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• W1986365874 abstract "The major core promoter-binding factor in polymerase II transcription machinery is TFIID, a complex consisting of TBP, the TATA box-binding protein, and 13 to 14 TBP-associated factors (TAFs). Previously we found that the histone H2A-like TAF paralogs TAF4 and TAF4b possess DNA-binding activity. Whether TAF4/TAF4b DNA binding directs TFIID to a specific core promoter element or facilitates TFIID binding to established core promoter elements is not known. Here we analyzed the mode of TAF4b·TAF12 DNA binding and show that this complex binds DNA with high affinity. The DNA length required for optimal binding is ∼70 bp. Although the complex displays a weak sequence preference, the nucleotide composition is less important than the length of the DNA for high affinity binding. Comparative expression profiling of wild-type and a DNA-binding mutant of TAF4 revealed common core promoter features in the down-regulated genes that include a TATA-box and an Initiator. Further examination of the PEL98 gene from this group showed diminished Initiator activity and TFIID occupancy in TAF4 DNA-binding mutant cells. These findings suggest that DNA binding by TAF4/4b-TAF12 facilitates the association of TFIID with the core promoter of a subset of genes. The major core promoter-binding factor in polymerase II transcription machinery is TFIID, a complex consisting of TBP, the TATA box-binding protein, and 13 to 14 TBP-associated factors (TAFs). Previously we found that the histone H2A-like TAF paralogs TAF4 and TAF4b possess DNA-binding activity. Whether TAF4/TAF4b DNA binding directs TFIID to a specific core promoter element or facilitates TFIID binding to established core promoter elements is not known. Here we analyzed the mode of TAF4b·TAF12 DNA binding and show that this complex binds DNA with high affinity. The DNA length required for optimal binding is ∼70 bp. Although the complex displays a weak sequence preference, the nucleotide composition is less important than the length of the DNA for high affinity binding. Comparative expression profiling of wild-type and a DNA-binding mutant of TAF4 revealed common core promoter features in the down-regulated genes that include a TATA-box and an Initiator. Further examination of the PEL98 gene from this group showed diminished Initiator activity and TFIID occupancy in TAF4 DNA-binding mutant cells. These findings suggest that DNA binding by TAF4/4b-TAF12 facilitates the association of TFIID with the core promoter of a subset of genes. Two types of DNA elements regulate transcription of protein-encoding genes in eukaryotes. Enhancer elements, which may be localized proximally or distally relative to the transcription initiation site, are the binding sites for gene-specific transcription factors. A core promoter, situated close to the transcription start site (TSS), 2The abbreviations used are: TSStranscription start siteTBPTATA-binding proteinTAFTBP-associated factorHFDhistone-fold domainAdMLadenovirus major lateEMSAelectrophoretic mobility shift assayHAhemagglutininChIPchromatin immunoprecipitation. 2The abbreviations used are: TSStranscription start siteTBPTATA-binding proteinTAFTBP-associated factorHFDhistone-fold domainAdMLadenovirus major lateEMSAelectrophoretic mobility shift assayHAhemagglutininChIPchromatin immunoprecipitation. serves as the site on which RNA polymerase II and the general transcription factors bind and assemble into a pre-initiation complex (1Juven-Gershon T. Hsu J.Y. Kadonaga J.T. Biochem. Soc. Trans. 