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• W1995303485 abstract "Article1 September 1998free access Cloning and characterization of mCtBP2, a co-repressor that associates with basic Krüppel-like factor and other mammalian transcriptional regulators Jeremy Turner Jeremy Turner Department of Biochemistry, G08, University of Sydney, NSW, Australia, 2006 Search for more papers by this author Merlin Crossley Corresponding Author Merlin Crossley Department of Biochemistry, G08, University of Sydney, NSW, Australia, 2006 Search for more papers by this author Jeremy Turner Jeremy Turner Department of Biochemistry, G08, University of Sydney, NSW, Australia, 2006 Search for more papers by this author Merlin Crossley Corresponding Author Merlin Crossley Department of Biochemistry, G08, University of Sydney, NSW, Australia, 2006 Search for more papers by this author Author Information Jeremy Turner1 and Merlin Crossley 1 1Department of Biochemistry, G08, University of Sydney, NSW, Australia, 2006 *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:5129-5140https://doi.org/10.1093/emboj/17.17.5129 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Basic Krüppel-like factor (BKLF) is a zinc finger protein that recognizes CACCC elements in DNA. It is expressed highly in erythroid tissues, the brain and other selected cell types. We have studied the activity of BKLF and found that it is capable of repressing transcription, and have mapped its repression domain to the N-terminus. We carried out a two-hybrid screen against BKLF and isolated a novel clone encoding murine C-terminal-binding protein 2 (mCtBP2). mCtBP2 is related to human CtBP, a cellular protein which binds to a Pro-X-Asp-Leu-Ser motif in the C-terminus of the adenoviral oncoprotein, E1a. We show that mCtBP2 recognizes a related motif in the minimal repression domain of BKLF, and the integrity of this motif is required for repression activity. Moreover, when tethered to a promoter by a heterologous DNA-binding domain, mCtBP2 functions as a potent repressor. Finally, we demonstrate that mCtBP2 also interacts with the mammalian transcripition factors Evi-1, AREB6, ZEB and FOG. These results establish a new member of the CtBP family, mCtBP2, as a mammalian co-repressor targeting diverse transcriptional regulators. Introduction Basic Krüppel-like factor (BKLF) (Crossley et al., 1996) is a zinc finger protein that belongs to the subfamily of Krüppel-like proteins, which includes erythroid Krüppel-like factor (EKLF) (Miller and Bieker, 1993), lung Krüppel-like factor (LKLF) (Anderson et al., 1995) and gut-enriched Krüppel-like factor (Shields et al., 1996) [also known as endothelial zinc finger protein, EZF (Garrett-Sinha et al., 1996; Yet et al., 1998)]. Members of this subfamily contain three characteristic Cys–Cys: His–His Krüppel-like zinc fingers and recognize CACCC motifs in the promoters and enhancers of various genes. The founding member of the family, EKLF, is expressed in erythroid and mast cells and specifically recognizes the CACCC-box in the β-globin promoter (Miller and Bieker, 1993). Naturally occurring mutations which inhibit the binding of EKLF to this element are associated with clinical β-thalassaemia (Feng et al., 1994), and knockout studies in mice have demonstrated that EKLF is required for transcription of the β-globin gene (Nuez et al., 1995; Perkins et al., 1995). Specific target genes regulated by other members of the family have not been as clearly defined. LKLF was identified originally as a CACCC-box protein that was expressed abundantly in the lung, as well as in a number of other tissues (Anderson et al., 1995). Targeted mutation of the LKLF gene in mice has revealed important roles for this protein in T cell activation (Kuo et al., 1997b) and in vascular endothelial cells (Kuo et al., 1997a). GKLF/EZF is also expressed in vascular endothelial cells (Yet et al., 1998), as well as in the gut (Shields et al., 1996) and in the epidermal layer of skin (Garrett-Sinha et al., 1996). It is believed to be instrumental in controlling the proliferation of epithelial tissues, and there is some evidence that it can act as either an activator or a repressor of transcription (Yet et al., 1998). Like EKLF, BKLF is expressed at high levels in erythroid