A chromatin immunoprecipitation screen in mouse keratinocytes reveals Runx1 as a direct transcriptional target of ΔNp63

Abstract

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.

ΔNp63α Confers Tumor Cell Resistance to Cisplatin through the AKT1 Transcriptional Regulation

“…A ChIP kit (Upstate Cell Signaling Solutions) along with the p63 (4A4) antibody was used for ChIP analysis as previously described (19, 32, 33). Primers used for ChIP assay shown in Suppl.…”

Abnormal hair follicle development and altered cell fate of follicular keratinocytes in transgenic mice expressing ΔNp63α

“…Moreover, crossing our BG animals to the well-characterized TOPGAL transgenic mice confirmed a loss of Wnt signaling in the mutant hair follicles, suggesting that the observed defects in the IRS and hair shaft compartments are a direct result of reduced Wnt signaling. We posit that the hair abnormalities might be further augmented by other key regulators of hair shaft differentiation including Dlx3 and Runx1, both of which are established p63 target genes and show dramatic downregulation in the transgenic animals based on microarray data (Hwang et al, 2008;Ortt et al, 2008;Osorio et al, 2008;Raveh et al, 2006). Indeed, a careful evaluation of the microarray data shows significant alterations in a number of genes associated with additional signaling pathways [insulin growth factor-binding protein 5 (Igfbp5) and fibroblast growth factor 5 (Fgf5) for example], which might, in part, contribute to some aspects of the observed BG hair phenotype such as the cycling defects (Hebert et al, 1994;Schlake, 2005;Schneider et al, 2009).…”

Role of p63/p73 in epithelial remodeling and their response to steroid treatment in nasal polyposis

“…Based on IPA analysis and literature reviews, [16][17][18][19][20][21][22][23][24][25][26][27][28][29] Table E1 (available in this article's Online Repository at www. jacionline.org) summarizes 2 upstream and 77 downstream genes of p63 that were differentially expressed at a more than 1.5-fold level in untreated (GC-naive) patients with NP versus control subjects based on our microarray analysis.…”