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• W2084821274 abstract "The Kruppel-associated box (KRAB) domain is a 75-amino acid transcriptional repressor module commonly found in eukaryotic zinc finger proteins. KRAB-mediated gene silencing requires binding to the RING-B box-coiled-coil domain of the corepressor KAP-1. Little is known about the biochemical properties of the KRAB domain or the KRAB·KAP-1 complex. Using purified components, a combination of biochemical and biophysical analyses has revealed that the KRAB domain from the KOX1 protein is predominantly a monomer and that the KAP-1 protein is predominantly a trimer in solution. The analyses of electrophoretic mobility shift assays, GST association assays, and plasmon resonance interaction data have indicated that the KRAB binding to KAP-1 is direct, highly specific, and high affinity. The optical biosensor data for the complex was fitted to a model of a one-binding step interaction with fast association and slow dissociation rates, with a calculated K d of 142 nm. The fitted R max indicated three molecules of KAP-1 binding to one molecule of the KRAB domain, a stoichiometry that is consistent with quantitative SDS-polyacrylamide gel electrophoresis analysis of the complex. These structural and dynamic parameters of the KRAB/KAP-1 interaction have implications for identifying downstream effectors of KAP-1 silencing and the de novo design of new repression domains. The Kruppel-associated box (KRAB) domain is a 75-amino acid transcriptional repressor module commonly found in eukaryotic zinc finger proteins. KRAB-mediated gene silencing requires binding to the RING-B box-coiled-coil domain of the corepressor KAP-1. Little is known about the biochemical properties of the KRAB domain or the KRAB·KAP-1 complex. Using purified components, a combination of biochemical and biophysical analyses has revealed that the KRAB domain from the KOX1 protein is predominantly a monomer and that the KAP-1 protein is predominantly a trimer in solution. The analyses of electrophoretic mobility shift assays, GST association assays, and plasmon resonance interaction data have indicated that the KRAB binding to KAP-1 is direct, highly specific, and high affinity. The optical biosensor data for the complex was fitted to a model of a one-binding step interaction with fast association and slow dissociation rates, with a calculated K d of 142 nm. The fitted R max indicated three molecules of KAP-1 binding to one molecule of the KRAB domain, a stoichiometry that is consistent with quantitative SDS-polyacrylamide gel electrophoresis analysis of the complex. These structural and dynamic parameters of the KRAB/KAP-1 interaction have implications for identifying downstream effectors of KAP-1 silencing and the de novo design of new repression domains. Kruppel-associated box RING finger, B boxes, and coiled-coil region nitrilotriacetic acid polyacrylamide gel electrophoresis electrophoretic mobility shift assay baculovirus zinc finger protein polyoxyethylene 5-octyl ether glutathioneS-transferase HP1 binding domain Transcriptional regulation of gene expression is mediated primarily by DNA sequence-specific transcription factors, which are generally composed of a DNA-binding domain and one or more separable effector domains that either activate or repress transcriptional initiation (1.Tjian R. Maniatis T. Cell. 1994; 77: 5-8Abstract Full Text PDF PubMed Scopus (955) Google Scholar, 2.Maldonado E. Hampsey M. Reinberg D. Cell. 1999; 99: 455-458Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 3.Zawel L. Reinberg D. Annu. Rev. Biochem. 