Purification and Characterization of a Protein Binding to the SP6 κ Promoter
1998; Elsevier BV; Volume: 273; Issue: 30 Linguagem: Inglês
10.1074/jbc.273.30.18881
ISSN1083-351X
AutoresMats Bemark, Henric Olsson, Dick Heinegård, Tomas Leanderson,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoA protein interacting with an A-T-rich region that is a positive control element within the SP6 κ promoter was purified and identified as CArG-box binding factor-A. The purified protein was shown to interact specifically with the coding strand of single-stranded DNA and, with lower affinity, with double-stranded DNA. A mutation that inhibited binding of the protein to the A-T-rich region also aborted the transcriptional stimulatory effect of the region. Two Ets proteins, PU.1 and elf-1, that have previously been shown to bind to an adjacent DNA element were shown to physically interact with CArG-box binding factor-A. An antiserum raised against the protein recognized two different forms indicating either that different splice-forms of CArG-box binding factor-A are expressed, or that the protein is subject to post-translational modification. A protein interacting with an A-T-rich region that is a positive control element within the SP6 κ promoter was purified and identified as CArG-box binding factor-A. The purified protein was shown to interact specifically with the coding strand of single-stranded DNA and, with lower affinity, with double-stranded DNA. A mutation that inhibited binding of the protein to the A-T-rich region also aborted the transcriptional stimulatory effect of the region. Two Ets proteins, PU.1 and elf-1, that have previously been shown to bind to an adjacent DNA element were shown to physically interact with CArG-box binding factor-A. An antiserum raised against the protein recognized two different forms indicating either that different splice-forms of CArG-box binding factor-A are expressed, or that the protein is subject to post-translational modification. The control of transcriptional initiation in eucaryotes is a multilayered process. At one end of the spectrum are the very specific interactions between distinct sequence motifs and defined transcription factors that can interact with a specific sequence only. Secondary protein-protein interactions between such DNA-binding factors and so-called transcriptional adapter molecules also show molecular specificity (1Tansey W.P. Herr W. Cell. 1997; 88: 729-732Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 2Strubin M. Newell J.W. Matthias P. Cell. 1995; 80: 497-506Abstract Full Text PDF PubMed Scopus (350) Google Scholar, 3Lou Y. Roeder R.G. Mol. Cell. Biol. 1995; 15: 4115-4124Crossref PubMed Scopus (65) Google Scholar, 4Gstaiger M. Knoepfel L. Georgiev O. Schaffner W. Hovens C.M. Nature. 1995; 373: 360-362Crossref PubMed Scopus (287) Google Scholar) and can easily be envisioned to have a defined role in the control of expression of a given gene. The mechanism of action and the specificity of other control elements pertinent to gene expression are of more general nature. Here, one may distinguish between control mechanisms of gene expression from a given locus and those that are acting downstream of this decisive event to facilitate efficient transcriptional initiation and elongation induced by the sequence-specific transcription factors mentioned above. Among the former are locus control regions (5Davie J.R. Int. Rev. Cytol. 1995; 162A: 191-250PubMed Google Scholar), matrix attachment regions (6Boulikas T. J. Cell. Biochem. 1993; 52: 14-22Crossref PubMed Scopus (173) Google Scholar), histone acetylation (7Wolffe A.P. Science. 1996; 272: 371-372Crossref PubMed Scopus (277) Google Scholar), and the action of the SWI/SNF complex (8Pazin M.J. Kadonaga J.T. Cell. 1997; 88: 737-740Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). The latter include specific DNA bending proteins (9Werner M.H. Burley S.K. 