The ΔNp63α Phosphoprotein Binds the p21 and 14-3-3σ Promoters In Vivo and Has Transcriptional Repressor Activity That Is Reduced by Hay-Wells Syndrome-Derived Mutations

Abstract

p63 is a recently identified homolog of p53 that is found in the basal layer of several stratified epithelial tissues such as the epidermis, oral mucosa, prostate, and urogenital tract. Studies with p63 ؊/؊ mice and analysis of several human autosomal-dominant disorders with germ line p63 mutations suggest p63 involvement in maintaining epidermal stem cell populations. The p63 gene encodes six splice variants with reported transactivating or dominant-negative activities. The goals of the current study were to determine the splice variants that are expressed in primary human epidermal keratinocytes (HEKs) and the biochemical activity p63 has in these epithelial cell populations. We found that the predominant splice variant expressed in HEKs was ⌬Np63␣, and it was present as a phosphorylated protein. During HEK differentiation, ⌬Np63␣ and p53 levels decreased, while expression of p53 target genes p21 and 14-3-3 increased. ⌬Np63␣ had transcriptional repressor activity in vitro, and this activity was reduced in ⌬Np63␣ proteins containing point mutations, corresponding to those found in patients with Hay-Wells syndrome. Further, we show that ⌬Np63␣ and p53 can bind the p21 and 14-3-3 promoters in vitro and in vivo, with decreased binding of p63 to these promoters during HEK differentiation. These data suggest that ⌬Np63␣ acts as a transcriptional repressor at select growth regulatory gene promoters in HEKs, and this repression likely plays an important role in the proliferative capacity of basal keratinocytes.

Downregulation of 14-3-3σ in ovary, prostate and endometrial carcinomas is associated with CpG island methylation

“…In addition, repression of 14-3-3s expression was seen by a tumor-associated p63 splice variant (Delta Np63) in vitro. 34,35 However, again no correlation was found between the p63 status and 14-3-3s expression in hyperproliferative skin disease. 20 These studies all suggest independence of 14-3-3s expression of p53 and its homologues p63 and p73.…”

p63 consensus DNA-binding site: identification, analysis and application into a p63MH algorithm

“…In the p63 ChIP, we observed significant enrichment for the putative PTPN14 and ING1 REs relative to background (Figure 4b). Constitutive p63 binding was also observed at the known p63-responsive elements p21 site 1 (Westfall et al, 2003) and JAG1 (Sasaki et al, 2002). After ADR treatment, p63 binding to all four REs decreased.…”

“…As a source for p53, H1299 cells were transiently transfected with pCEP4 or pCEP4-p53 expression vectors. Immunoprecipitations were carried out as described previously (Westfall et al, 2003), using an a-myc antibody for myc-DNp63a and a combination of the PAb1801 (Santa Cruz, Santa Cruz, CA, USA) and PAb421 (Calbiochem, San Diego, CA, USA) antibodies for p53.…”

“…Transfections and the Dual Luciferase Reporter Assay System (Promega) were performed as described previously (Westfall et al, 2003).…”

Proliferation and cornification during development of the mammalian epidermis

“…14-3-3 is expressed exclusively in epithelial cells and induces cell cycle arrest (Hermeking et al, 1997). ⌬Np63␣ binds directly to the p21 WAF/Cip1 and 14-3-3 promoters (Westfall et al, 2003) and inhibits their transcription, thus favoring cell proliferation. Another mechanism by which ⌬Np63 isoforms may promote proliferative potential is to counteract the antiproliferative activity of TAp63␥ (see Fig.…”