Yoshida Tomonori, Ohkumo Tsuyoshi, Ishibashi Shoko, Yasuda Kunio
Graduate School of Biological Sciences, Nara Institute of Science and Technology Takayama 8916-5, Ikoma, Nara, 630-0101, Japan.
Nucleic Acids Res. 2005 Jun 21;33(11):3465-78. doi: 10.1093/nar/gki653. Print 2005.
The Maf family of proteins are a subgroup of basic region-leucine zipper (bZIP) transcription factors, which recognize a long palindromic DNA sequence [TGCTGAC(G)TCAGCA] known as the Maf recognition element (MARE). Interestingly, the functional target enhancer sequences present in the alphaA-crystallin gene contain a well-conserved half-site of MARE rather than the entire palindromic sequence. To resolve how Maf proteins bind to target sequences containing only MARE half-sites, we examined their binding activities using electrophoretic gel mobility shift assays as well as in vitro and in vivo reporter assays. Our results indicate that the 5'-flanking region of the MARE half-site is required for Maf proteins to bind both in vitro and in vivo. The critical 5'-flanking sequences for c-Maf were determined by a selection and amplification binding assay and show a preference for AT-rich nucleotides. Furthermore, sequence analysis of the regulatory regions of several target genes also suggests that AT-rich sequences are important. We conclude that Maf can bind to at least two types of target sequences, the classical MARE (palindrome type) and a 5'-AT-rich MARE half-site (half-site type). Our results provide important new insights into the DNA binding and site selection by bZIP transcription factors.
Maf蛋白家族是碱性区域-亮氨酸拉链（bZIP）转录因子的一个亚组，它能识别一种被称为Maf识别元件（MARE）的长回文DNA序列[TGCTGAC(G)TCAGCA]。有趣的是，αA-晶状体蛋白基因中存在的功能性靶增强子序列包含一个高度保守的MARE半位点，而非完整的回文序列。为了弄清楚Maf蛋白如何与仅含MARE半位点的靶序列结合，我们使用电泳凝胶迁移率变动分析以及体外和体内报告基因分析来检测它们的结合活性。我们的结果表明，MARE半位点的5'侧翼区域是Maf蛋白在体外和体内结合所必需的。通过筛选和扩增结合分析确定了c-Maf的关键5'侧翼序列，结果显示其对富含AT的核苷酸具有偏好性。此外，对几个靶基因调控区域的序列分析也表明富含AT的序列很重要。我们得出结论，Maf可以与至少两种类型的靶序列结合，即经典的MARE（回文型）和富含5'-AT的MARE半位点（半位点型）。我们的结果为bZIP转录因子的DNA结合和位点选择提供了重要的新见解。
