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. 2000 Aug 1;28(15):2969-76.
doi: 10.1093/nar/28.15.2969.

Gut-enriched Krüppel-like factor represses cyclin D1 promoter activity through Sp1 motif

J L Shie  1 , Z Y Chen, M Fu, R G Pestell, C C Tseng
Affiliations

Gut-enriched Krüppel-like factor represses cyclin D1 promoter activity through Sp1 motif

J L Shie et al. Nucleic Acids Res. .

Abstract

Cancer cells differ from normal cells in many characteristics including loss of differentiation and uninhibited cell proliferation. Recent studies have focused on the identification of factors contributing to cell growth and differentiation. Gut-enriched Krüppel-like factor (GKLF or KLF4) is a newly identified eukaryotic transcription factor and has been shown to play a role in regulating growth arrest. We have previously shown that GKLF mRNA levels were significantly decreased in colon cancer tissues, and that over-expression of GKLF in colonic adenocarcinoma cells (HT-29) resulted in reduction of cyclin D1 (CD1) mRNA and protein levels. The current study was undertaken to determine the mechanisms by which GKLF inhibited CD1 expression. In a transient transfection system, GKLF suppressed CD1 promoter activity by 55%. Sequential deletion and site-directed mutation analysis of the CD1 promoter have identified the sequence between -141 and -66, a region containing an Sp1 response element, to be essential for GKLF function. By electrophoretic mobility gel shift assay, recombinant GKLF and nuclear extracts from HT-29 cells were found to bind to the Sp1 motif on the CD1 promoter. The inhibitory effect of GKLF on the CD1 promoter activity was completely abolished by excessive amount of Sp1 DNA and GKLF significantly reduced the stimulatory function of Sp1 suggesting that GKLF and Sp1 may compete for the same binding site on the CD1 promoter. These results indicate that GKLF is a transcriptional repressor of the CD1 gene and that the inhibitory effect of GKLF is, in part, mediated by interaction with the Sp1 binding domain on its promoter.

