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. 2002 Jun 1;30(11):2270-9.
doi: 10.1093/nar/30.11.2270.

Synergistic activation of the rat laminin gamma1 chain promoter by the gut-enriched Kruppel-like factor (GKLF/KLF4) and Sp1

Yuji Higaki  1 , Daniel Schullery, Yasunobu Kawata, Maria Shnyreva, Christine Abrass, Karol Bomsztyk
Affiliations

Synergistic activation of the rat laminin gamma1 chain promoter by the gut-enriched Kruppel-like factor (GKLF/KLF4) and Sp1

Yuji Higaki et al. Nucleic Acids Res. .

Abstract

Laminin is a multifunctional heterotrimeric protein present in extracellular matrix where it regulates processes that compose tissue architecture including cell differentiation. Laminin gamma1 is the most widely expressed laminin chain and its absence causes early lethality in mouse embryos. Laminin gamma1 chain gene (LAMC1) promoter contains several GC/GT-rich motifs including the bcn-1 element. Using the bcn-1 element as a bait in the yeast one-hybrid screen, we cloned the gut-enriched Kruppel-like factor (GKLF or KLF4) from a rat mesangial cell library. We show that GKLF binds bcn-1, but this binding is not required for the GKLF-mediated activation of the LAMC1 promoter. The activity of GKLF is dependent on a synergism with another Kruppel-like factor, Sp1. The LAMC1 promoter appears to have multiple GKLF- and Sp1-responsive elements which may account for the synergistic activation. We provide evidence that the synergistic action of GKLF and Sp1 is dependent on the promoter context and the integrity of GKLF activation and DNA-binding domain. GKLF is thought to participate in the switch from cell proliferation to differentiation. Thus, the Sp1-GKLF synergistic activation of the LAMC1 promoter may be one of the avenues for expression of laminin gamma1 chain when laminin is needed to regulate cell differentiation.

