Chromatin immunoprecipitation for identifying transcription factor targets in keratinocytes.

Protein-DNA interactions, such as those that are necessary for transcription, are critical in regulating cellular function and behavior. The identification of DNA sequences that interact with transcriptional regulatory proteins is an important step necessary to better understand the molecular mechanisms regulating gene expression. Chromatin immunoprecipitation (ChIP) is one such procedure that provides a snapshot of which transcription factors are occupying specific DNA sequences.

An active role of the DeltaN isoform of p63 in regulating basal keratin genes K5 and K14 and directing epidermal cell fate.

Background: One 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.

A chromatin immunoprecipitation screen in mouse keratinocytes reveals Runx1 as a direct transcriptional target of DeltaNp63.

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.

Novel in vivo targets of DeltaNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach.

Background: p63 is a transcription factor that plays an important role in skin epidermal development and differentiation. The p63 gene encodes for two major protein isoforms, those containing an amino-terminal trans-activation domain (TAp63) and those lacking this domain (DeltaNp63). Both the TA and DeltaN transcripts are also alternatively spliced at the 3' end producing proteins with unique C-termini that are designated as alpha, beta and gamma isoforms.

Ovol1 represses its own transcription by competing with transcription activator c-Myb and by recruiting histone deacetylase activity.

Ovol1 belongs to a family of evolutionarily conserved zinc finger proteins that act downstream of key developmental signaling pathways such as Wnt and TGF-beta/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.

Derivation of the consensus DNA-binding sequence for p63 reveals unique requirements that are distinct from p53.

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.