2006; 34: 1047-1050Crossref PubMed Scopus (62) Google Scholar, 2Smale S.T. Genes Dev. 2001; 15: 2503-2508Crossref PubMed Scopus (194) Google Scholar). Enhancer-bound transcription factors activate transcription by modulating chromatin structure or by recruiting the transcription machinery to the core promoter. transcription start site TATA-binding protein TBP-associated factor histone-fold domain adenovirus major late electrophoretic mobility shift assay hemagglutinin chromatin immunoprecipitation. transcription start site TATA-binding protein TBP-associated factor histone-fold domain adenovirus major late electrophoretic mobility shift assay hemagglutinin chromatin immunoprecipitation. The major core promoter-binding factor within the general transcription apparatus is TFIID, a large complex composed of the TATA-binding protein (TBP) and about 14 TBP-associated factors (TAFs) (for recent reviews see Refs. 3Smale S.T. Kadonaga J.T. Annu. Rev. Biochem. 2003; 72: 449-479Crossref PubMed Scopus (780) Google Scholar, 4Matangkasombut O. Auty R. Buratowski S. Adv. Protein Chem. 2004; 67: 67-92Crossref PubMed Scopus (37) Google Scholar). Within TFIID TBP is responsible for recognition and binding of TATA-containing promoters. The TAFs are also important for core promoter recognition, and they bind primarily to non-TATA-box elements, interacting with sequences upstream and downstream to the TATA box (5Sawadogo M. Roeder R.G. Cell. 1985; 43: 165-175Abstract Full Text PDF PubMed Scopus (715) Google Scholar, 6Nakatani Y. Horikoshi M. Brenner M. Yamamoto T. Besnard F. Roeder R.G. Freese E. Nature. 1990; 348: 86-88Crossref PubMed Scopus (81) Google Scholar, 7Kaufmann J. Smale S.T. Genes Dev. 1994; 8: 821-829Crossref PubMed Scopus (212) Google Scholar, 8Oelgeschlager T. Chiang C.M. Roeder R.G. Nature. 1996; 382: 735-738Crossref PubMed Scopus (149) Google Scholar, 9Verrijzer C.P. Tjian R. Trends Biochem. Sci. 1996; 21: 338-342Crossref PubMed Scopus (318) Google Scholar, 10Emanuel P.A. Gilmour D.S. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 8449-8453Crossref PubMed Scopus (40) Google Scholar, 11Wang J.C. Van Dyke M.W. Biochim. Biophys. Acta. 1993; 1216: 73-80Crossref PubMed Scopus (32) Google Scholar, 12Purnell B.A. Emanuel P.A. Gilmour D.S. Genes Dev. 1994; 8: 830-842Crossref PubMed Scopus (172) Google Scholar, 13Zhou Q. Lieberman P.M. Boyer T.G. Berk A.J. Genes Dev. 1992; 6: 1964-1974Crossref PubMed Scopus (288) Google Scholar). In addition certain TAF sub-complexes have been reported to specifically bind different core promoter elements. The TAF1·TAF2 complex binds to the Initiator element (14Chalkley G.E. Verrijzer C.P. EMBO J. 1999; 18: 4835-4845Crossref PubMed Scopus (175) Google Scholar) and Drosophila TAF6 and TAF9 cross-linked to the downstream promoter element in the context of TFIID (15Burke T.W. Kadonaga J.T. Genes Dev. 1997; 11: 3020-3031Crossref PubMed Scopus (395) Google Scholar), and, as a reconstituted complex, these were shown to associate with a downstream promoter element-containing promoter (16Shao H. Revach M. Moshonov S. Tzuman Y. Gazit K. Albeck S. Unger T. Dikstein R. Mol. Cell. Biol. 2005; 25: 206-219Crossref PubMed Scopus (52) Google Scholar). A feature common to 9 of the 14 TAFs is the histone-fold domain (HFD) (17Hoffmann A. Chiang C.M. Oelgeschlager T. Xie X. Burley S.K. Nakatani Y. Roeder R.G. Nature. 1996; 380: 356-359Crossref PubMed Scopus (159) Google Scholar, 18Selleck W. Howley R. Fang Q. Podolny V. Fried M.G. Buratowski S. Tan S. Nat. Struct. Biol. 2001; 8: 695-700Crossref PubMed Scopus (61) Google Scholar, 19Werten S. Mitschler A. Romier C. Gangloff Y.G. Thuault S. Davidson I. Moras D. J. Biol. Chem. 2002; 277: 45502-45509Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 20Xie X. Kokubo T. Cohen S.L. Mirza U.A. Hoffmann A. Chait B.T. Roeder R.G. Nakatani Y. Burley S.K. Nature. 1996; 380: 316-322Crossref PubMed Scopus (228) Google Scholar). The presence of histone-fold TAFs within TFIID led to the proposal that there is a nucleosomal-like interaction between HFD TAFs and DNA (21Hoffmann A. Oelgeschlager T. Roeder R.G. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 8928-8935Crossref PubMed Scopus (76) Google Scholar). Recently we reported that the H4-H3-like TAF6 and TAF9 have intrinsic DNA-binding activity that lies outside the HFD. However, when complexed through their HFDs, they show enhanced DNA-binding activity to the core promoter motif downstream promoter element (16Shao H. Revach M. Moshonov S. Tzuman Y. Gazit K. Albeck S. Unger T. Dikstein R. Mol. Cell. Biol. 2005; 25: 206-219Crossref PubMed Scopus (52) Google Scholar). We also found that human, Drosophila, and yeast TAF4 is capable of DNA binding, which we mapped to its H2A-like histone-fold motif and a unique spacer domain that is not present in histone H2A. The interaction of the H2A-like TAF4b with the H2B-like TAF12 increased the stability of the DNA-bound complex (16Shao H. Revach M. Moshonov S. Tzuman Y. Gazit K. Albeck S. Unger T. Dikstein R. Mol. Cell. Biol. 2005; 25: 206-219Crossref PubMed Scopus (52) Google Scholar). The ability of many TAFs to bind DNA suggests that they facilitate core promoter binding by TFIID. How TAF4/TAF4b·TAF12 accomplishes this is unknown. In the present study we report on the unique biochemical and molecular features of the interaction of the histone-like pair TAF4b·TAF12 with DNA. We show that it binds DNA with high affinity most likely through one TAF4b·TAF12 heterodimer forming several contacts with one DNA molecule. For optimal binding the complex requires the DNA to have a length of ∼70 bp. The complex displays a weak sequence preference for the adenovirus major late (AdML) promoter, but the nucleotide composition of the DNA is less important than its length for high affinity binding. Expression profiling revealed a gene set that is down-regulated when TAF4 DNA binding is impaired. The vast majority of genes in this group have an Initiator core promoter element around the TSS and a high prevalence of the TATA-box. Examination of the Pel98 promoter from this group demonstrated that TAF4 DNA binding was critical for the core promoter function. Our findings suggest that, in this subset of genes, TAF4 can facilitate the binding of TFIID to the core promoter by providing additional contacts with DNA. TAF4b and TAF12 were expressed in Escherichia coli BL-21 DE3 strain, refolded either alone or as complexes, and then purified as previously described (16Shao H. Revach M. Moshonov S. Tzuman Y. Gazit K. Albeck S. Unger T. Dikstein R. Mol. Cell. Biol. 2005; 25: 206-219Crossref PubMed Scopus (52) Google Scholar) and further purified on a Sephadex 200 column. TAF4CRII and TAF4CRIImDB were expressed and refolded as previously described (16Shao H. Revach M. Moshonov S. Tzuman Y. Gazit K. Albeck S. Unger T. Dikstein R. Mol. Cell. Biol. 2005; 25: 206-219Crossref PubMed Scopus (52) Google Scholar). For EMSA DNA was end-labeled using [γ-32P]ATP (Amersham Biosciences) and polynucleotide kinase. The DNA probe (4 ng) was incubated with the TAF4b·TAF12 complex in a 20-μl reaction volume in DNA binding buffer containing 10 mm Tris, pH 8.0, 75 mm KCl, 2.5 mm dithiothreitol, 10% glycerol, and 0.05% Nonidet P-40 for 20 min at 25 °C. The samples were loaded onto a 5% native polyacrylamide gel containing 0.5× TBE buffer (89 mm Tris-HCl, 89 mm boric acid, 2 mm EDTA) and run at 4 °C for 2 h. The gel was dried and visualized using a phosphorimaging device (Fuji BAS 2500). For DNA cellulose binding assays the proteins were incubated with either empty cellulose beads or DNA-containing cellulose beads (0.25 μg of double-stranded calf thymus DNA (Sigma) per reaction) for 45 min, at room temperature, in binding buffer composed of 10 mm Tris, pH 8.0, 50 mm KCl, 2.5 mm dithiothreitol, 0.1 mg/ml bovine serum albumin, 