Ortt Kori, Sinha Satrajit
Department of Biochemistry, State University of New York at Buffalo, 14214, USA.
FEBS Lett. 2006 Aug 7;580(18):4544-50. doi: 10.1016/j.febslet.2006.07.004. Epub 2006 Jul 25.
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' and 3' 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.
p63是p53蛋白家族的成员之一，在上皮细胞发育和分化过程中发挥重要作用。尽管已经在上皮基因调控元件中鉴定出一些p63结合位点，但该转录因子的最佳DNA结合序列尚未确定。在此，我们通过进行体外DNA筛选实验来鉴定p63的首选DNA结合位点。我们的分析表明，最佳的p63 DNA结合共有基序由一个CA(T)TG核心以及富含AT的5'和3'侧翼序列组成。凝胶迁移和竞争实验表明，存在特定的序列要求赋予p63高DNA结合亲和力，并且与共有序列的显著偏差会导致结合不佳或无结合。重组p63和角质形成细胞核提取物中存在的内源性蛋白的这种DNA结合模式相似。此外，我们表明共有序列与p53的共有序列明显不同，特别是在侧翼序列方面。p63共有DNA结合序列的鉴定将有助于验证体内介导多种靶基因转录调控的p63反应元件。