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Several of these single-stranded DNA-binding proteins are identical or related to proteins that are found within the heterogeneous nuclear ribonucleoprotein (hnRNP) 1The abbreviations used are: hnRNP, heterogeneous nuclear ribonucleoprotein; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; pd, pentadecamer; pdLMW, pd element low molecular weight shift; pdMMW, pd element medium molecular weight shift. complex (11Duncan R. Bazar L. Michelotti G. Tomonaga T. Krutzsch H. Avigan M. Levens M. Genes Dev. 1994; 8: 465-480Crossref PubMed Scopus (292) Google Scholar, 12Tomonaga T. Levens D. J. Biol. Chem. 1995; 270: 4875-4881Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar,23Kamada S. Miwa T. Gene ( Amst. ). 1992; 119: 229-236Crossref PubMed Scopus (86) Google Scholar, 24Tay N. Chan S.-H. Ren E.-C. J. 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Although each rearranged gene has a distinct promoter, distal enhancers are found in introns and 3′ of the genes (32Nelsen B. Sen R. Int. Rev. Cytol. 1992; 133: 121-149Crossref PubMed Scopus (39) Google Scholar). Within these enhancers, a variety of DNA elements involved in the transcriptional control have been defined (32Nelsen B. Sen R. Int. Rev. Cytol. 1992; 133: 121-149Crossref PubMed Scopus (39) Google Scholar). Furthermore, close to the intron enhancers, matrix attachment regions are found, and recent evidence indicates that these function as locus control regions (33Forrester W.C. van Genderen C. Jenuwein T. Grosschedl R. Science. 1994; 265: 1221-1225Crossref PubMed Scopus (210) Google Scholar, 34Jenuwein T. Forrester W.C. Fernández-Herrero L.A. Laible G. Dull M. Grosschedl R. Nature. 1997; 385: 269-272Crossref PubMed Scopus (226) Google Scholar). Ig κ promoters show sequence conservation within but not between V gene subgroups (35Schäble K.F. Zachau H.-G. Biol. Chem. Hoppe-Seyler. 1993; 374: 1001-1022Crossref PubMed Scopus (140) Google Scholar, 36Bemark M. Liberg D. Leanderson T. Immunogenetics. 1998; 47: 183-195Crossref PubMed Scopus (27) Google Scholar). Detailed functional studies have shown that κ promoters contain several DNA elements involved in transcriptional regulation (37Sigvardsson M. Bemark M. Leanderson T. Eur. J. Immunol. 1995; 25: 298-301Crossref PubMed Scopus (17) Google Scholar, 38Falkner F.G. Zachau H.G. Nature. 1984; 310: 71-74Crossref PubMed Scopus (437) Google Scholar, 39Högbom E. Magnusson A.-C. Lenderson T. Nucleic Acids Res. 1991; 19: 4347-4354Crossref PubMed Scopus (24) Google Scholar, 40Sigvardsson M. Leanderson T. Mol. Immunol. 1994; 31: 1005-1016Crossref PubMed Scopus (13) Google Scholar, 41Sigvardsson M. Bemark M. Leanderson T. Mol. Cell. Biol. 1995; 15: 1343-1352Crossref PubMed Google Scholar, 42Sigvardsson M. Åkerblad P. Leanderson T. J. Immunol. 1996; 156: 3788-3796PubMed Google Scholar, 43Bemark M. Leanderson T. Eur. J. Immunol. 1997; 27: 1308-1318Crossref PubMed Scopus (12) Google Scholar, 44Atchison M.L. Delmas V. Perry R., P. EMBO J. 1990; 9: 3109-3117Crossref PubMed Scopus (40) Google Scholar, 45Schwarzenbach H. Newell J.W. Matthias P. J. Biol. Chem. 1995; 270: 898-907Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The octamer, which is a binding site for the ubiquitous OCT1 and the B cell-specific OCT2 transcription factors, is a key control element in κ promoters and when mutated the promoter is inactivated (37Sigvardsson M. Bemark M. Leanderson T. Eur. J. Immunol. 1995; 25: 298-301Crossref PubMed Scopus (17) Google Scholar). However, the promoter is dependent on other, octamer-dependent, transcriptional control elements for proper function (37Sigvardsson M. Bemark M. Leanderson T. Eur. J. Immunol. 1995; 25: 298-301Crossref PubMed Scopus (17) Google Scholar). Such elements have been identified both 5′ and 3′ of the octamer and examples thereof are the κ-Y element (44Atchison M.L. Delmas V. Perry R., P. EMBO J. 1990; 9: 3109-3117Crossref PubMed Scopus (40) Google Scholar), the pentadecamer (pd) element containing an E-box of the E2A type (-CAGNTG-) (38Falkner F.G. Zachau H.G. Nature. 1984; 310: 71-74Crossref PubMed Scopus (437) Google Scholar, 41Sigvardsson M. Bemark M. Leanderson T. Mol. Cell. Biol. 1995; 15: 1343-1352Crossref PubMed Google Scholar), and the CCCT element (39Högbom E. Magnusson A.-C. Lenderson T. Nucleic Acids Res. 1991; 19: 4347-4354Crossref PubMed Scopus (24) Google Scholar, 42Sigvardsson M. Åkerblad P. Leanderson T. J. Immunol. 