BackgroundOne major defining characteristic of the basal keratinocytes of the stratified epithelium is the expression of the keratin genes K5 and K14. The temporal and spatial expression of these two genes is usually tightly and coordinately regulated at the transcriptional level. This ensures the obligate pairing of K5 and K14 proteins to generate an intermediate filament (IF) network that is essential for the structure and function of the proliferative keratinocytes. Our previous studies have shown that the basal-keratinocyte restricted transcription factor p63 is a direct regulator of K14 gene.Methodology/Principal FindingsHere we provide evidence that p63, specifically the ΔN isoform also regulates the expression of the K5 gene by binding to a conserved enhancer within the 5′ upstream region. By using specific antibodies against ΔNp63, we show a concordance in the expression between basal keratins and ΔNp63 proteins but not the TAp63 isoforms during early embryonic skin development. We demonstrate, that contrary to a previous report, transgenic mice expressing ΔNp63 in lung epithelium exhibit squamous metaplasia with de novo induction of K5 and K14 as well as transdifferentiation to the epidermal cell lineage. Interestingly, the in vivo epidermal inductive properties of ΔNp63 do not require the C-terminal SAM domain. Finally, we show that ΔNp63 alone can restore the expression of the basal keratins and reinitiate the failed epidermal differentiation program in the skin of p63 null animals.SignificanceΔNp63 is a critical mediator of keratinocyte stratification program and directly regulates the basal keratin genes.

p63 is a member of the p53 family of proteins and plays an important role in epithelial development and differentiation. Although some p63 binding sites in the regulatory elements of epithelial genes have been identified, the optimal DNA-binding sequence has not been ascertained for this transcription factor. Here, we identify the preferred DNA-binding site of p63 by performing in vitro DNA selection experiments. Our analysis shows that the optimal p63 DNA-binding consensus motif consists of a CA(T)TG core and an AT-rich 5 0 and 3 0 flanking sequence. Gel shift and competition experiments demonstrate that there are specific sequence requirements that confer high DNA-binding affinity for p63 and that significant deviation from the consensus sequences result in poor or no binding. This pattern of DNA-binding is similar for both recombinant p63 and the endogenous protein present in keratinocyte nuclear extracts. Furthermore, we show that the consensus sequence is distinctly different from that of p53, particularly in the flanking sequences. Identification of the p63 consensus DNA-binding sequence will facilitate the validation of in vivo p63-responsive elements that mediate transcriptional regulation of a wide variety of target genes.

Ovol1 belongs to a family of evolutionarily conserved zinc finger proteins that act downstream of key developmental signaling pathways such as Wnt and TGF-β/BMP. It plays important roles in epithelial and germ cell development, particularly by repressing c-Myc and Id2 genes and modulating the balance between proliferation and differentiation of progenitor cells. In this study, we show that Ovol1 negatively regulates its own expression by binding to and repressing the activity of its promoter. We further demonstrate that Ovol1 uses both passive and active repression mechanisms to auto-repress: (1) it antagonizes transcriptional activation of c-Myb, a known positive regulator of proliferation, by competing for DNA binding; (2) it recruits histone deacetylase activity to the promoter via an N-terminal SNAG repressor domain. At Ovol1 cognate sites in the endogenous Ovol1 promoter, c-Myb binding correlates with increased histone acetylation, whereas the expression of Ovol1 correlates with a displacement of c-Myb from the DNA and decreased histone acetylation. Collectively, our data suggest that Ovol1 restricts its own expression by counteracting c-Myb activation and histone acetylation of the Ovol1 promoter.

Development of the skin epidermis and appendages such as hair follicles involves coordinated processes of keratinocyte proliferation and differentiation. The transcription factor p63 plays a critical role in these steps as evident by a complete lack of these structures in p63 null mice. The p63 gene encodes for two proteins TAp63 and DeltaNp63, the latter being the more prevalent and dominant isoform expressed in keratinocytes. Although numerous p63 target genes have been identified, these studies have been limited to transformed human keratinocyte cell lines. Here, we have employed a genomic screening approach of chromatin immunoprecipitation (ChIP) coupled with an enrichment strategy to identify DeltaNp63 response elements in primary mouse keratinocytes. Analysis of p63-ChIP-derived DNA segments has revealed more than 100 potential target genes including novel as well as mouse counterparts of established human p63 targets. Among these is Runx1, a transcription factor important for hair follicle development. We demonstrate that DeltaNp63 binds to a p63-response element located within a well-conserved enhancer of the Runx1 gene. Furthermore, siRNA mediated reduction of DeltaNp63 in mouse keratinocytes reduces Runx1 expression. Consistent with this, endogenous Runx1 levels are lower in the skin of p63(+/-) animals as compared to wild type animals. Lastly, we demonstrate that DeltaNp63 and Runx1 are co-expressed in specific compartments of the hair follicle in a dynamic fashion. Taken together our data demonstrate that p63 directly regulates Runx1 gene expression through a novel enhancer element and suggests a role for these two transcription factors in dictating skin keratinocyte and appendage development.