cells, but it is found additionally in other tissues, most notably the brain (Crossley et al., 1996). In vitro binding studies have demonstrated that like EKLF, it binds to the CACCC motifs found in the β-globin promoter as well as to CACCC-boxes in the globin locus control regions, and several of the heme biosynthetic genes. The exact target genes regulated by BKLF, however, have not been defined. BKLF knockout mice have been generated, and initial analysis indicates that they suffer from a myeloproliferative disorder, and thus it seems likely that BKLF plays a role in hematopoiesis (Perkins et al., 1997). It has been demonstrated previously that BKLF can activate transcription from a minimal promoter containing a single BKLF-binding site, although activation was significantly weaker than achieved by other Krüppel family proteins and was only observed with very high levels of BKLF (Crossley et al., 1996). In this study, we have extended our analysis of the functional activity of BKLF and demonstrate that it can also act as a potent repressor. We show that the minimal repression domain maps to a 74 amino acid region within the N-terminus, and that this domain retains its activity when fused to a heterologous DNA-binding domain. In an effort to understand the mechanism by which BKLF represses transcription, we carried out a yeast two-hybrid screen against the repression domain of BKLF, and identified a novel cofactor protein which we refer to as murine C-terminal-binding protein 2 (mCtBP2). mCtBP2 is a new member of the CtBP family of related proteins. The first member of the family, human CtBP (now hCtBP1), was identified initially as a cellular protein that bound to the C-terminal region of the adenovirus E1a oncoprotein (Boyd et al., 1993; Schaeper et al., 1995). It was shown that deletion of this region dramatically enhanced the tumorigenecity of E1a, suggesting that the binding of hCtBP1 significantly modulated the effect of E1a in vivo. Subsequently, it was demonstrated that an equivalent deletion to a gal4–E1a fusion protein dramatically enhanced its ability to activate transcription (Sollerbrant et al., 1996). This result suggested that hCtBP1 might be involved in the negative regulation of transcription. Recently, a Drosophila homologue of human CtBP1 has been identified as a partner protein of the transcriptional repressors Hairy, Snail, Knirps and Enhancer of split [E(spl)] mδ (Nibu et al., 1998; Poortinga et al., 1998). It has also been suggested that dCtBP binds the repressor Krüppel (Nibu et al., 1998). The in vivo studies in Drosophila indicate that dCtBP is a genuine co-repressor protein that plays an important role in repressing gene expression during development. Human CtBP1 recognizes signature Pro-X-Asp-Leu-Ser motifs in the C-terminus of E1a (Boyd et al., 1993; Schaeper et al., 1995; Sollerbrant et al., 1996). Drosophila CtBP has been shown to bind similar motifs found within the C-terminus of Hairy and E(spl)mδ (Poortinga et al., 1998), and within the body of the Snail and Knirps proteins (Nibu et al., 1998). The mammalian BKLF protein contains a similar motif within its repression domain. We show that mCtBP2 associates with BKLF through this element and that it is instrumental in mediating the repressor activity of BKLF. We searched the sequences of previously reported mammalian repressor proteins and found that potential CtBP recognition motifs occurred in several of these proteins, most notably in the zinc finger proteins Evi-1 (Morishita et al., 1988), AREB6 (Watanabe et al., 1993) and ZEB (Genetta et al., 1994). The signature motif also occurs in Friend of GATA (FOG) (Tsang et al., 1997), and a related Drosophila repressor protein, U-shaped (Haenlin et al., 1997). We used the two-hybrid system to demonstrate that mCtBP2 is able to associate specifically with the mammalian proteins AREB6, ZEB, Evi-1 and FOG. Our results indicate that mCtBP2 is a co-repressor protein that can associate with a number of mammalian transcription factors. Results BKLF can act as a potent repressor of transcription In order to assess the function of BKLF, we carried out co-transfection assays in Drosophila Schneider line 2 (SL2) cells. These cells are used conventionally for the study of CACCC-box factors since they are devoid of