15% glycerol, and 0.2% Nonidet P-40. The beads were washed three times with binding buffer, and the proteins were eluted with 30 μl of binding buffer containing 1 m NaCl. 20% of the eluted bound proteins were analyzed by SDS-PAGE and visualized by silver staining. Mass spectrometry was performed under non-denaturing conditions on a QToF Q-Star XL (MDS Sciex, Concord, Ontario, Canada) mass spectrometer, modified for improved transmission of large, non-covalent complexes. The instrument was fitted with a high m/z quadrupole. In addition, the pressure regime in the early vacuum stage of the instrument was modulated to improve large ion transmission, by a flow-restricting sleeve surrounding part of the first quadrupole ion guide of the Sciex instrument (Chernushevich IV, Thomson BA; collisional cooling of large ions in electrospray mass spectrometry). Construction of pGEX-TAF4CRII was previously described (Shao et al. 16Shao H. Revach M. Moshonov S. Tzuman Y. Gazit K. Albeck S. Unger T. Dikstein R. Mol. Cell. Biol. 2005; 25: 206-219Crossref PubMed Scopus (52) Google Scholar). To construct the pGEX-TAF4CRIImDB two DNA fragments corresponding to TAF4 amino acids 828–1010 and 1052–1083 (end) were generated by PCR and sequentially inserted into pGEX-2TK. To generate expression plasmids for HA-tagged TAF4CRII and TAF4CRIImDB, DNAs encoding TAF4CRII and TAF4CRIImDB were amplified from the pGEX-TAF4CRII and pGEX-TAF4CRIImDB plasmids, respectively, by PCR with the oligonucleotides 5′-CCCCCCTCTAGAGACGATGATGACATTAATGA and 5′-GGGGGGGGATCCCCGGGAGCTGCATGTGTCAGAGG, and the fragments were first cloned into the pCGN expression vector downstream to the HA-epitope. The pCGN was then cut with SnaBI and EcoRI to generate DNA fragments that included TAF4CRII and TAF4CRIImDB with the HA tag in their N-terminal, and these fragments were cloned into pEIRES-P at NheI that was blunted by Klenow and EcoRI. The PEL98 promoter from −488 to +10 was amplified by PCR from mouse genomic DNA using primers 5′-CCCCCCGGTACCCCAAGGTCCCTCCTGACTTG and 5′-CCCCCCCTCGAGAGAGAGGTTTGGGGAGAGCC and cloned into the promoter-less reporter gene pGL3-basic (Promega, Madison, WI) at the KpnI and XhoI sites of the multiple cloning site. The Initiator mutant was generated by PCR using the same forward primer and the reverse primer 5′-CCCCCCCTCGAGAGAGAGCACACCGGAGAGCC. TAF4−/− cells, and their derivatives were grown in Dulbecco's minimal essential media supplemented with 10% fetal calf serum. TAF4CRII and TAF4CRIImDB expression plasmids were transfected into the TAF4−/− fibroblasts, and stable clones were picked out following puromycin selection. Poly-l-lysine-coated glass microarrays containing >23,000 different probes (mouse oligonucleotide set, Compugen) were purchased from the Center for Applied Genomics, New Jersey. The microarrays were probed with a mixture of cyanine 3- or cyanine 5-labeled cDNAs, generated from total RNA (100 μg) that was prepared from TAF4CRII and TAF4CRIImDB cell lines. The cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase (Promega) with aminoallyl-modified dUTP nucleotide (Ambion) at a 4:1 aminoallyl-modified dUTP-to-dTTP ratio and labeled with an N-hydroxysuccinimide-activated cyanine 3 or cyanine 5 fluorescent probe (Amersham Biosciences) through aminoallyl-modified dUTP. These labeled cDNAs from TAF4CRII and TAF4CRIImDB cells were mixed with equivalent amounts of fluorescent dye (100 pmol each) in 2× SSC (1× SSC is 0.15 m NaCl plus 0.015 m sodium citrate), 0.08% SDS, 6 μl of blocking solution (Amersham Biosciences), and water to 100 μl. This target mixture was denatured at 95 °C for 3 min, chilled, and applied between a raised coverslip (LifterSlip, Erie Scientific Co.) and the array. The slide was then sealed in a microarray hybridization chamber and submerged in a darkened