1995; 64: 533-561Crossref PubMed Scopus (391) Google Scholar). Much progress has been made in understanding how activation and repression domains of a DNA-bound protein transmit signals that modulate transcription via the basal transcriptional machinery. Both activation and repression domains may function by directly interacting with components of the basal transcriptional machinery to modulate transcription, or these domains function through cofactors that regulate transcription via a network of protein/protein interactions to regulate downstream targets (for review, see Refs. 2.Maldonado E. Hampsey M. Reinberg D. Cell. 1999; 99: 455-458Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholarand 4.Guarente L. Trends Biochem. Sci. 1995; 20: 517-521Abstract Full Text PDF PubMed Scopus (116) Google Scholar, 5.Torchia J. Glass C. Rosenfeld M.G. Curr. Opin. Cell Biol. 1998; 10: 373-383Crossref PubMed Scopus (515) Google Scholar, 6.Fondell J.D. Roy A.L. Roeder R.G. Genes Dev. 1993; 7: 1400-1410Crossref PubMed Scopus (234) Google Scholar, 7.Fondell J.D. Brunel F. Hisatake K. Roeder R.G. Mol. Cell. Biol. 1996; 16: 281-287Crossref PubMed Google Scholar, 8.Baniahmad A. Leng X. Burris T.P. Tsai S.Y. Tsai M.J. O'Malley B.W. Mol. Cell. Biol. 1995; 15: 76-86Crossref PubMed Google Scholar, 9.Sauer F. Fondell J.D. Ohkuma Y. Roeder R.G. Jackle H. Nature. 1995; 375: 162-164Crossref PubMed Scopus (131) Google Scholar, 10.Knoepfler P.S. Eisenman R.N. Cell. 1999; 99: 447-450Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). The paradigm is now well established for coactivators and corepressors to function as bridging molecules between transcription factors and either the basal transcription apparatus or chromatin components, resulting in the regulation of target genes (11.Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar). Modular transferable repression domains have emerged as a set of highly conserved structural motifs in families of transcription factors. These conserved repression domains include the BTB/POZ, WRPW, SNAG, SCAN, and Kruppel-associated box (KRAB)1 (12.Albagli O. Dhordain P. Deweindt C. Lecocq G. Leprince D. Cell Growth Differ. 1995; 6: 1193-1198PubMed Google Scholar, 13.Bellefroid 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 (345) Google Scholar, 14.Dawson S.R. Turner D.L. Weintraub H. Parkhurst S.M. Mol. Cell. Biol. 1995; 15: 6923-6931Crossref PubMed Scopus (183) Google Scholar, 15.Grimes H.L. Gilks C.B. Chan T.O. Porter S. Tsichlis P.N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14569-14573Crossref PubMed Scopus (97) Google Scholar, 16.Williams A.J. Khachigian L.M. Shows T. Collins T. J. Biol. Chem. 1995; 270: 22143-22152Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). We have focused on the KRAB domain as a model system for the analysis of repression modules (17.Friedman 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 (546) Google Scholar, 18.Margolin J.F. Friedman J.R. Meyer W.K. Vissing H. Theisen H.J. Rauscher III, F.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4509-4513Crossref PubMed Scopus (522) Google Scholar, 19.Peng H. Begg G.E. Schultz D.C. Friedman J.R. Jensen D.E. Speicher D.W. Rauscher III, F.J. J. Mol. Biol. 2000; 295: 1139-1162Crossref PubMed Scopus (162) Google Scholar) (Fig. 1). The KRAB domain was originally identified as a conserved motif at the NH2 terminus of zinc finger proteins (ZFPs) (13.Bellefroid 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 (345) Google Scholar) and was shown to be a potent, DNA binding-dependent transcriptional repression module (18.Margolin J.F. Friedman J.R. Meyer W.K. Vissing H. Theisen H.J. Rauscher III, F.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4509-4513Crossref PubMed Scopus (522) Google Scholar,20.Vissing H. Meyer W.K. Aagaard L. Tommerup N. Thiesen H.J. FEBS Lett. 1995; 369: 153-157Crossref PubMed Scopus (128) Google Scholar, 21.Witzgall R. O'Leary E. Leaf A. Onaldi D. Bonventre J.V. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4514-4518Crossref PubMed Scopus (317) Google Scholar). KRAB-ZFPs have been primarily described in higher vertebrate species, where their functions are largely unknown. Among the estimated 500–700 human Kruppel-type Cys2-His2 ZFPs (22.Klug A. Schwabe J.W. FASEB J. 1995; 9: 597-604Crossref PubMed Scopus (546) Google Scholar), one-third contain KRAB domains (13.Bellefroid 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 (345) Google Scholar). The KRAB domain homology consists of approximately 75 amino acid residues and is predicted to fold into two amphipathic helices that are involved in protein/protein interactions (13.Bellefroid 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 (345) Google Scholar, 23.Rosati M. Marino M. Franze A. Tramontano A. Grimaldi G. Nucleic Acids Res. 1991; 19: 5661-5667Crossref PubMed Scopus (75) Google Scholar). The minimal repression module is approximately 45 amino acid residues, and substitutions for conserved residues abolish repression (18.Margolin J.F. Friedman J.R. Meyer W.K. Vissing H. Theisen H.J. Rauscher III, F.