ubiquitous CACCC proteins (such as Sp1) which complicate interpretations of experiments in mammalian cell lines. It previously has been reported that CACCC-box factors can synergize with the glucocorticoid receptor (GR) on appropriately configured composite CACCC-glucorticoid response elements (GREs) (Schüle et al., 1988; Strähle et al., 1988). With a view to assessing whether BKLF would co-operate with or quench the activity of GRs, we prepared a test promoter containing three copies of a composite CACCC-GRE site [which had a spacing that previously had been reported to give maximal synergy (Strähle et al., 1988)] (Figure 1). First we tested the activity of BKLF alone, and in the absence of the glucocorticoid dexamethasone, and found it reduced the expression of this reporter gene slightly (Figure 1A, columns 1–4). In contrast, EKLF potently activated transcription (columns 5–7). This result suggested that BKLF was capable of repressing transcription. We next co-expressed increasing amounts of BKLF together with a constant amount of EKLF and found that BKLF could silence the activation mediated by EKLF (Figure 1B). We noted that this inhibition occurred with relatively low levels of BKLF (gel retardation data not shown), suggesting that the effect was not due entirely to competition between BKLF and EKLF for the three CACCC-boxes and that it might be due to an active repression domain within BKLF. We therefore tested a deletion derivative of BKLF, in which the entire N-terminus of the protein had been removed, leaving only the zinc finger DNA-binding domain (Figure 1C). This truncated protein was able to compete with and inhibit EKLF-mediated activation to some extent, but was significantly less effective than full-length BKLF. These results raised the possibility that BKLF contained a repression domain, capable of repressing activated transcription. Figure 1.BKLF represses transcription of a (CACCC-GRE)3-CAT reporter gene in SL2 cells, whilst EKLF activates transcription. (A) BKLF represses basal expression in a dose-dependent manner, whereas EKLF activates. In addition to 500 ng of reporter plasmid in all columns, 0, 60 and 250 ng, and 1 μg of pPac-BKLF and 60 ng, 250 ng and 4 μg of pPac-EKLF were used in columns 1–4 and 5–7, respectively. (B) Increasing amounts of BKLF compete with and inhibit activation by EKLF. pPac-EKLF (500 ng) was used alone in column 1 and together with 30, 60 and 250 ng, and 1 μg of pPac-BKLF in columns 2–5, respectively. (C) Increasing amounts of BKLF zinc finger DNA-binding domain alone are less effective than full-length BKLF but can compete with and inhibit activation by EKLF at high concentrations. pPac-EKLF (500 ng) was used alone in column 1 and together with 30, 60 and 250 ng, and 1 μg of pPac-BKLF-finger domain in columns 2–5, respectively. (D) Increasing amounts of BKLF repress transcription activated by the GR, whereas EKLF synergistically activates transcription. pPac-GR (500 ng) was used alone in column 1 and together with 10, 30 and 60 ng of pPac-BKLF in columns 2–4, and with 60 ng, 250 ng and 1 μg of pPac-EKLF in columns 5–7, respectively. (E) Deletion analysis demonstrates that the first 74 amino acids of BKLF are required for repression of GR-activated transcription. pPac-BKLF deletion constructs (60 ng), as shown, were used together with 500 ng of pPac-GR. Download figure Download PowerPoint We then proceeded to assess whether BKLF could repress activation mediated by the GR in the presence of dexamethasone. We found that BKLF potently repressed GR-activated transcription (Figure 1D, columns 1–4). In contrast, EKLF (and Sp1) (Figure 1D, columns 5–7, and data not shown) efficiently synergized with the GR on our promoter. We also tested the deletion derivative of BKLF, that only contains the zinc fingers, and found that it synergized weakly with the GR (Figure 1E). We conclude that BKLF contains a repression domain in its N-terminus that can silence both EKLF- and GR-activated transcription. In order to map this repression domain more precisely, we prepared a number of additional deletion constructs. We first used gel shift assays to verify that these mutants were all expressed at equivalent levels (data not shown), and then tested their ability to silence GR-activated transcription (Figure 