water bath set at 55 °C for hybridization. After 12 h, the slide was washed for 5 min in 2× SSC-0.5% SDS at 55 °C, 5 min in 0.5× SSC at room temperature, and 5 min in 0.05× SSC at room temperature. It was then quickly dried by centrifuging for 3 min at 1000 rpm and stored in the dark until scanned. Each cell line was represented by dye-swap microarray replicates. To correct for dye bias Lowess normalization was performed. Bad spots were flagged out before normalization. Average log intensities were calculated using the R package Limma (22Smyth G.K. Stat. Appl. Genet. Mol. Biol. 2004; 3 (Article3)Crossref PubMed Scopus (8979) Google Scholar). Linear models and empirical Bayes methods were used for assessing differential expression in microarray experiments. All genes with <1.9-fold changes were excluded from the list. Total RNA was prepared using the RNeasy Mini kit (Qiagen), according to the manufacturer's instructions. RNA preparations were treated with RQ1 DNase I (Promega) to avoid contamination by genomic DNA. First strand cDNA was synthesized from 1 μg of total RNA using an oligo(dT)15 primer and SuperScript II reverse transcriptase (Invitrogen). 1 μl of a 1/50 cDNA dilution was used for PCR. The real-time PCR was performed in 20-μl tubes using a SYBR Green PCR master mix (Applied Biosystems), according to the manufacturer's instructions in a 7300 real-time PCR system and was analyzed using 7300 system software. The oligonucleotides used for real-time PCR were as follows: β-actin, 5′-CCCTAAGGCCAACCGTGAA and 5′-TTGAAGGTCTCAAACATGATCTGG; mouse THBS1, 5′-CCATGAAGAGTTCCTTGGGTTT and 5′-TCTGGCTCTGTGAGTAAGGCAG; CYR61, 5′-TCAGGGACTAAGTGCCTCCAG and 5′-GCAAGGCACCATTCATCCTC; FGF7, 5′-AGGTCATGCTTCCACCTCGT and 5′-GGGCTGGAACAGTTCACACTC; SFRP2, 5′-TCCCAGTGGGTGGCTTCTC and 5′-TAGCTTTCCCGGACTGTGCTT; PEL98, 5′-AGCTGCATTCCAGAAGGTGA and 5′-ACATCATGGCAATGCAGGAC; OMD, 5′-TTCAGACACTCCAGAAGAGGGAG and 5′-CGACTGCTCTTCCGAAGGTC; MSLN, 5′-GAATGGCTGCAACACATCTCC and 5′-GTCGGAACCTTGGGTGTATGA; and IL1RL1, 5′-GAATGGGACTTTGGGCTTTG and 5′-CAGGACGATTTACTGCCCTCC. Transfections into TAF4CRII and TAF4CRIImDB cell lines were performed using the jetPEI transfection reagent (PolyPlus Transfection) according to the manufacturer's instructions. For reporter assays, subconfluent cells were transfected in a 6-well plate using 1000 ng of the firefly luciferase reporter vector, 50 ng of Rous sarcoma virus-Renilla control reporter vector (containing Renilla luciferase), and 50 ng of cytomegalovirus-green fluorescent protein. 4 h after transfection the medium was replaced. 24 h after transfection luciferase and Renilla activities were measured. ChIP assays were carried out as described (23Ainbinder E. Amir-Zilberstein L. Yamaguchi Y. Handa H. Dikstein R. Mol. Cell. Biol. 2004; 24: 2444-2454Crossref PubMed Scopus (42) Google Scholar). Equivalent amounts of cross-linked chromatin extract were used for immunoprecipitation. The input and the ChIP data were quantified by densitometric analysis using Quantity One one-dimensional analysis software (Bio-Rad), and the ChIP results were normalized to the input and the enrichment relative to the control antibodies was calculated. The ChIP primers were: PMM2 core promoter (forward, 5′-ACCGGTGTTCTGTGAACCAT; reverse, 5′-CCATCCATGTCGAAGAGACA), PEL98 promoter (forward, 5′-GCAAGAGCACAGTATCCATG; reverse, 5′-AGCAGTGCTATCAGACCAAC), and PEL98–1000 bp promoter upstream region (forward, 5′-CCTTTATGCTCCTTACTACTG; reverse, 5′-GTCTCATCTGATAGGACACG). Different promoter fragments were generated by PCR using the primers as follows: IκB (forward, 5′-TCTGGTCTGACTGGCTTGG; reverse, 5′-GGACTGCTGTGGGCTCTG) and A20 (forward, 5-GAAATCCCCGGGCCTACAAC; reverse, 5-CAAGCTCGCTTGGCCCGCC). The template for the control fragment was the pTZ57R plasmid (forward, 5′-GACTCACTATAGGGAAAGCTTGC; reverse, 