The Krüppel-associated Box (KRAB)-Zinc Finger Protein Kid-1 and the Wilms' Tumor Protein WT1, Two Transcriptional Repressor Proteins, Bind to Heteroduplex DNA

Abstract

Zinc finger proteins of the Cys2His2 class represent a large group of DNA-binding proteins. A major subfamily of those proteins, the Krüppel-associated box (KRAB) domain-containing Cys2His2-zinc finger proteins, have been described as potent transcriptional repressors. So far, however, no DNA-binding sites for KRAB domain-containing zinc finger proteins have been isolated. Using a polymerase chain reaction-based selection strategy with double- and single-stranded DNA, we failed to reveal a binding site for Kid-1, one member of KRAB-zinc finger proteins. Binding of Kid-1 both to single- and homoduplex double-stranded DNA was negligible. We now present evidence that Kid-1 binds to heteroduplex DNA. Similar to Kid-1, the non-KRAB-zinc finger protein WT1 also bound avidly to heteroduplex DNA (both the −KTS and +KTS splice variant of WT1), whereas the POU domain protein Oct-6, the ets domain protein Ets-1 and the RING finger of BRCA-1 did not bind to heteroduplex DNA. Binding of WT1 to heteroduplex DNA was markedly reduced in naturally occurring mutants. The recognition of certain DNA structures by transcriptional repressor proteins may therefore represent a more common phenomenon than previously thought. Zinc finger proteins of the Cys2His2 class represent a large group of DNA-binding proteins. A major subfamily of those proteins, the Krüppel-associated box (KRAB) domain-containing Cys2His2-zinc finger proteins, have been described as potent transcriptional repressors. So far, however, no DNA-binding sites for KRAB domain-containing zinc finger proteins have been isolated. Using a polymerase chain reaction-based selection strategy with double- and single-stranded DNA, we failed to reveal a binding site for Kid-1, one member of KRAB-zinc finger proteins. Binding of Kid-1 both to single- and homoduplex double-stranded DNA was negligible. We now present evidence that Kid-1 binds to heteroduplex DNA. Similar to Kid-1, the non-KRAB-zinc finger protein WT1 also bound avidly to heteroduplex DNA (both the −KTS and +KTS splice variant of WT1), whereas the POU domain protein Oct-6, the ets domain protein Ets-1 and the RING finger of BRCA-1 did not bind to heteroduplex DNA. Binding of WT1 to heteroduplex DNA was markedly reduced in naturally occurring mutants. The recognition of certain DNA structures by transcriptional repressor proteins may therefore represent a more common phenomenon than previously thought. DNA-binding proteins are essential for the utilization of genetic information. They take part in the replication of the genome, in transcription of DNA into mRNA and in the repair of the genome. In these processes the interaction with DNA takes place in a variety of ways. Interaction with sequence-specific DNA is a common and well established phenomenon. There are many examples of proteins binding to a specific sequence of double-stranded DNA. In contrast, sequence-specific interaction with single-stranded DNA seems to occur more rarely. An example for such an interaction would be YB-1, a member of the Y box family of transcription factors, which binds to a region of single-stranded DNA spanning the X box of the major histocompatibility class II DRA promotor (1MacDonald G.H. Itoh-Lindstrom Y. Ting J.P.-Y. J. Biol. Chem. 1995; 270: 3527-3533Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Interaction independent of a specific sequence is another possibility, but so far only a few sequence-independent, structural motifs have been described as binding sites. Poly(ADP-ribose) polymerase and DNA-dependent protein kinase are essential elements of the cellular response to DNA damage (for reviews see, Refs. 2de Murcia G. de Murcia J.M. Trends Biochem. Sci. 1994; 19: 172-176Abstract Full Text PDF PubMed Scopus (753) Google Scholar and 3Weaver D.T. Trends Genet. 1995; 11: 388-392Abstract Full Text PDF PubMed Scopus (191) Google Scholar). Whereas poly(ADP-ribose) polymerase binds to DNA containing single-strand breaks, DNP-PK binds to double-strand DNA breaks, and both do so independently of the DNA sequence. In the case of single-strand DNA breaks, the DNA molecule assumes a V-shaped conformation, which is recognized by poly(ADP-ribose) polymerase. Other examples of protein/DNA interaction based on structural characteristics of the DNA molecule can be found in the recognition of cruciform DNA by the high-mobility group protein HMG-1 (4Bianchi M.E. Beltrame M. Paonessa G. Science. 