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4509-4513Crossref PubMed Scopus (522) Google Scholar). More than 10 independently encoded KRAB domains have been demonstrated to be potent repressors of transcription, suggesting that this activity is a common property of this domain. Thus, the KRAB-ZFP family represents a large class of transcriptional repressor molecules. KRAB-ZFPs appear to play important regulatory roles during cell differentiation and development. The KRAB-ZFPs ZNF43 and ZNF91 exhibit expression that is mainly restricted to lymphoid cells, suggesting roles as transcriptional regulators specific for lymphoid cell differentiation (24.Bellefroid E.J. Marine J.C. Ried T. Lecocq P.J. Riviere M. Amemiya C. Poncelet D.A. Coulie P.G. de Jong P. Szpirer C. Ward D.C. Martial J.A. EMBO J. 1993; 12: 1363-1374Crossref PubMed Scopus (128) Google Scholar, 25.Lovering R. Trowsdale J. Nucleic Acids Res. 1991; 19: 2921-2928Crossref PubMed Scopus (26) Google Scholar). Other KRAB-ZFPs, such as HPF4, HTF10, and HTF34, are down-regulated during myeloid differentiation (13.Bellefroid 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 (345) Google Scholar). SZF1, a KRAB-ZFP specific to CD34 stem cells, is down-regulated in differentiated hematopoietic derived cell lines (26.Liu C. Levenstein M. Chen J. Tsifrina E. Yonescu R. Griffin C. Civin C.I. Small D. Exp. Hematol. 1999; 27: 313-325Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). A number of KRAB-ZFPs are candidate genes for human diseases based on their chromosomal locations (27.Tommerup N. Aagaard L. Lund C.L. Boel E. Baxendale S. Bates G.P. Lehrach H. Vissing H. Hum. Mol. Genet. 1993; 2: 1571-1575Crossref PubMed Scopus (45) Google Scholar, 28.Crew A.J. Clark J. Fisher C. Gill S. Grimer R. Chand A. Shipley J. Gusterson B.A. Cooper C.S. EMBO J. 1995; 14: 2333-2340Crossref PubMed Scopus (407) Google Scholar). There are more than 40 KRAB-ZFP-encoding genes that have been identified on human chromosome 19p13 and more than 10 KRAB-ZFP genes clustered on chromosome 19q13 (29.Shannon M. Ashworth L.K. Mucenski M.L. Lamerdin J.E. Branscomb E. Stubbs L. Genomics. 1996; 33: 112-120Crossref PubMed Scopus (40) Google Scholar, 30.Bellefroid E.J. Lecocq P.J. Benhida A. Poncelet D.A. Belayew A. Martial J.A. DNA. 1989; 8: 377-387Crossref PubMed Scopus (196) Google Scholar), many of which exhibit hematopoietic specific expression (31.Han Z.G. Zhang Q.H. Ye M. Kan L.X. Gu B.W. He K.L. Shi S.L. Zhou J. Fu G. Mao M. Chen S.J., Yu, L. Chen Z. J. Biol. Chem. 1999; 274: 35741-35748Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Some of these KRAB-ZFPs are selectively expressed in certain leukemia cell lines representing different lineages (31.Han Z.G. Zhang Q.H. Ye M. Kan L.X. Gu B.W. He K.L. Shi S.L. Zhou J. Fu G. Mao M. Chen S.J., Yu, L. Chen Z. J. Biol. Chem. 1999; 274: 35741-35748Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). We hypothesized that KRAB domain repression may be mediated by a common cellular co-factor. We identified and cloned a protein, KAP-1, that binds to the KRAB repression domain using affinity chromatography (17.Friedman 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 (546) Google Scholar). This protein was subsequently identified by other investigators in yeast two-hybrid screens and designated TIF1β and KRIP-1 (32.Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (249) Google Scholar, 33.Kim 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 (249) Google Scholar). KAP-1 is a member of an emerging superfamily of transcriptional co-regulators, including TIF1α and TIF1γ (17.Friedman 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 (546) Google Scholar, 34.Le Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat P. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (576) Google Scholar, 35.Venturini L. You J. Stadler M. Galien R. Lallemand V. Koken M.H. Mattei M.G. Ganser A. Chambon P. Losson R. de The H. Oncogene. 1999; 18: 1209-1217Crossref PubMed Scopus (136) Google Scholar). The TIF1 family encodes the signature RBCC motif that designates the RING-B box-coiled-coil tripartite structure. This motif probably functions as a cooperative protein/protein interaction motif (19.Peng H. Begg G.E. Schultz D.C. Friedman J.R. Jensen D.E. Speicher D.W. Rauscher III, F.J. J. Mol. Biol. 2000; 295: 1139-1162Crossref PubMed Scopus (162) Google Scholar, 36.Saurin A.J. Borden K.L. Boddy M.N. Freemont P.S. Trends Biochem. Sci. 1996; 21: 208-214Abstract Full Text PDF PubMed Scopus (613) Google Scholar, 37.Borden K.L. Biochem. Cell Biol. 1998; 76: 351-358Crossref PubMed Scopus (235) Google Scholar). The definitive element of the tripartite motif is the RING finger, which is found almost exclusively in the NH2-terminal position in RBCC proteins and is likely to contribute specificity and/or multimerization properties to the tripartite motif. Mutational analyses have confirmed the requirement for the RING finger for proper biological function (for reviews, see Refs. 36.Saurin A.J. Borden K.L. Boddy M.N. Freemont P.S. Trends Biochem. Sci. 1996; 21: 208-214Abstract Full Text PDF PubMed Scopus (613) Google Scholar, 37.Borden K.L. Biochem. Cell Biol. 1998; 76: 351-358Crossref PubMed Scopus (235) Google Scholar, 38.Boddy M.N. Duprez E. Borden K.L. Freemont P.S. J. Cell Sci. 1997; 110: 2197-2205Crossref PubMed Google Scholar, 39.Cao T. Borden K.L. Freemont P.S. Etkin L.D. J. Cell Sci. 1997; 110: 1563-1571Crossref PubMed Google Scholar). The second signature motif of the RBCC domain is the B-box (40.Reddy B.A. Etkin L.D. Nucleic Acids Res. 1991; 19: 6330Crossref PubMed Scopus (79) Google Scholar). Two B-box motifs are often found immediately COOH-terminal to the RING finger in the RBCC domain. The third RBCC signature motif is a coiled-coil domain that displays helical amphipathic character and is probably required for homo- or heterotypic interaction. Many biological functions of RBCC proteins have been shown to be dependent on multimerization via this coiled-coil region (36.Saurin A.J. Borden K.L. Boddy M.N. Freemont P.S. Trends Biochem. Sci. 1996; 21: 208-214Abstract Full Text PDF PubMed Scopus (613) Google Scholar). KAP-1 appears to function as a universal corepressor for KRAB domain proteins (17.Friedman 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 (546) Google Scholar). The RBCC domain of KAP-1 is both necessary and sufficient for the KRAB domain binding (19.Peng H. Begg G.E. Schultz D.C. Friedman J.R. Jensen D.E. Speicher D.W. Rauscher III, F.J. J. Mol. Biol. 2000; 295: 1139-1162Crossref PubMed Scopus (162) Google Scholar). Oligomerization of the KAP-1-RBCC is required for binding KRAB domain, and all three components of the tripartite motif appear to cooperate in KRAB recognition. The central region of KAP-1 contains the HP1 binding domain (HP1BD), which directly binds to mammalian homologues of the heterochromatin protein, HP1 (41.Ryan R.F. Schultz D.C. Ayyanathan K. Singh P.B. Friedman J.R. Fredericks W.J. Rauscher III, F.J. Mol. Cell. Biol. 1999; 19: 4366-4378Crossref PubMed Scopus (316) Google Scholar). A stable quaternary complex can be formed between DNA, a KRAB-ZFP, KAP-1, and HP1. The COOH terminus of KAP-1 includes a plant homeo-domain finger and bromodomain, and this region is able to repress transcription when tethered to DNA using a heterologous DNA-binding domain. 2D. Schultz, unpublished data. Thus, KAP-1 is composed of an independent KRAB-recognition domain and at least two independent repression domains. It has been firmly established that KAP-1 is required for KRAB domain-mediated transcriptional repression. The evidence includes the following. 