Regulation of KLF5 involves the Sp1 transcription factor in human epithelial cells Ceshi Chen a , Yingfa Zhou a , Zhongmei Zhou a , Xiaodong Sun a , Kristen B. Otto a , Rosalie M. Uht b , Jin-Tang Dong a, * a Department of Oncology and Hematology, Winship Cancer Institute, Emory University School of Medicine, 1365-C Clifton Road, Atlanta, GA 30322, USA b Department of Pathology, Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Box 800214, Charlottesville, VA 22908, USA Received 24 October 2003; received in revised form 11 December 2003; accepted 15 January 2004 Received by A.J. van Wijnen Abstract Human Kruppel-like factor 5 (hKLF5) is a transcription factor with a potential tumor suppressor function in prostate and breast cancers. In the majority of cancer samples examined, a significant loss of expression for KLF5 has been detected. Whereas hemizygous deletion appears to be responsible for KLF5’s reduced expression in about half of the cases, the mechanism for reduction is unknown in the remaining half; gene promoter methylation does not appear to be involved. In this report, we studied the regulation of KLF5 and cloned and functionally characterized a 1944-bp fragment of the 5V-flanking region of the hKLF5 gene. Several mitogens as well as global demethylation induced the expression of KLF5, implicating multiple factors in the regulation of KLF5. KLF5’s promoter lacks a TATA box and has a GC-rich region. Deletion mapping in combination with promoter activity assay showed that multiple cis-elements are involved in the transcriptional regulation of KLF5, some of which may play a repressor role whereas some others play an enhancer role. The Sp1 site between position  239 and  219 is essential for a basal promoter activity. Deletion or mutations of this Sp1 site significantly reduced promoter activity in several epithelial cell lines. Electrophoretic mobility shift assays (EMSAs) revealed that the Sp1 site binds Sp1 protein in nucleic extracts of different cell lines. In addition, overexpression of Sp1 protein transactivates KLF5 promoter activity. These findings suggest that Sp1 is a key transcription factor in KLF5’s dynamic transcriptional regulation. D 2004 Elsevier B.V. All rights reserved. Keywords: hKLF5; Promoter; Gene expression; Cancer cell line 1. Introduction Human Kruppel-like factor 5 (KLF5/ IKLF/ BTEB2) belongs to the Kruppel-like family of transcription factors, which are characterized by three zinc-finger domains (Sogawa et al., 1993). More than 18 members of the KLF family form a network to regulate a diverse range of biological processes, including cellular proliferation, differ- entiation, cell cycle regulation, apoptosis, and angiogenesis (Dang et al., 2000; Black et al., 2001). Similar to other members, KLF5 binds to a GC box in promoter DNA. Consequently, the expression of many genes including smooth muscle myosin heavy chain B (Watanabe et al., 1999), SM22-a (Adam et al., 2000), lactoferrin (Teng et al., 1998; Shi et al., 1999), KLF4 (Dang et al., 2002), platelet- derived growth factor a (PDGF-a), transforming growth factor h (TGF-b) (Shindo et al., 2002), and T cell receptor (TCRb) (Yang et al., 2003) is regulated by KLF5. Function- ally, KLF5-mediated transcriptional regulation appears to be context-dependent: although it stimulates the growth of mouse NIH-3T3 fibroblasts (Sun et al., 2001) and smooth 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.01.014 Abbreviations: KLF5, Kruppel-like factor 5; KLF4, Kruppel-like factor 4; PDGFa, platelet-derived growth factor a; TGF-b, transforming growth factor b; bFGF, basic fibroblast growth factor; TCR, T cell receptor; SMC, smooth muscle cells; Egr-1, early growth response 1; PMA, phorbol 12- myristate 13-acetate; MAPK, mitogen-activated protein kinase; aza-dC, 5- aza-2V-deoxycytidine; BSA, bovine serum albumin; PBS, phosphate- buffered saline; DDT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; BAC, bacterial artificial chromosome; PCR, polymerase chain reaction; EMSA, electrophoretic mobility shift assay. * Corresponding author. Tel.: +1-404-712-2568; fax: +1-404-712- 2571. E-mail address: jdong2@emory.edu (J.-T. Dong). www.elsevier.com/locate/gene Gene 330 (2004) 133 – 142