1989; 243: 1056-1059Crossref PubMed Scopus (542) Google Scholar), the tumor suppressor protein p53 (5Lee S. Cavallo L. Griffith J. J. Biol. Chem. 1997; 272: 7532-7539Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), and the transcriptional regulator complex SWI-SNF (6Quinn J. Fyrberg A.M. Ganster R.W. Schmidt M.C. Peterson C.L. Nature. 1996; 379: 844-847Crossref PubMed Scopus (170) Google Scholar). Transcription factors as a subclass of DNA-binding proteins play an important role during growth and differentiation of cells and organs. Several families of transcription factors have been described, one of which is the family of zinc finger proteins (for recent reviews, see Refs. 7Berg J.M. Shi Y. Science. 1996; 271: 1081-1085Crossref PubMed Scopus (1646) Google Scholar and 8Klug A. Schwabe J.W.R. FASEB J. 1995; 9: 597-604Crossref PubMed Scopus (531) Google Scholar). So far, however, binding sites have been characterized for only a subset of zinc finger proteins. “Classical” zinc finger proteins like Sp1, Egr-1, and WT1 have been shown to recognize sequence-specific double-stranded DNA (e.g. Ref. 9Berg J.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11109-11110Crossref PubMed Scopus (164) Google Scholar). One large subfamily of zinc finger proteins is characterized by the presence of a highly conserved motif, the so-called KRAB-A domain or FBP (10Bellefroid E.J. Poncelet D.A. Lecocq P.J. Revelant O. Martial J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3608-3612Crossref PubMed Scopus (343) Google Scholar, 11Rosati M. Marino M. Franze A. Tramontano A. Grimaldi G. Nucleic Acids Res. 1991; 19: 5661-5667Crossref PubMed Scopus (75) Google Scholar), which has been identified as a potent transcriptional repressor domain (12Margolin J.F. Friedman J.R. Meyer W.K.-H. Vissing H. Thiesen H.-J. Rauscher III, F.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4509-4513Crossref PubMed Scopus (506) Google Scholar, 13Pengue G. Calabrò V. Bartoli P.C. Pagliuca A. Lania L. Nucleic Acids Res. 1994; 22: 2908-2914Crossref PubMed Scopus (119) Google Scholar, 14Vissing H. Meyer W.K.-H. Aagaard L. Tommerup N. Thiesen H.-J. FEBS Lett. 1995; 369: 153-157Crossref PubMed Scopus (125) Google Scholar, 15Witzgall R. O'Leary E. Leaf A. Önaldi D. Bonventre J.V. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4514-4518Crossref PubMed Scopus (309) Google Scholar). Because the KRAB-A domain is present in approximately one-third of the large number of Cis2His2-zinc finger proteins (10Bellefroid E.J. Poncelet D.A. Lecocq P.J. Revelant O. Martial J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3608-3612Crossref PubMed Scopus (343) Google Scholar) (there are several hundred genes coding for those proteins in the mammalian genome (16Bellefroid E.J. Lecocq P.J. Benhida A. Poncelet D.A. Belayew A. Martial J.A. DNA (N. Y.). 1989; 8: 377-387Crossref PubMed Scopus (195) Google Scholar)), it represents a very important paradigm of transcriptional repression. The identification of the KRAB-A domain as a transcriptional repressor motif was soon followed by the cloning of proteins that interact with the KRAB-A domain (17Friedman J.R. Fredericks W.J. Jensen D.E. Speicher D.W. Huang X.-P. Neilson E.G. Rauscher III, F.J. Genes Dev. 1996; 10: 2067-2078Crossref PubMed Scopus (523) Google Scholar, 18Kim S.-S. Chen Y.-M. O'Leary E. Witzgall R. Vidal M. Bonventre J.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15299-15304Crossref PubMed Scopus (243) Google Scholar, 19Moosmann P. Georgiev O. Le Douarin B. Bourquin J.-P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (245) Google Scholar), but up to this point no DNA-binding site for a KRAB-zinc finger protein could be identified. Only in the case of the human KRAB-zinc finger protein ZNF74 has an affinity for RNA homopolymers been described (20Grondin B. Bazinet M. Aubry M. J. Biol. Chem. 1996; 271: 15458-15467Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Kid-1 is a 66-kDa protein with 13 Cis2His2-zinc fingers at its COOH terminus. The zinc fingers are clustered in two groups of four and nine zinc fingers and are separated by a 32-amino acid spacer (21Witzgall R. O'Leary E. Gessner R. Ouellette A.J. Bonventre J.V. Mol. Cell. Biol. 1993; 13: 1933-1942Crossref PubMed Scopus (90) Google Scholar). The Kid-1 protein also contains a KRAB-A domain at its NH2 terminus. Here we demonstrate binding of the zinc finger region of Kid-1 to heteroduplex DNA. Another transcriptional repressor protein, the Wilms' tumor protein WT1, also binds to heteroduplex DNA. This function is impaired in naturally occurring mutants of WT1. In the case of Kid-1 and WT1, recombinant plasmids encoding fusion proteins with glutathione S-transferase were constructed using the plasmid pGEX-KG (22Guan K. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1635) Google Scholar). Details are available from the authors upon request. The recombinant proteins were purified from bacterial extracts according to standard protocols (23$$Google Scholar). The binding site selections were carried out using the random oligonucleotide 5′-CTT GAT CCT AAG ATT CCC TG-N15-AGG CTC AAA GCT GAA TTA CT-3′ and the PCR 1The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s); GST, glutathioneS-transferase; KRAB, Krüppel-associated box; WT1, Wilms' tumor protein 1. primers 5′-CTT GAT CCT AAG ATT CCC TG-3′ and 5′-AGT AAT TCA GCT TTG AGC CT -3′, the random oligonucleotide 5′-CTT GAT CCT AAG ATT CCC TG-N35-AGG CTC AAA GCT GAA TTA CT-3′ and the same PCR primers mentioned before, and the random oligonucleotide 5′-GAT CCT AAG ATT CCC TGT CGA C-N75-GTC GAC TCC CTT TAG TGA GGG TT-3′ and the PCR primers 5′-CTT GAT CCT AAG ATT CCC TG-3′ and 5′-ATT AAC CCT CAC TAA AGG GA-3′. Heteroduplex DNA with a bubble of 25 nucleotides consisted either of the oligonucleotides ss1 5′-CCT CAC TAA AGG GAG TCG AC T ACC TTT GAA ATC GAA AGG CGC CTC GGA TCC AGG GAA TCT TAG GA-3′ and ss2 5′-TCC TAA GAT TCC CTG GAT CC T CTT ATA AGG GAT CGG GAC CCT TGC GTC GAC TCC CTT TAG TGA GG-3′ or of oligonucleotides ss3 5′-AAG ACT GAG TTC ACC GGA GC A TTA TCC CCG TTG CGT AAC CGA GAA GCT TTA CGC AAG TGG AAG AC-3′ and ss4 5′-GTC TTC CAC TTG CGT AAA GC G GAG ATT GGC ATA CCA TTT TCG CCG GCT CCG GTG AAC TCA GTC TT-3′ (noncomplementary nucleotides are bold and italic). When a homoduplex of the same length was used, oligonucleotide ss1 was substituted with an oligonucleotide fully complementary to oligonucleotide ss2. The structure of heteroduplex ss1/ss2 was corroborated by digests with restriction enzymes cutting either in the double-stranded region or in the bubble. AvaII recognizes a site in the bubble region of ss2 and in the homoduplex (5′-GGACC-3′), this site is cut only in the homoduplex. SalI (5′-GTCGAC-3′) and BamHI (5′-GGATCC-3′) recognize sites in the double-stranded region of the heteroduplex immediately adjacent to the bubble, these enzymes are able to cut the heteroduplex DNA, although less efficiently than the homoduplex (the lower efficiency probably results from the close location to the bubble). The heteroduplex with a bubble of 7 nucleotides consisted of the oligonucleotides ss5 5′-CCT CAC TAA AGG GAG TCG AC T ACC TTT GGA TCC AGG GAA TCT TAG GA-3′ and ss6 5′-TCC TAA GAT TCC CTG GAT CC C CCT TGC GTC GAC TCC CTT TAG TGA GG-3′ (noncomplementary nucleotides are bold and italic). The oligonucleotide with the oct-binding site had the sequence 5′-CTG AGC AAA ACA CCA CCT GGG TA A TTT GCA T TT CTA AAA TAA G-3′ (the octamer motif is bold and italic), the oligonucleotide with the ets-binding site had the sequence 5′-TGA ATA TTT TGT AA T TTC CT A GTC TTG-3′ (the ets-binding site is bold and italic), and the oligonucleotide with the Egr-1/WT1-binding site had the sequence 5′-CGC CCT CGC CCC CGC GCC GGG-3′ (the Egr-1/WT1-binding site is bold and italic); the oligonucleotides containing the octamer motif, the ets-binding site and the Egr-1/WT1-binding site were regular homoduplex DNA. Homoduplex and heteroduplex DNA were prepared by mixing equimolar amounts of both strands, heating to 80–85 °C in a water bath, and subsequent slow cooling to room temperature. The selection was carried out according to already published protocols with minor modifications (24Pierrou S. Enerbäck S. Carlsson P. Anal. Biochem. 1995; 229: 99-105Crossref PubMed Scopus (25) Google Scholar). Approximately 1 μg of purified protein and 100–200 ng of selected and PCR-amplified oligonucleotide were used for each round of selection. To produce single-stranded oligonucleotide, a second PCR reaction was run with the reverse primer only after having precipitated the double-stranded PCR product with ethanol and ammonium acetate. Gel shifts were performed essentially as described previously (24Pierrou S. Enerbäck S. Carlsson P. Anal. Biochem. 