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, 28 (9), 1955-62

Human Krüppel-like Factor 8: A CACCC-box Binding Protein That Associates With CtBP and Represses Transcription

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Human Krüppel-like Factor 8: A CACCC-box Binding Protein That Associates With CtBP and Represses Transcription

J van Vliet et al. Nucleic Acids Res.

Abstract

CACCC-boxes are recognised by transcription factors of the Sp/Krüppel-like Factor (Sp1/KLF) family. Here we describe one member of this family, KLF8/ZNF741/BKLF3 (KLF8). KLF8 contains a characteristic C-terminal DNA-binding domain comprised of three Krüppel-like zinc fingers, but also has limited homology to another family member, KLF3/Basic Krüppel-like Factor (KLF3/BKLF), in its N-terminus. Most significantly, it shares with KLF3/BKLF a Pro-Val-Asp-Leu-Ser/Thr motif. In KLF3/BKLF this motif mediates contact with the co-repressor protein C-terminal Binding Protein (CtBP). We demonstrate that the KLF8 Pro-Val-Asp-Leu-Ser motif also contacts CtBP. We show that the N-terminus of KLF8 functions as a repression domain and that its activity relies on the integrity of the CtBP recognition motif. We demonstrate that the zinc fingers of KLF8 recognize CACCC elements in DNA and that full-length KLF8 can repress a CACCC-dependent promoter. Finally we determine that KLF8 is broadly expressed in human tissues. These results establish KLF8 as a CACCC-box binding protein that associates with CtBP and represses transcription.

