Novel in vivo targets of ΔNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach

被引:24
作者
Birkaya, Barbara [1 ]
Ortt, Kori [1 ]
Sinha, Satrajit [1 ]
机构
[1] SUNY Buffalo, Dept Biochem, Buffalo, NY 14214 USA
来源
BMC MOLECULAR BIOLOGY | 2007年 / 8卷
关键词
D O I
10.1186/1471-2199-8-43
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 (Delta Np63). Both the TA and Delta N 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 Delta Np63 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 Delta Np63-specific antibodies. We included an additional step in the ChIP procedure to enrich for Delta Np63 targets by screening the library of immunoprecipitated DNA for its ability to bind recombinant GST-Delta Np63. Cloning of Delta Np63-ChIP-derived DNA fragments identified more than 60 potential Delta Np63 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 Delta Np63 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 Delta Np63 levels in keratinocytes are reduced by siRNA, further confirming that these are bona fide targets. Conclusion: This unbiased genomic approach has allowed us to uncover functional targets of Delta Np63 and serves as the initial step in further analysis of the transcriptional regulatory mechanisms that are governed by p63 in keratinocytes.
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