Shields J M, Yang V W
Department of Medicine and Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Nucleic Acids Res. 1998 Feb 1;26(3):796-802. doi: 10.1093/nar/26.3.796.
The gut-enriched Krüppel-like factor (GKLF) is a recently identified eukaryotic transcription factor that contains three C2H2zinc fingers. The amino acid sequence of the zinc finger portion of GKLF is closely related to several Krüppel proteins, including the lung Krüppel-like factor (LKLF), the erythroid Krüppel-like factor (EKLF) and the basic transcription element binding protein 2 (BTEB2). The DNA sequence to which GKLF binds has not been definitively established. In the present study we determined the DNA binding sequence of GKLF using highly purified recombinant GKLF in a target detection assay of an oligonucleotide library consisting of random sequences. Upon repeated rounds of selection and subsequent characterization of the selected sequences by base-specific mutagenesis a DNA with the sequence 5'-G/AG/AGGC/TGC/T-3' was found to contain the minimal essential binding site for GKLF. This sequence is present in the promoters of two previously characterized genes: the CACCC element of the beta-globin gene, which interacts with EKLF, and the basic transcription element (BTE) of the CYP1A1 gene, which interacts with Sp1 and several Sp1-like transcription factors. Moreover, the selected GKLF binding sequence was capable of mediating transactivation of a linked reporter gene by GKLF in co-transfection experiments. Our results establish GKLF as a sequence-specific transcription factor likely involved in regulation of expression of endogenous genes.
肠道富集型锌指蛋白（GKLF）是最近发现的一种真核转录因子，含有三个C2H2锌指结构。GKLF锌指部分的氨基酸序列与几种锌指蛋白密切相关，包括肺锌指蛋白（LKLF）、红系锌指蛋白（EKLF）和碱性转录元件结合蛋白2（BTEB2）。GKLF结合的DNA序列尚未明确确定。在本研究中，我们在由随机序列组成的寡核苷酸文库的靶标检测试验中，使用高度纯化的重组GKLF确定了GKLF的DNA结合序列。经过多轮筛选，并通过碱基特异性诱变对所选序列进行后续表征，发现一个序列为5'-G/AG/AGGC/TGC/T-3'的DNA包含GKLF的最小必需结合位点。该序列存在于两个先前已表征基因的启动子中：与EKLF相互作用的β-珠蛋白基因的CACCC元件，以及与Sp1和几种Sp1样转录因子相互作用的CYP1A1基因的碱性转录元件（BTE）。此外，在共转染实验中，所选的GKLF结合序列能够介导GKLF对连接的报告基因的反式激活。我们的结果表明，GKLF是一种序列特异性转录因子，可能参与内源性基因表达的调控。