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Figures

Figure 1
Figure 1
The deduced amino acid sequence of rat GKLF and its similarity to the mouse and human sequences. The acidic residue clusters within the activation domain (arrows) and the predicted three tandem zinc finger motifs at the carboxyl end of the C2H2 type found in the Kruppel family of proteins (32) (bold) are identical in the three species.
Figure 2
Figure 2
Binding of recombinant GKLF to the bcn-1 motif in vitro. (A) The sequence of double-stranded synthetic oligonuleotide containing either wild-type or mutated bcn-1 element that was used as a competitor in the binding reaction. (B) Autoradiograph of the gel from EMSA. DNA-binding reaction was carried out in 10 µl binding buffer containing recombinant rat GST–GKLF and 32P-labeled double-stranded oligonucleotide containing wild-type bcn-1 element in the presence of either no competitor (none, lane 1), or 500-fold molar excess of double-stranded oligonucleotide containing either wild-type or mutated bcn-1 sequences as shown in (A). (C) Graph of the densitometric measurements of the shifted bands (B) expressed in digital light units (DLU).
Figure 3
Figure 3
GKLF-mediated activation of LAMC1 promoter in rat mesangial and human HeLa cells. Subconfluent rat mesangial and human HeLa cells were transiently transfected with firefly luciferase gene driven by the rat –1077/–20 LAMC1 promoter fragment with either pMt3 expression vector [(–) GKLF] or expression vector containing GKLF cDNA, pMt3-GKLF [(+) GKLF]. Renilla luciferase plasmid was used as a control for transfection efficiency. Forty-eight hours following transfection, cells were harvested and firefly and renilla luciferase activities were measured. Data are shown as mean ± SD of the ratios of firefly to renilla luciferase activities (n = 4).
Figure 4
Figure 4
The bcn-1 element is not required for the GKLF-mediated activation of the LAMC1 promoter in human HeLa cells. (A) Diagram of firefly luciferase reporter gene constructs that were used in transfection of HeLa cells. The sequences of the wild-type (wt-bcn-1) and mutated (mt-bcn-1-M4) bcn-1 located at –495 to –479 within the LAMC1 promoter are shown. (B) Firefly luciferase gene driven by LAMC1 promoter containing either wild-type (wt-bcn-1) or mutated (mt-bcn-1-M4) bcn-1 element fragments was co-transfected in HeLa cells with either pMt3 expression vector [(–) GKLF] or expression vector containing GKLF cDNA, pMt3-GKLF [(+) GKLF]. The renilla luciferase plasmid was used as a control for transfection efficiency. Forty-eight hours following transfection, cells were harvested and firefly and renilla luciferase activities were measured. Data are shown as mean ± SD of the ratios of firefly to renilla luciferase activities (n = 6 ).
Figure 5
Figure 5
Synergistic activation of rat LAMC1 promoter by GKLF and Sp1. The Drosophila SL2 cells were transiently co-transfected with rat –1077/–20 LAMC1 promoter-luciferase reporter plasmid with given amounts (ng) of either pPac-GKLF (GKLF), pPac-Sp1 (Sp1) or both expression plasmids. The expression plasmid, pPac-O, containing no insert, was used to adjust the total amount of DNA. Renilla luciferase reporter gene was also included to correct for transfection efficiency. Forty-eight hours following transfections, cells were harvested and luciferase activities in cell lysates were measured. (A) Cells transfected with increasing amounts (0, 50, 100, 1000 ng) of either pPac-GKLF (open columns) or pPac-Sp1 (closed columns). (B) Cells transfected with either pPac-O alone, pPac-GKLF (50 ng), pPac-Sp1 (50 ng) or pPac-Sp1 (50 ng) plus increasing amounts of pPac-GKLF (50–1000 ng). Data are shown as mean ± SD of the ratios of firefly to renilla luciferase activities (n = 4).
Figure 6
Figure 6
Functional mapping of the region responsible for the Sp1–GKLF-mediated synergistic activation of rat LAMC1 promoter in SL2 cells. The Drosophila SL2 cells were transiently co-transfected with a series of LAMC1 promoter deletion constructs in combination with 50 ng of either pPac-O (pPac), pPac-GKLF (GKLF), pPac-Sp1 (Sp1) or both expression (GKLF&Sp1) plasmids. Forty-eight hours following transfections, cells were harvested and both firefly and renilla luciferase activities in the cell lysates were measured. Activities of the promoter constructs were calculated as ratios of firefly to renilla luciferase activity (mean of n = 4). The numbers shown for each construct represent values that were normalized by dividing each firefly luciferase activity by the renilla luciferase activity measured in pPac alone. Synergism was calculated as the activity obtained when cells were co-transfected with both Sp1 and GKLF divided by the sum of the activities measured when Sp1 and GKLF were expressed alone, GKLF&Sp1/GKLF + Sp1, where a ratio >1.0 reflects synergistic activation.
Figure 7
Figure 7
Comparison of the Sp1–GKLF-mediated synergistic activation of LAMC1, K19 and SV40 promoters in SL2 cells. SL2 cells were transiently transfected with expression vector containing no insert pPac-O, pPac-GKLF or pPac-Sp1 together with firefly luciferase reporter gene driven by either LAMC1 or K19 promoters (A), or SV40 promoter with or without three tandem repeats of the bcn-1 element (B). The tables below the graphs show amounts of pPac-GKLF and pPac-Sp1 expression plasmids used (ng). Data are shown as mean ± SD of ratios of firefly to renilla luciferase activities (n = 4).
Figure 8
Figure 8
The GKLF activation and zinc finger domains are required for the synergistic activation of LAMC1 promoter in SL2 cells. (A) Diagram of rat GKLF. Putative activation domain (AD), nuclear localization signal (NLS) and the three zinc fingers (Zn) are shown. (B) pPac-Sp1 (50 ng) and a series of pPac-GKLF mutants were co-transfected with the LAMC1 promoter-firefly and renilla luciferase reporter gene constructs. Activity of the LAMC1 promoter upstream of firefly luciferase was assessed in transient co-transfection experiments in SL2 cells as before. Results are shown as the mean ratio of firefly to renilla luciferase activity ± SD (n = 4).
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