1) KAP-1 binds to multiple KRAB repression domains bothin vitro and in vivo; 2) KRAB domain mutations that abolish repression decrease or eliminate KAP-1 binding; 3) overexpression of KAP-1 enhances KRAB-mediated repression; 4) the KRAB domain does not repress in cells that lack KAP-1. These results support a model in which KRAB-ZFPs repress transcription by recruiting the KAP-1 corepressor via the RBCC domain. To understand the interaction between the KRAB domain and KAP-1 in more detail, we have purified the KRAB domain and employed a comprehensive set of biochemical and biophysical approaches to analyze the complex. The plasmids pQE30 GAL4-KRAB-(1–90), pQE30 KOX1-KRAB, GST-KRAB, GST-KRAB(DV), pQE30 KAP-1-RBCC, and pVL1392 KAP-1-RBCC have been described previously (17.Friedman 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 (546) Google Scholar, 18.Margolin J.F. Friedman J.R. Meyer W.K. Vissing H. Theisen H.J. Rauscher III, F.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4509-4513Crossref PubMed Scopus (522) Google Scholar, 19.Peng H. Begg G.E. Schultz D.C. Friedman J.R. Jensen D.E. Speicher D.W. Rauscher III, F.J. J. Mol. Biol. 2000; 295: 1139-1162Crossref PubMed Scopus (162) Google Scholar). The pQE30 KOX1-(1–161) plasmid was generated via polymerase chain reaction using KOX1 cDNA as a template. A 5′ oligonucleotide, which incorporated a BamHI site 5′ to the initiation methionine, and a 3′ oligonucleotide, which incorporated a stop codon preceding a HindIII site after amino acid 161 of KOX1, were used to amplify the desired sequence. The polymerase chain reaction product was digested and cloned into the corresponding sites of the pQE30 vector (Qiagen). The protein thus contains the NH2-terminal amino acid residues MRGSHHHHHHGS, followed by residues 1–161 of the KOX1 protein. The pQE30 KAP-1-(22–618) plasmid was generated by subcloning an XmaI/XmaI fragment encoding residues 22–618 of human KAP-1 from a pBluescript II SK+ that contained the full-length human KAP-1 cDNA (17.Friedman 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 (546) Google Scholar) into the corresponding sites of pQE30 (Qiagen). The protein contains the NH2-terminal amino acid residues MRGSHHHHHHGSACELGT, followed by residues 22–618 of the KAP-1 protein and then the COOH-terminal sequence GRPAAKLN encoded by the vector. DNA sequencing of both strands confirmed all of the plasmids generated by polymerase chain reaction. The purification of KOX1-KRAB protein was performed at room temperature under denaturing conditions followed by renaturation on the column (42.Shi P.Y. Maizels N. Weiner A.M. BioTechniques. 1997; 23: 1036-1038Crossref PubMed Scopus (38) Google Scholar) as described previously (19.Peng H. Begg G.E. Schultz D.C. Friedman J.R. Jensen D.E. Speicher D.W. Rauscher III, F.J. J. Mol. Biol. 2000; 295: 1139-1162Crossref PubMed Scopus (162) Google Scholar). The KAP-1-RBCC protein expressed from bacterial and baculovirus vectors was purified using nondenaturing conditions as described previously (19.Peng H. Begg G.E. Schultz D.C. Friedman J.R. Jensen D.E. Speicher D.W. Rauscher III, F.J. J. Mol. Biol. 2000; 295: 1139-1162Crossref PubMed Scopus (162) Google Scholar). To reconstitute the complex, the KAP-1-RBCC and the KOX1-(1–161) proteins were first purified under denaturing conditions and then eluted from the Ni2+-NTA-agarose with 300 mmimidazole and pH 4.5. The eluted KOX1-(1–161) and KAP-1-RBCC proteins were then mixed at a 1:1 molar ratio in a volume of 20 ml of buffer containing 8 m urea, 0.1 m sodium phosphate, 0.01 m Tris-HCl, pH 8.0, 10% glycerol, 20 μmZnSO4, and 0.5 mm dithiothreitol. These proteins were then renatured during dialysis by a stepwise 1:1 serial dilution of urea from 4 to 0 m in P300 buffer (10 mm Na2HPO4, 1.4 mmKH2PO4, 2.7 mm KCl, 450 mm NaCl, pH 7.0, 1 mm phenylmethylsulfonyl fluoride) plus 10% glycerol, 20 μm ZnSO4, and 0.5 mm dithiothreitol using five changes of the buffer over a 2-day period. After dialysis, the insoluble protein was removed by centrifugation, and the soluble fraction was concentrated in an Amicon concentrator. The soluble KOX1-KRAB protein derived from on-column renaturation was chromatographed on a Superdex 200 HR 10/30 column equilibrated in P300 buffer plus 10% glycerol and 10 mm polyoxyethylene 5-octyl