1996; 156: 3788-3796PubMed Google Scholar). All three elements interact with distinct proteins in electrophoretic mobility shift assay (EMSA) and interact functionally with the octamer (42Sigvardsson M. Åkerblad P. Leanderson T. J. Immunol. 1996; 156: 3788-3796PubMed Google Scholar, 43Bemark M. Leanderson T. Eur. J. Immunol. 1997; 27: 1308-1318Crossref PubMed Scopus (12) Google Scholar). So far, only the proteins that interact with the κ-Y element have been identified, PU.1 and elf-1, both of which belong to the Ets family of transcription factors (43Bemark M. Leanderson T. Eur. J. Immunol. 1997; 27: 1308-1318Crossref PubMed Scopus (12) Google Scholar, 45Schwarzenbach H. Newell J.W. Matthias P. J. Biol. Chem. 1995; 270: 898-907Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In this study, we purified a protein that interacts with a functionally important A-T-rich region within the SP6 κ promoter pd element and identified it as CArG-box binding factor-A (23Kamada S. Miwa T. Gene ( Amst. ). 1992; 119: 229-236Crossref PubMed Scopus (86) Google Scholar). J558 plasmacytoma cells were grown in large cell culture flasks that were harvested every 2–3 days, and nuclear extracts were prepared according to a modification of the Dignam method using buffer C′ (46Dignam J.D. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9684) Google Scholar, 47Dignam J.D. Methods Enzymol. 1990; 182: 194-203Crossref PubMed Scopus (226) Google Scholar). The nuclear extracts were pooled and precipitated sequentially with increasing amounts of ammonium sulfate, and the pellets were dissolved in buffer Z (25 mm HEPES (pH 7.6), 100 mm KCl, 12.5 mm MgCl2, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 20% glycerol, and 0.1% Nonidet P-40) (48Kadonaga J.T. Methods Enzymol. 1991; 208: 10-23Crossref PubMed Scopus (99) Google Scholar). The material that precipitated between 70 and 90% ammonium sulfate saturation was dialyzed against buffer Z and was loaded onto a native DNA cellulose column (Amersham Pharmacia Biotech) equilibrated in buffer Z. After washing, bound proteins were eluted stepwise in 100 mm steps up to 1 m KCl in buffer Z. Fractions containing the pdLMW binding activity (41Sigvardsson M. Bemark M. Leanderson T. Mol. Cell. Biol. 1995; 15: 1343-1352Crossref PubMed Google Scholar) (500 mm and 600 mm KCl) were pooled and dialyzed against buffer A (20 mm Tris (pH 8.0), 25 mmKCl, 5 mm MgCl2, 1 mmdithiothreitol, and 0.1 mm phenylmethylsulfonyl fluoride) and loaded onto a MonoQ™ (Amersham Pharmacia Biotech) column equilibrated in buffer A. After washing with buffer A, bound proteins were eluted with a continuos gradient of buffer B (as A but 500 mm KCl). SDS-PAGE and electroblotting were performed according to standard procedures (49Sambrook L. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar), and blotted proteins were immunodetected by chemiluminescence (50Nesbitt S.A. Horton M.A. Anal. Biochem. 1992; 206: 267-272Crossref PubMed Scopus (59) Google Scholar). For blocking of the antisera, 3 μl of protein G purified sera was preincubated with 50 μg of recombinant protein for 30 min on ice before it was added to the membranes. For amino acid sequencing, the protein band was identified in the gel after Coomassie Blue staining, excised, and sent to SLU (Bo Ek, Uppsala, Sweden) for trypsin digestion, peptide purification by reversed phase high performance liquid chromatography, and amino acid sequencing. Northern blots were performed according to standard procedures using 0.5 μg of poly(A) RNA per lane (49Sambrook L. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The RNA was purified using a QuickPrep™ micro mRNA purification kit (Amersham Pharmacia Biotech). CArG-box binding factor-A was cloned from a HybriZAP cDNA library made from murine B cells that had been stimulated with lipopolysaccharide for 72 h, according to the manufacturer's instructions (Stratagene), using an end-labeled oligonucleotide (5′-ATCAAGGTTGCCCAGCCCAAAGAG-3′) according to standard procedures (49Sambrook L. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). A clone that contained the complete 5′ sequence but lacking a part of the 3′ untranslated region was cloned (23Kamada S. Miwa T. Gene ( Amst. ). 