1995; 229: 99-105Crossref PubMed Scopus (25) Google Scholar). When competition experiments were performed, a 5-, 25-, and 125-fold molar excess of unlabeled oligonucleotide was added to the protein together with the labeled binding site. When the zinc finger structure was to be disrupted, a combination of 10 mm EDTA, pH 8.0, and 0.1 mm 1,10-phenanthroline (final concentrations) was added to the binding buffer. The 1,10-phenanthroline was added from a 20 mm stock in ethanol; as a negative control, an equal amount of ethanol was added to the binding buffer. To define a binding site for the KRAB-zinc finger protein Kid-1, we performed a PCR-based selection strategy with double-stranded DNA and various portions of the Kid-1 zinc finger domain. Binding site selections with the four-zinc finger cluster and a 15-bp random oligonucleotide, with the nine-zinc finger cluster and a 35-bp random oligonucleotide, and finally with all 13 zinc fingers of Kid-1 and a 75-bp random oligonucleotide did not result in the isolation of a binding site for any of the zinc finger constructs. We therefore also initiated a binding site selection with single-stranded DNA and the 13-zinc finger construct of Kid-1. Although we noticed a weak but consistent shift throughout the selection process, we were unable to define a consensus sequence. The observation of a weak shift even with nonselected single-stranded DNA prompted us to try a gel shift with heteroduplex DNA. Heteroduplex DNA occurs as a result of “bubble formation” during transcription of DNA into RNA, when the double-stranded DNA is melted so that RNA polymerase II can read the template strand, and may therefore serve as a natural substrate for transcriptional repressor proteins. A heteroduplex DNA with 20 paired nucleotides at the 5′- and 3′-end and a bubble of 25 nucleotides in the middle was designed randomly, consisting of single-stranded oligonucleotides ss1 and ss2. Care was taken that the nucleotides facing each other in the bubble consisted of pyrimidine·pyrimidine and purine·purine base pairs, to avoid non-Watson/Crick base pairs. Such a heteroduplex DNA was shifted efficiently by a fusion protein between the 13 zinc fingers of Kid-1 and glutathioneS-transferase (GST), whereas either single-stranded oligonucleotide was only barely shifted (Fig.1 A). When the 13-zinc finger construct was incubated with homoduplex DNA consisting of the single-stranded oligonucleotide ss2 used in the heteroduplex DNA, and another fully complementary single-stranded oligonucleotide, no shift could be detected (Fig. 1 B). A heteroduplex DNA with the same paired sequences at either end but only a bubble of 7 nucleotides in the middle could not be shifted as efficiently as the heteroduplex DNA with a bubble of 25 nucleotides (Fig. 1 B). GlutathioneS-transferase could not shift any of the DNA molecules just described (Fig. 1, A and B). To further demonstrate the specificity of the interaction between Kid-1 and heteroduplex DNA, competition experiments were performed with the 13 zinc fingers of Kid-1 and a radiolabeled heteroduplex oligonucleotide containing the 25 nucleotide bubble. The shift was competed out by a 125-fold molar excess of the heteroduplex oligonucleotide with the 25 nucleotide bubble, whereas a homoduplex oligonucleotide and a single-stranded oligonucleotide of the same length had no effect on the shift (Fig.2 A). We then tested Kid-1 with a second heteroduplex DNA containing a 25-nucleotide bubble. This second heteroduplex consisted of single-stranded oligonucleotides ss3 and ss4, which were obtained by scrambling oligonucleotides ss1 and ss2, respectively. Incubation of the 13 zinc fingers of Kid-1 with this second large heteroduplex again resulted in an efficient shift (data not shown). To control for the role of the zinc fingers, we incubated the fusion protein between GST and the 13 zinc fingers of Kid-1 with a combination of EDTA, a divalent cation chelator, and 1,10-phenanthroline, a Zn2+-chelating agent, before performing a gel shift with heteroduplex DNA. This resulted in the complete abrogation of the shift. Prior incubation of the protein with an equal volume of ethanol, the solvent for 1,10-phenanthroline, did not influence binding of Kid-1 to heteroduplex DNA, thus again attesting to the specificity of the shift (Fig. 2 B). Another set of experiments was carried out to