1E). Deletion of the first 74 amino acids dramatically reduced repression activity, suggesting that the relevant domain included this region. The N-terminal domain of BKLF interacts with the co-repressor mCtBP2 In order to identify possible cofactors which might bind to this domain and mediate BKLF's ability to repress transcription, we carried out a yeast two-hybrid screen using a murine erythroleukaemia (MEL) cell library (Tsang et al., 1997) and a gal4 DNA-binding domain (DBD)–BKLF(1–268) fusion bait protein. This bait protein contains the repression domain of BKLF and adjoining sequences, but does not contain the BKLF zinc fingers. The analysis of ∼1.2×106 colonies led to the isolation of 110 that were His+, and 35 that were both His+ and β-galactosidase positive. Sequencing of these isolates revealed a full-length clone that encodes a member of the CtBP family of proteins. This protein is most highly related to a sequence generated through the assembly of database expressed sequence tags (ESTs) termed mCtBP2 (Katsanis and Fisher, 1998); however, it differs at four amino acids, and contains an additional 25 amino acids at the N-terminus. The sequences of recognized CtBP family proteins are shown in Figure 2. Figure 2.The amino acid sequences of existing members of the CtBP family. Conserved residues are indicated by asterisks. The putative active site histidine conserved in certain dehydrogenase enzymes is shown in bold, with an arrowhead. Accession numbers are as follows: mCtBP2, AF059735; hCtBP1, G1063638; hCtBP2, G2909777; rCtBP1, g1585432; dCtBP, G2950374/G2982720; cCtBP, Q20596. Download figure Download PowerPoint Finally we showed that as well as interacting with gal4DBD–BKLF(1–268), mCtBP2 interacted with gal4DBD–full-length BKLF (Figure 3), but did not interact with three control baits, p53(72–390) (Iwabuchi et al., 1993), the N-finger of GATA-1 (Tsang et al., 1997) or human lamin C(66–230) (Bartel et al., 1993). We also exchanged the bait and prey, cloning BKLF(1–268) in-frame with the gal4 activation domain (AD) and mCtBP2 in-frame with the gal4DBD. A strong interaction was also observed in this experiment (Figure 3). Figure 3.mCtBP2 recognizes a Pro-Val-Asp-Leu-Thr motif in the repression domain of BKLF. A deletion series of gal4DBD–BKLF fusions was assayed for binding to a gal4AD–mCtBP2 fusion using the yeast two-hybrid system. Mutation of the core CtBP-binding site from Asp–Leu to Ala–Ser impairs the interaction. An interaction was also observed when the bait and prey were exchanged. A gal4DBD–mCtBP2 fusion interacts with a gal4AD–BKLF(1–268) fusion, but not the mutant gal4AD–BKLF(1–268)mut, where the core CtBP-binding motif is mutated as above. The rate of yeast growth on His/Leu/Trp-deficient medium observed after 30 h incubation is shown on the right. Download figure Download PowerPoint The repression domain and mCtBP2-binding domain of BKLF co-localize We next used the two-hybrid assay to determine whether mCtBP2 bound to the region of BKLF previously implicated in transcriptional repression. We constructed a panel of gal4DBD–BKLF deletion constructs and used the two-hybrid system to test which of these interacted with mCtBP2 (Figure 3). Significantly, deletion of the N-terminal repression domain abolished the interaction with mCtBP2, but not with other BKLF-interacting proteins isolated from the library screen (data not shown). This result suggests that mCtBP2 binds within the BKLF repression domain. Within this N-terminal repression domain of BKLF, we noted the motif Pro-Val-Asp-Leu-Thr, which is closely related to a recognized hCtBP1-binding site in E1a (Pro-Val-Asp-Leu-Ser) (Schaeper et al., 1995). It previously has been reported that mutation of the core Asp–Leu residues to Ala–Ser abolished the interaction between E1a and hCtBP1 (Schaeper et al., 1995). We therefore used the two-hybrid system to test whether the analogous mutation in either the original BKLF(1–268) bait or in the full-length BKLF bait affected their interaction with mCtBP2. In both cases, this mutation significantly impaired the interaction (Figure 3), suggesting that mCtBP2 directly targets this motif in BKLF. Furthermore, exchange of the bait and prey (as above) also demonstrated that this mutation impaired the interaction between