Mahatan C S, Kaestner K H, Geiman D E, Yang V W
Department of Medicine, The Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
Nucleic Acids Res. 1999 Dec 1;27(23):4562-9. doi: 10.1093/nar/27.23.4562.
Gut-enriched Krüppel-like factor (GKLF, KLF4) is an epithelial-specific transcription factor whose expression is associated with growth arrest. In order to understand the mechanisms regulating expression of the gene encoding GKLF, we isolated a genomic clone containing murine GKLF. The gene spans 5.3 kb and contains four exons. A major start site of transcription was mapped to an adenine residue 601 nt 5' of the translation initiation codon. An additional 1 kb of the 5'-flanking region was sequenced and found to contain multiple cis -elements homologous to the binding sites of several established transcription factors including Sp1, AP-1, Cdx, GATA, and USF. In particular, three closely spaced GC-boxes 5' of the TATA box resemble the established binding site for GKLF. DNase I protection and electrophoretic mobility shift assays verified that recombinant GKLF bound to each of the three GC-boxes. In co-transfection experiments, GKLF transactivated a reporter gene linked to the GKLF 1 kb 5'-flanking region, as did Sp1, Sp3 and Cdx-2. Mutations of one or both of the first and second GC-boxes in the promoter resulted in diminished transactivation by GKLF. These results demonstrate that the 5'-flanking sequence of the mouse GKLF gene functions as a promoter and is subject to autoregulation by its own gene product.
肠道富集型锌指蛋白（GKLF，KLF4）是一种上皮特异性转录因子，其表达与生长停滞相关。为了了解调控GKLF编码基因表达的机制，我们分离出了一个包含小鼠GKLF的基因组克隆。该基因跨度为5.3 kb，包含四个外显子。一个主要的转录起始位点被定位到翻译起始密码子5'端601 nt处的一个腺嘌呤残基。对另外1 kb的5'侧翼区域进行测序，发现其中包含多个与几种已确定的转录因子（包括Sp1、AP-1、Cdx、GATA和USF）结合位点同源的顺式元件。特别是，TATA框5'端三个紧密排列的GC框类似于已确定的GKLF结合位点。DNA酶I保护实验和电泳迁移率变动分析证实重组GKLF与这三个GC框中的每一个都结合。在共转染实验中，GKLF激活了与GKLF 1 kb 5'侧翼区域相连的报告基因，Sp1、Sp3和Cdx-2也有同样的作用。启动子中第一个和第二个GC框中的一个或两个发生突变会导致GKLF的反式激活作用减弱。这些结果表明，小鼠GKLF基因的5'侧翼序列起启动子的作用，并受其自身基因产物的自动调节。
