DNA binding over the wild type p63 that is also present in the cell, because the p63 protein is DNA-binding competent as a tetramer (Dotsch, Bernassola et al, 2010, Serber, Lai et al, 2002. These findings corroborate the dominant negative model of EEC p63 mutations that has been proposed by several previous studies using in vitro approaches (Browne et al, 2011, Celli et al, 1999 and allele-specific knockdown of p63 mutant (Novelli, Lena et al, 2016).…”

SOX2 Regulates P63 and Stem/Progenitor Cell State in the Corneal Epithelium

“…Cells were collected at indicated time points for different analyses. Clonogenicity test , corneal epithelial differentiation of human embryonic stem cells , and neural differentiation of mouse embryonic stem cells were carried out as previously reported. For cell proliferation assay, cells were transfected with indicated factors, and 48 hours later, cell proliferation was measured with alamar blue (Biorad) as detailed in the kit.…”