BKLF and mCtBP2 (Figure 3). mCtBP2 binds BKLF in vitro We next sought to determine whether the interaction between mCtBP2 and BKLF could be detected in vitro. We prepared GST–BKLF fusion proteins immobilized on agarose beads and tested their ability to retain in vitro translated 35S-radiolabelled mCtBP2. GST–full-length BKLF and GST–BKLF(1–268) fusions were able to bind mCtBP2 efficiently, whereas proteins containing the core Ala–Ser mutation, the zinc fingers of BKLF alone or GST alone could not retain mCtBP2 (Figure 4A). Thus, the interaction of mCtBP2 with BKLF in vitro requires the N-terminal repression domain, and is dependent on the integrity of the Pro-Val-Asp-Leu-Thr motif. Figure 4.mCtBP2 associates with BKLF in vitro. (A) GST pull-down experiments show that mCtBP2 recognizes the core motif in BKLF. The amounts of 35S-radiolabelled mCtBP2 retained by GST (lane 2), GST–BKLF fingers (lane 3), GST–BKLF(1–268) (lane 4), GST–mutant BKLF(1–268) (containing the core Ala–Ser mutation) (lane 5) and GST–full-length BKLF (lane 6) are shown. The input lane (lane 1) contains 50% of the 35S-radiolabelled mCtBP2 used in the binding assays. Equivalent amounts of GST fusion proteins were used in each lane. (B) Gel mobility shift experiments using BKLF and mutant BKLF (containing the core Ala-–Ser mutation) and mCtBP2 co-expressed in SL2 cells show that BKLF and mCtBP2 can form a complex on a double-stranded CACCC-box oligonucleotide. Lanes 1–16 contain SL2 cell nuclear extracts prepared from cells transfected with the following expression vectors: lane 1, 1 μg of pPac alone; lane 2, 1 μg of pPac-BKLF; lanes 3–6, 1 μg of pPac–BKLF and 10 and 100 ng, 1 and 5 μg of pPac-mCtBP2, respectively; lane 7, 1 μg of pPac-mutant BKLF; lanes 8–11, pPac-mutant BKLF and pPac–mCtBP2 as for lanes 3–6; lane 12, 1 μg of pPac; lane 13, 1 μg of pPac-BKLF and 100 ng of pPac-mCtBP2; lane 14, as for 13 but with anti-BKLF sera; lane 15, 1 μg of pPac-BKLF and 1 μg of pPac-mCtBP2; lane 16, as for 15 but with anti-BKLF antisera. The arrows indicate the site of migration of BKLF–DNA and the BKLF–mCtBP2–DNA complexes as indicated. Download figure Download PowerPoint BKLF, mCtBP2 and CACCC-box DNA form a ternary complex We also investigated whether mCtBP2 could associate with full-length BKLF bound to a CACCC sequence in DNA. We expressed full-length BKLF, or full-length BKLF containing the core Ala–Ser mutation, in SL2 cells and used the nuclear extracts in gel mobility shift experiments with the β-globin CACCC site as a probe (Figure 4B). Retarded complexes corresponding to BKLF and mutant BKLF can readily be observed (Figure 4B, lanes 2 and 7). However, when increasing amounts of mCtBP2 are co-expressed, the intensity of the BKLF–DNA complex diminishes, whereas the intensity of the mutant BKLF–DNA complex is unaffected (Figure 4B, compare lanes 2–6 and 7–11). The reduction in the observable BKLF–DNA complex suggests that either mCtBP2 is interfering with BKLF's ability to bind DNA, or that a higher molecular weight complex is formed but is obscured by another band. Additional experiments revealed the presence of a high molecular weight complex which co-migrates with a fainter endogenous band (Figure 4B, lanes 13 and 15). This complex is only observed when intact BKLF is co-expressed with mCtBP2, and addition of anti-BKLF antisera leads to the disruption of this complex and the formation of a new supershifted complex (Figure 4B, lanes 14 and 16). This latter complex is of only slightly lower mobility than the BKLF–mCtBP2–DNA complex, and co-migrates with the BKLF–Ab–DNA complex (data not shown), suggesting that the antibody displaces mCtBP2. These results indicate that BKLF and mCtBP2 can form a complex on a CACCC-box oligonucleotide. mCtBP2 is a co-repressor To determine whether BKLF was repressing transcription primarily by recruiting mCtBP2 (or a related family member) to the promoter, we tested whether the core Ala–Ser mutation [which reduces the binding of mCtBP2 to BKLF (Figures 3 and 4)] would impair BKLF's ability to repress transcription in the SL2 cell assay (Figure 5A). In this system, full-length BKLF repressed GR-mediated activation in a dose-dependent manner, to >35-fold (Figure 5A, columns 2–4). In contrast, the mutant version of BKLF (which contains