determine which portion of the Kid-1 zinc finger domain contributed to the shift. Fusion proteins between GST and the four-zinc finger cluster of Kid-1, between GST and the nine-zinc finger cluster, and between GST and the complete zinc finger domain of Kid-1 were used in gel shift assays with heteroduplex DNA containing a 25-nucleotide bubble. All three fusion proteins shifted the heteroduplex DNA (Fig.3), whereas no shift could be detected when the corresponding single-stranded oligonucleotides were used (data not shown). Kid-1 belongs to the zinc finger class of transcription factors, and we therefore wanted to know whether other zinc finger proteins and members of other classes of transcription factors also bound to heteroduplex DNA. The POU domain protein Oct-6 (25Suzuki N. Rohdewohld H. Neuman T. Gruss P. Schöler H.R. EMBO J. 1990; 9: 3723-3732Crossref PubMed Scopus (190) Google Scholar) and the ets domain protein Ets-1 (26Sieweke M.H. Tekotte H. Frampton J. Graf T. Cell. 1996; 85: Full Text Full Text PDF PubMed Scopus Google Scholar) bind to homoduplex DNA sequences of nucleotides and nucleotides in respectively. protein, however, bound to heteroduplex DNA when in a gel shift (Fig. The specificity of the shift by the Kid-1 zinc fingers was further by the of another zinc finger the RING finger domain of to bind to heteroduplex DNA (data not shown). The Wilms' tumor protein WT1 also belongs to the family of Cys2His2-zinc finger proteins, it contains four zinc fingers at its COOH terminus. Several naturally occurring splice of WT1 have been described with DNA binding (for a recent see and 1996; Google A binding site selection with the splice variant the between the and zinc finger of WT1 resulted in the isolation of a binding site that that of the zinc finger protein III, F.J. J.F. T. Science. 1990; PubMed Scopus Google Scholar). WT1, which not to the subfamily of KRAB-zinc finger proteins, has been found to as a transcriptional repressor in experiments (e.g. Refs. D.T. Rauscher F.J. T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: PubMed Scopus Google Scholar, J.F. A. Rauscher III, F.J. Science. 1991; PubMed Scopus Google Scholar, J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). When a fusion protein between GST and all four zinc fingers of WT1 was incubated with the a gel shift could be detected only with the splice variant the between the and zinc but not with the (Fig. both and bound to heteroduplex DNA consisting of single-stranded oligonucleotides ss1 and ss2 (Fig. Similar to Kid-1, and also bound to the second large heteroduplex consisting of oligonucleotides ss3 and ss4 (data not shown). The specificity of those gel shifts was by competition In the case of the splice competition was carried out with an excess of both unlabeled heteroduplex DNA and site A and B). The gel shift of the heteroduplex DNA by could only be competed by an excess of unlabeled heteroduplex DNA, but not binding site (Fig. Binding of and to homoduplex DNA consisting of one of the oligonucleotides used in the heteroduplex DNA and a fully complementary oligonucleotide was very weak (data not shown). To define the structural for the binding of WT1 to heteroduplex DNA, we of naturally occurring WT1 proteins. a a of zinc finger D.A. A.J. T. J. D.E. Cell. 1990; Full Text PDF PubMed Scopus Google Scholar) as well as a protein with a in zinc finger found in a with binding to heteroduplex DNA (Fig. This that zinc finger is in this The results described demonstrate for the the binding of a KRAB-zinc finger protein to DNA. it is DNA structure that is recognized by Kid-1, two heteroduplex with of 25 nucleotides are shifted with the same with the that another transcriptional repressor protein, WT1, also binds to heteroduplex DNA sequence this represents an important Although this DNA heteroduplex DNA, the which can be found when RNA polymerase II is a it that the transcription bubble as the natural recognition site. Kid-1 bound only to heteroduplex DNA with a bubble, a that the region of the transcription bubble not by RNA polymerase experiments are to those recognition sites are in the and are recognized in to We are for the of Ets-1 protein and ets-binding site from for the of Oct-6 protein and the octamer binding site from and for the of the plasmid pGEX-KG from We also to for the of a the WT1 The of is The of and in of the fusion proteins during a is highly The of and the with by also are