1992; 119: 229-236Crossref PubMed Scopus (86) Google Scholar). The cDNA was cut with NcoI, blunt-ended, and then cut with SacI and inserted between theSmaI and the SacI site in the pGEM3Z plasmid (Promega). This plasmid was completely digested with EcoRI and then partially digested with BamHI, and the CArG-box binding factor-A fragment derived in this manner was inserted in frame with the glutathione S-transferase gene between theBamHI and the EcoRI sites in the pGEX-2 vector (Amersham Pharmacia Biotech). The protein was expressed and purified on glutathione-Sepharose according to the manufacturer's instructions (Amersham Pharmacia Biotech), whereafter it was further purified on a MonoQ™ column (Amersham Pharmacia Biotech). The protein was cleaved by incubating with 0.5 units of thrombin (Sigma) in 200 μl of digestion buffer (20 mm Tris (pH 8.0), 150 mmNaCl, 2.5 mm CaCl2, and 10% glycerol) at room temperature for 2–3 h. The polyclonal antiserum was raised in rabbits according to standard procedures using the GST-CArG-box binding factor-A fusion protein. The IgG fraction was purified on a Protein G HiTrap™ column (Amersham Pharmacia Biotech) according to the manufacturer's instructions and dialyzed against phosphate-buffered saline. Variants 3, 7, 8, 9, and 10 were generated by PCR (15 cycles) using primers 1 and 5, 2 and 5, 3 and 5, 2 and 4, 3 and 4, respectively, and the cloned CBF-A cDNA as a template. Variants 4, 5, and 6 were generated by primary PCRs using primer 5 and 6, 7, or 8, respectively, or primer 1 and 4 for 15 cycles to generate fragment 1–4. The fragments were gel-purified, pre-PCR in the absence of primers was performed with fragment 4 and 1, 2, or 3 for 20 cycles, new Taq enzyme and primers 1 and 5 were added to the reactions, and 15 cycles more were performed. After digestion withBglII and EcoRI, the fragments were then cloned between the BamHI and EcoRI sites in the GST vector pGEX-2 (Amersham Pharmacia Biotech). All constructs were verified by sequencing of the first 250 base pairs using the pGEX 5′ sequencing primer (Amersham Pharmacia Biotech). The sequence of the primers used were: 1) 5′-GAGAGATCTCCGAGCGGGAACCAGA-3′; 2) 5′-GAGAGATCTGCGGGAAAAATGTTCGTT-3′; 3) 5′-GCTAGATCTATGAAGAAGGACCCTGTG 3′; 4) 5′-CCTCTGAATTCTAGGCAACCTTGATTTC 3′; 5) 5′-TCCGAATTCCTCTCAGTATGGCTTGTA-3′; 6) 5′-CGCCAGCAAGAACGAGGAGGACGCGGGAAAAATCTTTGTGGGAGGTCTAAACCCTG-3′; 7) 5′-GGCTATGGCTATGAAGAAGGACCCTGTGCAGCCCAAAGAGGTGTATCAGCAACAGC-3′; 8) 5′-CGCCAGCAAGAACGAGGAGGACGCGGGACAGCCCAAAGAGGTGTATCAGCAACAGC-3′. The deletion mutants were expressed in bacteria and purified on glutathione beads as described by the manufacturer (Amersham Pharmacia Biotech), and subsequently dialyzed against phosphate-buffered saline for 8 h with one buffer exchange. The amounts of the different variants were estimated by A 280 measurement. EMSAs, transfections, and CAT assays were performed as described (43Bemark M. Leanderson T. Eur. J. Immunol. 1997; 27: 1308-1318Crossref PubMed Scopus (12) Google Scholar), but for the EMSAs single-stranded probe was used unless stated different in the figure legends. Preimmune or immune serum was added before the addition of the probe, and the reactions were then preincubated for 30 min at room temperature. The sequences of the probes can be found in the figures. For the EMSA in Fig. 5 B, the promoter region of the SP6 κ gene was cloned between the PstI and the HindIII sites of the pGEM3Z vector (Promega). An end-labeled promoter probe was generated by Klenow fill-in labeling after XbaI digestion followed by digestion with HindIII. Native PAGE purified probe in bind buffer was denatured at 95 °C for 5 min and then rapidly cooled on ice. 6000 cpm of native or denatured probe was added to 25 μl of footprint binding buffer with different amounts of protein as indicated. The reactions were incubated for 5 min at 37 °C, whereafter an aliquot of 10 μl was separated on a 5% native PAGE gel in 1× TBE. Stop buffer (0.67% SDS, 30% glycerol, 0.3 mg/ml tRNA, 0.3 mg/ml proteinase K, and 0.3% bromphenol blue) (51Pontius B.W. Berg P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8403-8407Crossref PubMed Scopus (129) Google Scholar) was then added, and the reaction was continued for 5 min. The