the core Ala–Ser mutation) caused only a 5-fold repression, which was not dose dependent (Figure 5A, columns 5–7). This result strongly suggests that BKLF is repressing transcription by recruiting CtBP to the reporter gene promoter. In this case, we conclude that BKLF is associating directly with endogenous dCtBP. Figure 5.BKLF and CtBP associate to repress transcription (A) Mutation of the CtBP-binding motif in BKLF impairs BKLF′s ability to repress gene expression in SL2 cells. Increasing amounts of pPac-BKLF (columns 2–4) or pPac-mutant BKLF (containing the core Asp–Leu to Ala–Ser mutation) (columns 5–7) were co-transfected with 500 ng of pPac-GR and 500 ng of the (CACCC-GRE)3-CAT reporter vector. In addition to reporter, 500 ng of pPac-GR was used alone in column 1 and together with 10, 30 and 60 ng of pPac-BKLF in columns 2–4, and 10, 30 and 60 ng of pPac-mutant BKLF in columns 5–7. (B) When tethered to DNA as a BKLF zinc finger fusion, mCtBP2 can repress directly GR-activated transcription in SL2 cells. pPac-GR (500 ng) alone is used in column 1 and together with 1, 5, 10, 20 and 40 ng of pPac-mCtBP2–BKLF fingers in columns 2–6. Columns 7–11 contain 1, 5, 10, 20 and 40 ng of a plasmid encoding a protein which is identical except that the putative active site histidine, H321 in mCtBP2, is replaced by alanine. (C) The N-terminus of BKLF, when fused to the gal4DBD, functions as a repressor in NIH 3T3 cells but does not appear to require the CtBP-binding motif. Columns 1–4 contain 0, 5, 20 and 80 ng of pcDNA3-gal4DBD–BKLF(1–268). Columns 5–7 contain 5, 20 and 80 ng of pcDNA3-gal4DBD–BKLF(1–268) containing the core Ala–Ser mutation. (D) The minimal repression domain of BKLF requires CtBP to repress transcription. Columns 2–4 contain 10, 20 and 80 ng of pcDNA3-gal4DBD–BKLF(1–75). Columns 5–7 contain 10, 20 and 80 ng of pcDNA3-gal4DBD–BKLF(1–75) containing the core Ala–Ser mutation. (E) mCtBP2 can repress gene expression in NIH 3T3 cells when fused to the gal4DBD. Columns 1–6 contain 0, 20, 80 and 250 ng, 1 and 4 μg of pcDNA3-gal4DBD–mCtBP2; columns 7–11 contain 20, 80 and 250 ng, 1 and 4 μg of an equivalent plasmid, but in this case the putative catalytic site histidine 321 is altered to alanine. Download figure Download PowerPoint We carried out an additional experiment to demonstrate that mCtBP2 itself was capable of directly silencing gene expression. We constructed a chimaeric gene in which the coding sequence of mCtBP2 was fused directly to the zinc finger region of BKLF (Figure 5B, columns 1–6). In this case, the entire BKLF repression domain is removed and replaced by mCtBP2. mCtBP2 can therefore be targeted to the CACCC-box promoter, not by piggy-backing onto BKLF, but directly by means of the linked zinc finger domain. We tested the ability of this mCtBP2-finger chimaeric protein to repress GR-mediated transcriptional activation in SL2 cells. As shown in Figure 5B (columns 1–6), it repressed transcription efficiently in a dose-dependent manner. This result indicates that mCtBP2 is capable of repressing transcription, at least in Drosophila SL2 cells. Additional experiments in mammalian cells provide further evidence that BKLF and mCtBP2 associate to mediate repression. We constructed a gal4DBD–BKLF(1–268) fusion protein and tested its ability to repress a gal4-dependent promoter driving growth hormone expression in NIH 3T3 cells (Figure 5C). We chose these cells since they express both endogenous BKLF and mCtBP2 (unpublished results). As shown in Figure 5C (columns 1–4), this fusion protein efficiently silenced gene expression in a dose-dependent manner. This result is consistent with our previous conclusion that the N-terminus of BKLF contains a repression domain. In order to determine whether the abrogation of mCtBP2 binding influenced BKLF's ability to repress transcription, we tested whether a mutant gal4DBD–BKLF(1–268) fusion protein, containing the core Ala–Ser substitution, could also mediate repression (Figure 5C, columns 5–7). In contrast to the result in SL2 cells, in NIH 3T3 cells this mutation had no discernible effect on BKLF's repression activity. Reasoning that BKLF might possess an additional repression domain that functions in NIH 3T3 cells and compensates for the loss of CtBP-mediated repression, we