Yasuda Kunihiko, Hirayoshi Kazunori, Hirata Hiromi, Kubota Hiroshi, Hosokawa Nobuko, Nagata Kazuhiro
Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8397, Japan.
J Biol Chem. 2002 Nov 22;277(47):44613-22. doi: 10.1074/jbc.M208558200. Epub 2002 Sep 13.
In several cells and tissues the synthesis of HSP47, a collagen-specific molecular chaperone in the endoplasmic reticulum, is closely correlated with the synthesis of collagen. We previously reported that the Sp1 binding site at -210 bp in the promoter region and the first and second introns are required for the tissue-specific expression of HSP47 in transgenic mice (Hirata, H., Yamamura, I., Yasuda, K., Kobayashi, A., Tada, N., Suzuki, M., Hirayoshi, K., Hosokawa, N., and Nagata, K. (1999) J. Biol. Chem. 274, 35703-35710). Here, we analyze how these introns influence the transcriptional regulation of the hsp47 gene in BALB/c 3T3 cells, which produce high levels of HSP47. In vitro promoter analysis using a luciferase reporter and gel mobility shift analysis revealed that two cis-acting elements in the first and second introns, BS5-B and EP7-D, respectively, are required for the activation of hsp47 in BALB/c 3T3 cells. Several members of the Kruppel-like factor (KLF) family of proteins were identified as BS5-B-binding proteins by yeast one-hybrid analysis using these elements as baits. One of these proteins, KLF-6/Zf9, binds to the BS5-B element and activates expression of the reporter construct when transfected into cells. Chromatin immunoprecipitation assay analysis revealed that the endogenous KLF-6/Zf9 binds the BS5-B elements that contain the CACCC motif, which is a consensus recognition sequence for other proteins in the KLF family. We also showed that BS5-B and EP7-D are bound by two members of the Sp1 family, Sp2 and Sp3. These results suggest that at least three sequences are required for the constitutive expression of hsp47 in BALB/c 3T3 cells: the -210 bp Sp1 binding site, the BS5-B element in the first intron, and the EP7-D element in the second intron. We suggest that KLF proteins regulate the transcription of hsp47 by binding the BS5-B element in cooperation with Sp2 and/or Sp3.
在内质网中，HSP47是一种胶原蛋白特异性分子伴侣，在几种细胞和组织中，其合成与胶原蛋白的合成密切相关。我们之前报道过，转基因小鼠中HSP47的组织特异性表达需要启动子区域-210 bp处的Sp1结合位点以及第一和第二个内含子（平田浩、山村一、安田健、小林明、多田直、铃木真、平良浩史、细川直树和永田健（1999年）《生物化学杂志》274卷，35703 - 35710页）。在此，我们分析这些内含子如何影响在产生高水平HSP47的BALB/c 3T3细胞中hsp47基因的转录调控。使用荧光素酶报告基因进行的体外启动子分析和凝胶迁移率变动分析表明，第一和第二个内含子中的两个顺式作用元件，分别为BS5 - B和EP7 - D，是BALB/c 3T3细胞中hsp47激活所必需的。通过酵母单杂交分析，以这些元件为诱饵，鉴定出Kruppel样因子（KLF）家族的几种蛋白质为BS5 - B结合蛋白。其中一种蛋白质KLF - 6/Zf9，与BS5 - B元件结合，并在转染到细胞中时激活报告基因构建体的表达。染色质免疫沉淀分析表明，内源性KLF - 6/Zf9与包含CACCC基序的BS5 - B元件结合，该基序是KLF家族中其他蛋白质的共有识别序列。我们还表明，BS5 - B和EP7 - D被Sp1家族的两个成员Sp2和Sp3结合。这些结果表明，BALB/c 3T3细胞中hsp47的组成型表达至少需要三个序列：-210 bp的Sp1结合位点、第一个内含子中的BS5 - B元件和第二个内含子中的EP7 - D元件。我们认为，KLF蛋白通过与Sp2和/或Sp3协同结合BS5 - B元件来调节hsp47的转录。