Figures

Figure 1
Figure 1
Alignment of the primary amino acid sequences of KLF8, KLF3/BKLF and the related protein KLF12/Ap2-rep. KLF8 is identical to ZNF741, GenBank accession no. U28282, but contains a glutamate in place of a glycine at residue 263. Residues that are identical are marked with an asterisk below the sequence. The C-terminal zinc finger domain is overlined and the zinc coordinating cysteine and histidine residues are marked by arrows. Three additional stretches of homology are boxed and the CtBP contact motif Pro-Val-Asp-Leu-Ser/Thr is shaded.
Figure 2
Figure 2
KLF8 recognises the CACCC-box from the β-globin promoter. (a) A gel retardation experiment showing retarded complexes generated by the binding of proteins present in COS cell nuclear extracts to a radiolabelled double-stranded oligonucleotide containing a CACCC element. Lanes 1–4 contain nuclear extracts from: COS cells transfected with pcDNA3 vector alone; cells transfected with pMT2/(KLF3/BKLF); cells transfected with pcDNA3/KLF8; cells transfected with pcDNA3/KLF8(240–359) encompassing the entire zinc finger domain of KLF8. Lanes 5–8 also contain nuclear extracts from COS cells transfected with pcDNA3 vector alone and in addition contain antibodies as follows: lane 6, anti-Sp1; lane 7, anti-Sp3; lane 8, anti-Sp1 and anti-Sp3. The positions of bands generated by endogenous Sp1 and Sp3 are shown to the right of the figure, together with the complexes generated by KLF3/BKLF, KLF8 and the zinc fingers of KLF8 and the position of the unbound probe. (b) Competition experiments demonstrate that the KLF8 zinc fingers bind most tightly to the β-globin CACCC box. All lanes contain nuclear extract from COS cells transfected with pcDNA3/KLF8(240–359) as in lane 4 above, but in addition unlabelled competitor double-stranded oligonucleotides have been added as follows: lanes 1–4 contain 0, 10, 30 and 100 ng of the β-globin CACCC site; lanes 5–8 contain the same amounts of the γ-globin CACCC site; lanes 9–12 contain the GC-rich Sp1 site; and lanes 13–16 contain the mutant β-globin CACCC site. See Materials and Methods for the precise sequence of all oligonucleotides.
Figure 3
Figure 3
KLF8 physically interacts with CtBP and the Pro-Val-Asp-Leu-Ser motif is required for this interaction. (a) A yeast two-hybrid experiment showing the interaction between KLF8 and CtBP. On the left yeast strains harbouring the test plasmids indicated are shown growing on minimal media deficient in tryptophan and leucine. On the right the same yeast are shown on minimal media that also lacks histidine. As can be seen, neither the negative control strain, which harbours a plasmid encoding the Gal4 activation domain together with a plasmid encoding the Gal4 DNA-binding domain/mCtBP2 fusion, nor the strain which contains a plasmid encoding KLF8 with a Asp-Leu→Ala-Ser mutation in the core CtBP contact motif, grow on histidine deficient media. But in the remaining strain, the physical interaction of the Gal4 DNA-binding domain/mCtBP2 fusion and the intact Gal4 activation domain/KLF8(1–262) fusion allows the reconstitution of Gal4 activity, the activation of the HIS3 reporter and growth on media lacking histidine. (b) A GST-pulldown experiment comparing the ability of GST-mCtBP2 to retain either radiolabelled KLF8 or a mutant version of KLF8 containing an Asp-Leu→Ala-Ser mutation within the Pro-Val-Asp-Leu-Ser motif. GST-mCtBP2 fusion protein and control GST were immobilised on agarose beads and mixed with radiolabelled KLF8. The beads were then washed repeatedly, boiled in loading buffer and subjected to electrophoresis. The resulting gel was examined by Phosphorimaging for the presence of KLF8 and mutant KLF8 which had been retained by the GST-mCtBP2 fusion or GST alone. Lanes 1 and 4 show 20% of the total input radiolabelled KLF8 and mutant KLF8, respectively. Lanes 2 and 5 show the background amounts of intact and mutant KLF8 retained by GST alone. Lanes 3 and 6 show the amounts of intact and mutant KLF8 retained by GST-mCtBP2, respectively. As can be seen by comparing lanes 3 and 6, a significant amount of intact KLF8 is retained by GST-mCtBP2, whereas very little of the mutant KLF8 is retained, indicating that an intact Pro-Val-Asp-Leu-Ser motif is involved in the contact of KLF8 and mCtBP2 in vitro. The bottom panel shows the gel stained with Coomassie blue, with molecular weight size markers in lane 7, GST protein in lane 8 and GST-mCtBP2 in lane 9.
Figure 4
Figure 4
KLF8 represses transcription. (a) Full length KLF8 represses a CACCC-dependent promoter. The ability of full-length KLF8 to repress transcription was compared with a mutant version carrying an Asp-Leu→Ala-Ser mutation in the CtBP recognition motif. KLF8 expression plasmids were co-transfected together with a reporter containing three copies of the β-globin CACCC box upstream of the Herpes thymidine kinase promoter and a GH reporter gene. Reporter (0.5 µg) was used in all lanes, together with 1 and 4 µg of pcDNA3/KLF8 in lanes 2 and 3, or pcDNA3/KLF8 Asp-Leu→Ala-Ser in lanes 4 and 5. (b) The ability of the N-terminal domain of KLF8 to function as a repression domain was compared with that of a mutant version containing a Asp-Leu→Ala-Ser mutation in the core CtBP binding motif. Gal4 DNA-binding domain/KLF8(1–262) fusions were co-transfected into NIH 3T3 cells, together with a reporter containing five Gal4 recognition elements upstream of a Herpes thymidine kinase promoter and a CAT reporter gene. Reporter (2.5 µg) was used in all lanes, together with 0.5 and 4 µg of an expression plasmid encoding intact Gal4 DNA-binding domain-KLF8(1–262) in lanes 2 and 3, or the mutant version in lanes 4 and 5.
Figure 5
Figure 5
The expression of KLF8 mRNA in human tissues analysed by northern blotting. (a) The tissues from which the mRNA was derived are shown above the figure. The major bands migrating at 3, 6, 7 and 11 kb are indicated. (b) The blot was then reprobed with human β-actin in order to control for the loading and integrity of the mRNA samples.

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