The Hay Wells Syndrome-Derived TAp63αQ540L Mutant has Impaired Transcriptional and Cell Growth Regulatory Activity

Abstract

p63 mutations have been associated with several human hereditary disorders characterized by ectodermal dysplasia such as EEC (ectrodactyly, ectodermal dysplasia, clefting) syndrome, ADULT (acro, dermato, ungual, lacrimal, tooth) syndrome and AEC (ankyloblepharon, ectodermal dysplasia, clefting) syndrome (also called Hay-Wells syndrome). The location and functional effects of the mutations that underlie these syndromes reveal a striking genotype-phenotype correlation. Unlike EEC and ADULT that result from missense mutations in the DNA-binding domain of p63, AEC is solely caused by missense mutations in the SAM domain of p63. In this paper we report a study on the TAp63alpha isoform, the first to be expressed during development of the embryonic epithelia, and on its naturally occurring Q540L mutant derived from an AEC patient. To assess the effects of the Q540L mutation, we generated stable cell lines expressing TAp63alpha wt, DeltaNp63alpha or the TAp63alpha-Q540L mutant protein and used them to systematically compare the cell growth regulatory activity of the mutant and wt p63 proteins and to generate, by microarray analysis, a comprehensive profile of differential gene expression. We found that the Q540L substitution impairs the transcriptional activity of TAp63alpha and causes misregulation of genes involved in the control of cell growth and epidermal differentiation.

Regulation ofDlx5andDlx6gene expression by p63 is involved in EEC and SHFM congenital limb defects

“…ChIP was performed on sheared genomic DNA from cultured mouse keratinocytes and from H1299-tet-on ⌬Np63␣, using 2 g of the 4A4 antibody, as described (Ceribelli et al, 2006;LoIacono et al, 2006). IP…”

“…The procedure and expression vectors for luciferase assay have been previously described (Beretta et al, 2005). H1299 cells stably transfected with a tet-on ⌬Np63␣ expression plasmid were induced with Doxycyclin, as described (LoIacono et al, 2006).…”

“…To determine whether p63 binds to the Dlx5 and Dlx6 promoters, we carried out ChIP analysis for Dlx5 on genomic DNA from mouse keratinocytes, in which both ⌬Np63 and Dlx5 are expressed (Morasso et al, 1999), and for DLX6 on H1299 cells induced to express ⌬Np63␣ (LoIacono et al, 2006). PCR primers were designed to amplify genomic fragments from the Dlx5 and DLX6 promoters (Fig.…”

Microarray data analysis and mining approaches

“…A generally applicable filtering approach called the IQR filter, which eliminates genes that do not show sufficient variation in expression across all samples, as they tend to provide little discriminatory power, was proposed by von Heydebreck [32] and could be used routinely to reduce the number of hypothesis testing [40][41][42][43]. This filter is one of the filters implemented in the Bioconductor package genefilter (www.bioconductor.org) and allows the removal of genes that do not show an expression variation over all samples greater than a user defined threshold.…”

Hitting the Numbers: The Emerging Network of p63 Targets

“…These cautionary considerations notwithstanding, several groups performed profiling analysis in Saos2-or H1299-cells, in which TAp63 [13][14][15][16][17][18] or ΔNp63 19 were overexpressed. These experiments have generated a considerable amount of information: Jag1, 13,20 Pedf, 16 Hsp70, 21 Bpag, 22 Cd95, 15 Evpl, Smarcd3, 14 Vdr, 18 Gpx2, 23 Maspin 24,25 and Wnt4 26 are all genes duly validated by other methods (Fig.…”