ether (C8E5) (Sigma). The KAP-1-(22–618) protein and the KOX1-(1–161)·KAP-1-RBCC complex were purified using a Superose 6 HR 10/30 column equilibrated with the P300 buffer plus 10% glycerol. The columns were run at 4 °C at a flow rate of 0.5 ml/min, and 0.5- or 1-ml fractions were collected. Aliquots of each fraction were analyzed for protein content by SDS-PAGE and Coomassie Blue staining. EMSA was performed essentially as described previously (19.Peng H. Begg G.E. Schultz D.C. Friedman J.R. Jensen D.E. Speicher D.W. Rauscher III, F.J. J. Mol. Biol. 2000; 295: 1139-1162Crossref PubMed Scopus (162) Google Scholar, 43.Fredericks W.J. Galili N. Mukhopadhyay S. Rovera G. Bennicelli J. Barr F.G. Rauscher III, F.J. Mol. Cell. Biol. 1995; 15: 1522-1535Crossref PubMed Google Scholar). The purified recombinant GAL4-KRAB was incubated with purified Escherichia coli- or baculovirus-expressed KAP-1-RBCC protein for 15 min at 30 °C. In the competition assays, the KRAB protein was added to the reaction simultaneously with the GAL4-KRAB and KAP-1-RBCC proteins, or the KRAB protein was pre-incubated with the KAP-1-RBCC protein for 15 min at 30 °C. One μl of 32P-labeled GAL4 probe (105 cpm/μl) was then added, and the reaction was incubated for an additional 15 min at 30 °C. The DNA-protein complexes were resolved on native polyacrylamide gels by electrophoresis in 45 mm Tris borate, pH 8.3, 1 mm EDTA buffer at 4 °C. The EMSA gels were dried and visualized by autoradiography. The GAL4 probe was the double-stranded synthetic oligonucleotide 5′-GAT CCC GGA GGA CAG TAC TCC GT-3′, which was labeled with [32P]ATP as described (43.Fredericks W.J. Galili N. Mukhopadhyay S. Rovera G. Bennicelli J. Barr F.G. Rauscher III, F.J. Mol. Cell. Biol. 1995; 15: 1522-1535Crossref PubMed Google Scholar). The CD spectra (190–240 nm) were measured on a Jasco J-720 spectropolarimeter (Japan Spectroscopic Co.) at 25 °C. The CD spectra were recorded using a 100-μl cell containing a 0.2-mm path length. The sample was at a concentration of 1 mg/ml in P300 buffer plus 10% glycerol. Spectra were analyzed using the SOFTSPEC software supplied by the manufacturer. Prior to analytical ultracentrifugation, the proteins were purified by gel filtration. The KOX1-KRAB protein in P300 buffer plus 10% glycerol was incubated with 20 mm C8E5 for 1 h at 4 °C by rotation to attempt to dissociate aggregates before gel filtration. The KOX1-KRAB protein was then subjected to gel filtration in the same buffer containing 10 mm C8E5. The KAP-1-(22–618) protein was purified by gel filtration in P300 buffer plus 10% glycerol without detergent. Sedimentation equilibrium experiments were performed in an Optima XL-I ultracentrifuge, using either the absorbance (280 nm) optics (for KOX1-KRAB) or the interference optics (for KAP-1-(22–618)) to measure the protein concentration gradient. For each experiment, three cells were assembled with 12-mm double-sector centerpieces and quartz or sapphire windows, respectively. The cells were loaded with 110 μl of reference buffer (P300 plus 10% glycerol) or 110 μl of sample at three different protein concentrations. The experiments were performed at 4 °C and using various speeds between 16,000 and 43,400 rpm. The absorbance or fringe displacement data were collected every 6 h until equilibrium was reached, as determined by comparing successive scans using the MATCH program, and the data were edited using the REEDIT program. Analysis of sedimentation equilibrium data was performed using the NONLIN program (44.Johnson M.L. Correia J.J. Yphantis D.A. Halvorson H.R. Biophys. J. 1981; 36: 575-588Abstract Full Text PDF PubMed Scopus (778) Google Scholar). The partial specific volume of the protein was calculated according to Laue et al. (45.Laue T.M. Shah B. Ridgeway T.M. Pelletier S.L. Harding S.E. Rowe A.J. Horton L.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge1992Google Scholar). Three data sets from different loading concentrations were fitted simultaneously. Examination of the residuals and minimization of the variance determined the goodness of fit. The KOX1-(1–161)·KAP-1-RBCC complex, which was formed by co-renaturation as described above was purified by gel filtration, and the peak fraction of the complex was concentrated by deoxycholate-trichloroacetic acid precipitation (46.Peterson G.L. Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7141) Google Scholar). The precipitated proteins were resuspended in 30 μl of 0.1m NaOH and resolved on 10% SDS-PAGE with known amounts of KOX1-(1–161) and KAP-1-RBCC proteins. The proteins were visualized with Coomassie Blue stain and quantitated by densitometry on a MultiImage Light Cabinet instrument (Alpha Innotechnology). The data were analyzed with the program AlphaImager 2000 version 4.03. The preparation of the GST fusion proteins and the GST association assays were performed essentially as described previously (41.Ryan R.F. Schultz D.C. Ayyanathan K. Singh P.B. Friedman J.R. Fredericks W.J. Rauscher III, F.J. Mol. Cell. Biol. 1999; 19: 4366-4378Crossref PubMed Scopus (316) Google Scholar). Briefly, 5 μg of freshly prepared GST fusion protein immobilized on glutathione-Sepharose was incubated with 10 μg of Ni2+-NTA-purified recombinant His6-tagged protein in 100 μl of BB500 buffer (20 mm Tris, pH 7.9, 500 mm NaCl, 0.2 mm EDTA, 10% glycerol, 0.2% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 500 μg of bovine serum albumin (fraction V)) for 1 h at room temperature. The protein complexes were washed four times with BB750 (20 mm Tris, pH 7.9, 750 mm NaCl, 0.2 mm EDTA, 10% glycerol, 0.2% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride), and the bound proteins were eluted in 5× Laemmli buffer, resolved by SDS-PAGE, and visualized with Coomassie Blue stain. Kinetic studies of the direct protein/protein interaction between the KRAB domain and KAP-1 were done using optical biosensors-BIAX and BIA3000 Instruments (Biacore, Inc. Uppsala, Sweden). All experiments were done at 25 °C. The Biacore TM sensor surface CM5 was activated by a 7-min incubation with amine coupling reagent as recommended by the manufacturer. The amine coupling reagents were N-hydroxysuccinimide andN-ethyl-N′-(dimethylaminopropyl)carbodiimide (Biacore, Inc.). One hundred μl of anti-GST polyclonal antibody (Biacore, Inc.) at a concentration of 10–50 μg/ml in 10 mm sodium acetate, pH 5.0, was then injected at a flow rate of 5 μl/min, resulting in 4000 response units being captured. The remaining coupling sites were blocked by injection of 35 μl of ethanolamine-HCl (Biacore, Inc.). After equilibration with P300 buffer plus 10% glycerol, the purified recombinant GST-KRAB and GST-KRAB(DV-AA) proteins were captured via the GST tag and anti-GST antibody. The reference surfaces include both the anti-GST antibody surface and the captured GST-KRAB(DV-AA) surface, respectively. The GST-KRAB(DV-AA) surface displayed the same density as GST-KRAB. The purpose of two reference surfaces is to normalize the refractive index changes between reference and specific sensor surfaces and to measure the contribution of the anti-GST antibody to the total signal, respectively. The experimental GST-KRAB surface was 100 response units. A 300-μl sample of KAP-1-(22–618) protein at concentrations of 530 nm to 4 μm in P300 buffer plus 10% glycerol was added to the GST-KRAB and GST-KRAB(DV) surfaces and allowed to bind for 5 min. The flow cells then were washed with P300 buffer plus 10% glycerol. The binding surface of GST-KRAB was regenerated with injection of 0.2% SDS solution in 1% Me2SO and 15 mm of NaOH, pH 9.5, in two or three pulses by previously described methods (47.Andersson K. Hamalainen M. Malmqvist M. Biasymposium ‘98. Biacore, Edinburgh1998Google Scholar). The sample loop was then washed with P300 buffer plus 10% glycerol after regeneration steps to avoid “carry-over” of bulk signal. The data reported were obtained from two independent experiments. The data were collected automatically and analyzed subsequently with BIAevaluation" @default.
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• W2084821274 title "Biochemical Analysis of the Kruppel-associated Box (KRAB) Transcriptional Repression Domain" @default.
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