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. Recent research has suggested that DeltaNp63 is the predominant isoform expressed and active in keratinocytes.


RESULTS
To better elucidate the biological role of p63 in regulating gene expression in keratinocytes we performed chromatin immunoprecipitation (ChIP) experiments with DeltaNp63-specific antibodies. We included an additional step in the ChIP procedure to enrich for DeltaNp63 targets by screening the library of immunoprecipitated DNA for its ability to bind recombinant GST-DeltaNp63. Cloning of DeltaNp63-ChIP-derived DNA fragments identified more than 60 potential DeltaNp63 target loci that were located close to or embedded within known or predicted genes. Identity of these target genes suggests that they may participate in a myriad of cellular processes including transcriptional regulation, signaling and metabolism. Here we confirm the binding of DeltaNp63 to several of these genomic loci both by EMSA and replicate ChIP assays. Finally we show that the expression of many of these target genes is altered when DeltaNp63 levels in keratinocytes are reduced by siRNA, further confirming that these are bona fide targets.


CONCLUSIONS
This unbiased genomic approach has allowed us to uncover functional targets of DeltaNp63 and serves as the initial step in further analysis of the transcriptional regulatory mechanisms that are governed by p63 in keratinocytes.
Birkaya
Barbara
B
Department of Biochemistry, State University of New York at Buffalo, Buffalo, NY 14214, USA. bbirkaya@buffalo.edu <bbirkaya@buffalo.edu>
Ortt
Kori
K
Sinha
Satrajit
S
D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000502:physiology; D030561:Databases, Nucleic Acid; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D015603:Keratinocytes / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D034622:RNA Interference; D017422:Sequence Analysis, DNA; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000502:physiology; D014157:Transcription Factors; D015533:Transcriptional Activation; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000502:physiology
D004268:DNA-Binding Proteins; C115051:TP63 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D004247:DNA