New p63 targets in keratinocytes identified by a genome-wide approach

Abstract

p63 is a developmentally regulated transcription factor related to p53. It is involved in the development of ectodermal tissues, including limb, skin and in general, multilayered epithelia. The DNp63a isoform is thought to play a 'master' role in the asymmetric division of epithelial cells. It is also involved in the pathogenesis of several human diseases, phenotypically characterized by ectodermal dysplasia. Our understanding of transcriptional networks controlled by p63 is limited, owing to the low number of bona fide targets. To screen for new targets, we employed chromatin immunoprecipitation from keratinocytes (KCs) coupled to the microarray technology, using both CpG islands and promoter arrays. The former revealed 96 loci, the latter yielded 85 additional genes. We tested 40 of these targets in several functional assays, including: (i) in vivo binding by p63 in primary KCs; (ii) expression analysis in differentiating HaCaT cells and in cells overexpressing DNp63a; (iii) promoter transactivation and (iv) immunostaining in normal tissues, confirming their regulation by p63. We discovered several new specific targets whose functional categorization links p63 to cell growth and differentiation.

p63 in epithelial development

“…The accepted activity was its dominant negative effect exerted towards the TAp63 isoform, as suggested by co-transfection experiments in luciferase reporter assays [3]. However, in addition to the competition between TAp63 and Np63 isoforms, growing evidence indicated that these N-terminal isoforms had different transcriptional specificities a specificity confirmed by functional domain characterization [32][33][34], and microarray analysis showing that both isoforms can induce gene transcription [35,29]. As expected from genes important for developmental and differentiation processes, the more strongly represented classes of target genes are those concerned with cell adhesion, the cytoskeleton, and the cell cycle.…”

“…In this context, a genomewide approach led to the identification of several high-confidence target genes for Np63 [35] However, only a few of these have been demonstrated to have a defined impact on epidermal formation and differentiation in vivo.…”

Mutant p53 subverts p63 control over KLF4 expression in keratinocytes

“…HaCaT cells and first passage primary human adult keratinocytes (haKCs), derived from healthy individuals (breast skin biopsies), were grown and handled as previously described (Vigano`et al, 2006). Maintenance, differentiation and transfection of primary human neonatal keratinocytes (hnKCs; CellNtec, CH) were conducted as previously described (Pozzi et al, 2009).…”

“…Immunofluorescence analysis of frozen human adult-thigh skin sections and cultured cells was performed as previously described (Vigano`et al, 2006) with anti-p63, anti-KLF4 (Genespin, I) and anti-KRT1 (Covance, Princeton, NJ, USA) antibodies. Nuclei were counterstained with 4 0 ,6-diamidino-2-phenylindole (DAPI).…”

“…Our lab has been involved in the identification of p63 targets in human KCs, either by expression profiling or by ChIP (chromatin immunoprecipitation) on chip analysis (Vigano`et al, 2006;Testoni et al, 2006). In this report, we investigated the possible regulation between p63 and KLF4 in primary and immortalized human KCs harboring mutant alleles of p53.…”