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Figures

Figure 1
Figure 1
Effect of GKLF on CD1 promoter or CMV-Luc, SV40-Luc and TK-Luc reporters activity. (A) The sense (black bar), antisense (gray bar) GKLF or pCDNA-3 (open bar) expression vector was transfected with pCD1-1745 Luc reporter plasmid into L293 cells. The ratio of expression vector to reporter plasmid is shown on the abscissa. (B) The sense GKLF (black bar) or pCDNA-3 (open bar) DNA was transfected with CMV-Luc, SV40-Luc, or TK-Luc reporter plasmids (ratio of 1:100) into L293 cells. Luciferase activity was determined 48 h later. pCMV-βgal (0.05 µg) was also cotransfected with each construct to correct for differences in transfection efficiency. Data are expressed as means ± S.E. of three separate experiments. *P < 0.05, compared to pCDNA-3-transfected cells.
Figure 2
Figure 2
Repression of CD1 promoter activity by GKLF. (A) Schematic representation of the CD1 promoter showing the location of the DNA sequences resembling Sp1 and E2F binding sites as well as multiple CACCC motifs. (B) Sense GKLF (black bar) or pCDNA-3 (open bar) expression vector was cotransfected with the pCD1-1745 Luc reporter or an equal amount of each of the other 5′ promoter constructs into L293 cells. pCMV-βgal (0.05 µg) was also cotransfected with each construct to correct for differences in transfection efficiency. Data are expressed as means ± S.E. of four separate experiments. *P < 0.05, compared to pCDNA-3-transfected cells in each individual construct.
Figure 3
Figure 3
Effect of GKLF on the transcriptional activity of mutated CD1 and heterologous reporters plasmids. –163Sp1mutLuc, mutation of the Sp1 sequence within the context of the –163 bp fragment of the CD1 promoter; –163E2FmutLuc, mutation of the E2F sequence in the context of the –163 bp of the CD1 promoter; CD1(Sp1)Luc, heterologous reporter construct consisting of the CD1 Sp1 sequence; and CD1(E2F)Luc, heterologous reporter construct consisting of the CD1 E2F. Plasmids were cotransfected with sense GKLF (black bar) or pCDNA-3 (open bar) into L293 cells and luciferase activity was determined 48 h later. Data represent the mean ± S.E. of four separate experiments. *P < 0.05, compared to pCDNA-3-transfected cells in each individual construct.
Figure 4
Figure 4
Effect of fasting on CD1 promoter activity and GKLF mRNA levels. HT-29 cells were transfected with pCD1-1745 Luc and pCMV-βgal DNAs. Cells were then cultured in medium without serum for 48 h and luciferase activity (upper panel) and GKLF mRNA (lower panel) were examined. Luciferase data represent the mean ± S.E. of four separate experiments. *P < 0.05 compared to normal. Total RNA (20 µg) was extracted from HT-29 cells and analyzed by formaldehyde gel electrophoresis. RNA blots were hybridized with 32P-labeled GKLF probe and a representative blot is shown on the lower panel.
Figure 5
Figure 5
Histidine-tagged GKLF cDNA expressed in L293 cells yields a major 66-kDa and a minor 30-kDa fusion protein. L293 cells were transiently transfected with the pCDNA3.1/His or GKLF cDNAs. Fusion protein from L293 cells was harvested 48 h after transfection and was eluted through Ni-Chelated® columns. Protein (10 µg) from different eluted fractions was used for western blot analysis using GKLF-specific antibody. No GKLF immunoreactive protein was found in the pCDNA3.1/His-transfected cells.
Figure 6
Figure 6
Affinity-purified GKLF protein binds specifically to the Sp1 motif on the CD1 promoter. Gel mobility shift assays were performed with purified GKLF protein (fractions 8 and 9) and a 25-bp doubled-stranded Sp1 oligonucleotide probe. The DNA–protein complex is indicated by C. The binding reactions were performed in the presence of excessive amount of unlabeled Sp1 oligonucleotide (comp. fold indicated fold increases in the amount of cold Sp1). The complex C was supershifted by the addition of GKLF antiserum in the reaction (SS). F, free probe.
Figure 7
Figure 7
Gel mobility shift assays were performed with nuclear extracts from HT-29 cells and a 25-bp doubled-stranded Sp1 oligonucleotide probe in the absence (lane 1), or the presence of unlabeled wild-type (lane 2), or mutated Sp1 oligonucleotide (lane 3). The DNA–protein complex (C) was supershifted by the addition of GKLF antiserum in the reaction (SS, lane 4). Unlabeled competitors were added at 100-fold molar excess.
Figure 8
Figure 8
Effect of wild-type GKLF and truncated GKLF-1580, -1680 and -1780 on pCD1-963 Luc promoter activity. L293 cells were transfected with 1 µg/well of pCD1-963 Luc, 0.05 µg/well pCMV β-Gal and 0.01 µg/well wild-type or truncated GKLF DNA. Data are expressed as % of control (cells transfected with pCD1-963 Luc only) and presented as means ± S.E. of five separate experiments.
Figure 9
Figure 9
Effect of GKLF and Sp1 cotransfection on the CD1 promoter activity. L293 cells were transfected with pCD1-963 Luc construct and expression plasmids containing GKLF and Sp1 as indicated. Luciferase and β-galactosidase activities were determined 48 h later. Data were expressed as % of control (cells transfected with pCD1-963 Luc only) and presented as means ± S.E. of three separate experiments.
Figure 10
Figure 10
Induction of CD1 promoter activity by Sp1 was abolished by GKLF but not by truncated GKLF-1580. L293 cells were transfected with pCD1-963 Luc construct and expression plasmids containing GKLF and Sp1 as indicated. Luciferase and β-galactosidase activities were determined 48 h later. Data were expressed as fold increases over control (cells transfected with pCD1-963 Luc only) and presented as means ± S.E. of three separate experiments.
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