constructed a minimal gal4DBD–BKLF(1–75) fusion protein that contained only the previously defined minimal repression domain of BKLF (see Figure 1). This protein also repressed reporter gene expression efficiently in a dose-dependent manner (Figure 5D, columns 1–4). In the case of the minimal construct, however, we found that mutation of the core Asp–Leu residues, within the mCtBP2-binding site, severely impaired its ability to repress transcription (Figure 5D, columns 5–7). This result suggests that the gal4DBD–BKLF(1–75) fusion represses transcription by recruiting mCtBP2 (or another member of the CtBP family expressed in NIH 3T3 cells). Finally, in order to determine whether mCtBP2 itself was capable of repressing transcription in mammalian cells, we constructed a gal4DBD–mCtBP2 fusion and tested its activity against the gal4 site-dependent promoter. As shown in Figure 5E (columns 1–6), the gal4DBD–mCtBP2 chimaeric protein efficiently repressed the expression of the reporter gene in a dose-dependent manner. Taken together, these results demonstrate that mCtBP2 is a true co-repressor in that it is capable of repressing transcription when delivered to a target promoter, either by binding to BKLF or when provided with its own BKLF zinc finger or gal4DBD. While mutant versions of BKLF, unable to bind CtBP, are unable to repress transcription in SL2 cells, it is interesting that the long form of the mutant gal4DBD–BKLF fusion could still repress gene expression in NIH 3T3 cells. This result suggests that additional cofactors may exist in mammalian cells that compensate for the loss of direct CtBP binding. Thus, our results indicate that while BKLF and CtBP co-operate to repress transcription, additional proteins are also likely to be involved (see Discussion). The putative dehydrogenase activity of mCtBP2 is not required for transcriptional repression It has been noted that CtBP family proteins have significant homology to the family of D-isomer-specific 2-hydroxy acid dehydrogenases (Schaeper et al., 1995), and it has been suggested that dCtBP may repress transcription by means of this dehydrogenase activity (Nibu et al., 1998). Attempts to demonstrate dehydrogenase activity of hCtBP1, however, have been unsuccessful, and an alternative hypothesis that the homology indicates structural similarity only and may reflect a conserved dimerization domain has been proposed (Schaeper et al., 1995; Poortinga et al., 1998). The dehydrogenases most similar to CtBP family proteins function as homodimers in vivo (Goldberg et al., 1994), and it has also been shown that dCtBP can dimerize (Poortinga et al., 1998). We tested mCtBP2's ability to dimerize in both the yeast two-hybrid assay and in in vitro GST pull-down experiments, and found that mCtBP2 was also able to homodimerize efficiently (data not shown). Thus it is clear that like the dehydrogenase enzymes, CtBP family proteins can dimerize, and it is possible that the similarity to dehydrogenases arises primarily from conservation of the dimerization surfaces. Nevertheless, it is noteworthy that all reported CtBP family members contain a conserved histidine residue that appears to correspond to the active site histidine in the enzyme D-lactate dehydrogenase (H296 in D-lactate dehydrogenase, H321 in mCtBP2 and H314 in hCtBP1) (Taguchi and Ohta, 1993; Schaeper et al., 1995) (Figure 2). Thus it remains possible that CtBP proteins possess undetected dehydrogenase activity. It has been shown that mutation of H296 in D-lactate dehydrogenase severely impaired its enzymatic activity (Taguchi and Ohta, 1993). We therefore constructed a mutation of H321 in mCtBP2 and carried out experiments to determine whether this mutation influenced the ability of mCtBP2 to repress gene expression. We tested both mutant mCtBP2–BKLF zinc finger and gal4DBD–mCt" @default.
• W1995303485 created "2016-06-24" @default.
• W1995303485 creator A5000256814 @default.
• W1995303485 creator A5073904629 @default.
• W1995303485 date "1998-09-01" @default.
• W1995303485 modified "2023-09-26" @default.
• W1995303485 title "Cloning and characterization of mCtBP2, a co-repressor that associates with basic Kruppel-like factor and other mammalian transcriptional regulators" @default.
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