reactions were then separated by native PAGE as above. The footprint assays were performed according to standard procedures, and single-stranded or double-stranded probes (20 000 cpm) were used as indicated. After preincubation for 30 min in 50 μl of bind buffer (10 mm HEPES (pH 7.9), 50 mm KCl, 5 mmMgCl2, 0.5 mm EDTA, 0.5 mmspermidine, 0.5 mm dithiothreitol, and 10% glycerol) on ice, 50 μl of 5 mm CaCl2 and 10 mm MgCl2 was added and finally 0.25 μg of DNase was added. After incubation for 1 min the reaction was stopped by the addition of 100 μl of DNase stop buffer (200 mm NaCl, 20 mm EDTA, 1% SDS, 250 μg/ml yeast tRNA), whereafter the reaction was phenol-extracted and ethanol-precipitated. The reactions were separated on 20% denaturing PAGE gels. The ladders were made according to the rapid protocol for G/A Maxam-Gilbert sequencing (49Sambrook L. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The methylation interference assay was performed essentially as described in Ref. 41Sigvardsson M. Bemark M. Leanderson T. Mol. Cell. Biol. 1995; 15: 1343-1352Crossref PubMed Google Scholar, although a single-stranded probe was used and that the probe was gel-purified after methylation. Recombinant thrombin-cut CBF was used in EMSA with methylated probe (100,000 cpm), and about 50% of the probe was shifted. The bound and free probe was identified after autoradiography, excised and eluted over night. The reaction was separated on a 15% denaturing PAGE gel. Recombinant GST or GST/CArG-box binding factor fusion protein was bound to glutathione-Sepharose (Amersham Pharmacia Biotech) in NETN buffer (20 mm Tris (pH 8.0), 100 mm NaCl, 1 mmEDTA, 0.5% Nonidet P-40, and 0.1% bovine serum albumin), and the beads were then washed in NETN. After incubating 10 μl of beads with 1 μl of in vitro translated protein (43Bemark M. Leanderson T. Eur. J. Immunol. 1997; 27: 1308-1318Crossref PubMed Scopus (12) Google Scholar) labeled with [35S]methionine (Amersham Pharmacia Biotech) in 300 μl of NETN for 2 h at 4 °C, the beads were washed five times in NETN at room temperature, 10 μl of SDS load was added to the beads, and bound proteins were separated on a 10% SDS-PAGE after heating to 95 °C. The gels were either fixed (45% methanol and 10% HAc) and dried or transferred to nitrocellulose filters, and then autoradiographed. The pull-downs using the different deletion mutants were made as described above, and 0.006 OD units were loaded of each mutant to 10 μl of beads as described above. Quantifications were made in a phosphorimager (Fuji Bio-Imaging Analyzer BAS2000). The region 5′ of the octamer in the murine SP6 κ promoter has two sequence elements, the pd and κ-Y elements, that stimulate transcription by synergistic interactions with the octamer (41Sigvardsson M. Bemark M. Leanderson T. Mol. Cell. Biol. 1995; 15: 1343-1352Crossref PubMed Google Scholar, 43Bemark M. Leanderson T. Eur. J. Immunol. 1997; 27: 1308-1318Crossref PubMed Scopus (12) Google Scholar, 44Atchison M.L. Delmas V. Perry R., P. EMBO J. 1990; 9: 3109-3117Crossref PubMed Scopus (40) Google Scholar, 45Schwarzenbach H. Newell J.W. Matthias P. J. Biol. Chem. 1995; 270: 898-907Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The pd element can be further divided into two sites, a 5′ E-box of the E2A type and a 3′ A-T-rich region (41Sigvardsson M. Bemark M. Leanderson T. Mol. Cell. Biol. 1995; 15: 1343-1352Crossref PubMed Google Scholar). The individual function of these three sites was tested by transient transfections of CAT reporter constructs into lipopolysaccharide-stimulated B cells (41Sigvardsson M. Bemark M. Leanderson T. Mol. Cell. Biol. 1995; 15: 1343-1352Crossref PubMed Google Scholar). A promoter fragment containing the pd, the κ-Y, and the octamer element stimulated transcriptional initiation when placed upstream from a TATA-box, as compared with the TATA-box alone (Fig. 1 A; compare the first two lanes). When the octamer was mutated only limited transcriptional stimulation was observed (third lane); this confirmed earlier studies showing that the octamer is obligate for promoter function (37Sigvardsson M. Bemark M. Leanderson T. Eur. J. Immunol. 1995; 25: 298-301Crossref PubMed Scopus (17) Google Scholar). The individual function of the three sites 5′ of the octamer was tested by introducing mutations in the reporter construct. Deletion of the 5′ E-box of the pd element, or mutations in either the A-T-rich region or the κ-Y element all diminished the costimulatory function (fourth through sixth lanes). Thus, the interdependence of the three 5′ elements for octamer costimulation was confirmed (43Bemark M. Leanderson T. Eur. J. Immunol. 1997; 27: 1308-1318Crossref PubMed Scopus (12) Google Scholar). Next, the pd element was used as probe in an EMSA with nuclear extract from the plasmacytoma cell line J558L as protein source (Fig. 1 B). As described previously, two complexes were formed (pdMMW and pdLMW; Ref. 41Sigvardsson M. Bemark M. Leanderson T. Mol. Cell. Biol. 1995; 15: 1343-1352Crossref PubMed Google Scholar). When the A-T-rich region was mutated, no pdLMW complex was formed while the pdMMW complex was unaffected. A mutation of the E-box did not affect the pdLMW complex but the pdMMW complex was reduced in intensity. The binding of the pdMMW complex merits some comment, as it may seem to be in contrast to an earlier report where mutations of the E-box disrupted binding (41Sigvardsson M. Bemark M. Leanderson T. Mol. Cell. Biol. 1995; 15: 1343-1352Crossref PubMed Google Scholar). However, different methods were used to generate the nuclear extracts; the method of Schreiber et al. (52Schreiber E. Matthias P. Müller M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3968) Google Scholar) was used in the earlier report, whereas a modification of the Dignam method (47Dignam J.D. Methods Enzymol. 1990; 182: 194-203Crossref PubMed Scopus (226) Google Scholar) was used here. Thus, the pdMMW shift here appears to be composed of two overlapping complexes: a specific E-box binding complex that does not bind to the mutated E-box and a nonspecific binding complex that also interacts with single-stranded DNA (data not shown). To identify the A-T region interacting protein, we developed a three-step method that was used for purification (Fig. 2 A). Nuclear extract was made from the J558L plasmacytoma cell line according to a modification of the original Dignam method (46Dignam J.D. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9684) Google Scholar, 47Dignam J.D. Methods Enzymol. 1990; 182: 194-203Crossref PubMed Scopus (226) Google Scholar), and the extract was sequentially precipitated with ammonium sulfate in 10% steps. The pdLMW activity precipitated mainly in the 70–80% and 80–90% ammonium sulfate saturation fractions, while the pdMMW activity (and most other transcription factors) precipitated at concentrations below 40% saturation (data not shown). The 70–80% and 80–90% fractions were pooled and loaded onto a native DNA cellulose column, and while most contaminating proteins passed through the column at 100 mm salt concentration, the pdLMW activity was eluted in the 500 and 600 mm fractions (data not shown). These two fractions were pooled, loaded onto a MonoQ™ ion exchange column, and eluted with a continuos gradient of KCl. Every second fraction was tested for protein content by SDS-PAGE and for pdLMW binding activity in EMSA (Fig. 2 B, left panels). The activity eluted in one peak in fractions 24–26, as did an approximately 35-kDa protein, a molecular mass in agreement with a preliminary characterization of the pdLMW activity (41Sigvardsson M. Bemark M. Leanderson T. Mol. Cell. Biol. 1995; 15: 1343-1352Crossref PubMed Google Scholar). A contaminating protein of higher molecular mass eluted earlier than and overlapping with the 35-kDa protein. The purification steps were monitored by SDS-PAGE (Fig. 2 B, right panels). Although some contaminating proteins were evident after the DNA-cellulose step, only the higher molecular mass protein contaminated the purified protein after the MonoQ™ step. Hence, we conclude that a 35-kDa protein co-purified with the pdLMW forming activity during the three purification steps. To identify the protein, fraction 25 was acetone-precipitated and separated on SDS-PAGE. After staining with Coomassie Blue to identify the position of the protein, the band was
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