5-CGACGTTGTAAAACGACGGC). For the AdML promoter the primers were: forward, 5′-GTGACCGGGTGTTCCTGAAGGGGGGC; reverse, 5′-CCATGATTACGCCAAGCTTGCATG; the AdML promoter was generated by PCR and then digested with ApaI restriction enzyme to get the 74-bp fragment containing the core promoter. In other experiments the AdML core promoter was generated by annealing two oligonucleotides and filling in with Klenow. Primers were forward, 5′-GTGACCGGGTGTTCCTGAAGGGGGGCTATAAAAGGGGGTGGGGGC; reverse, 5-CGGAAGAGAGTGAGGACGAACGCGCCCCCACCCCCTTTTATAGCC. The AdML promoter derivatives were generated by annealing synthetic oligonucleotides with the following sequences: AdML Δ5′ (forward, 5′-GTTCCTGAAGGGGGGCTATAAAAGGGGGTGGGGGCGCGTT; reverse, 5-CGGAAGAGAGTGAGGACGAACGCGCCCCCACCCCCTTTTATAGCC), AdML Δ3′ (forward, 5′-GTGACCGGGTGTTCCTGAAGGGGGGCTATAAAAGGGGGTGGGGGC; reverse, 5′-AACGCGCCCCCACCCCCTTTTATAGCCCCCCTTCAGGAAC), AdML Δ5′+Δ3′ (forward, 5-GTTCCTGAAGGGGGGCTATAAAAGGGGGTGGGGGCGCGTT; reverse, 5-AACGCGCCCCCACCCCCTTTTATAGCCCCCCTTCAGGAAC), AdML 5′ mutant (forward, 5′-TGTCAATTTGGTTCCTGAAGGGGGGCTATAAAAGGGGGTGGGGGC; reverse, 5′-CGGAAGAGAGTGAGGACGAACGCGCCCCCACCCCCTTTTATAGCC), AdML 3′-1 mutant (forward, 5′-GTGACCGGGTGTTCCTGAAGGGGGGCTATAAAAGGGGGTGGGGGC; reverse, 5′-ATTCCTCTAGTGAGGACGAACGCGCCCCCACCCCCTTTTATAGCC), and AdML 3′-2 mutant (forward, 5′-GTGACCGGGTGTTCCTGAAGGGGGGCTATAAAAGGGGGTGGGGGC; reverse, 5′-CGGAAGAGCTGTCTTCCGAACGCGCCCCCACCCCCTTTTATAGCC). The H2A-like TAF4 and TAF4b interact with the H2B-like TAF12 in vitro and in native TFIID. Both TAF4/4b and TAF12 possess intrinsic DNA-binding activity, and their interaction through the HFD is important for stable DNA binding (16Shao H. Revach M. Moshonov S. Tzuman Y. Gazit K. Albeck S. Unger T. Dikstein R. Mol. Cell. Biol. 2005; 25: 206-219Crossref PubMed Scopus (52) Google Scholar). To characterize TAF4b·TAF12 DNA binding further we first determined the composition of the TAF4b·TAF12 complex. When the TAF4b C-terminal DNA-binding domain (amino acids 561–769) and His6-TAF12 are either co-expressed in E. coli or expressed separately and then combined and purified on nickel beads, they co-purify, indicating complex formation (Fig. 1A). The TAF4b·TAF12 complex was loaded onto a Sephadex 200 gel filtration column, and it eluted in a single peak. The presence of TAF4b and TAF12 in the peak fraction was verified by mass spectrometry (data not shown). We performed a special mass spectrometric analysis to elucidate the oligomeric state of the TAF4b·TAF12 complex (24Sharon M. Robinson C.V. Annu. Rev. Biochem. 2007; 76: 167-193Crossref PubMed Scopus (312) Google Scholar). This technique preserves non-covalent interactions between proteins, allowing determination of the molecular mass of the complex and the subunits ratio. The measured mass of the TAF4b·TAF12 complex was 41,233 ± 11 Da corresponding to a 1:1 stoichiometry between TAF4b and TAF12 (supplemental Fig. S1). To confirm the composition of the complex, tandem mass spectrometry experiments were conducted. The complex dissociates into two subunits with measured masses of 23,367 ± 2 and 17,794 ± 1 Da, which is in close agreement with the calculated masses of TAF4b and TAF12, respectively, without their first methionine (supplemental Fig. S1). This experiment confirms that TAF4b and TAF12 form a stable complex and suggests that the complex consists of a heterodimer. To examine the DNA-binding activity of this complex we employed the EMSA in which the ratio of DNA to TAF4b·TAF12 dimer was gradually increased up to 1:2 (Fig. 1B). The DNA used in this experiment is the AdML promoter that binds TAF4b·TAF12 preferentially (see below). Under conditions of excess protein, formation of the DNA·protein complex is inefficient and unstable, because it tends to dissociate during electrophoresis (lane 1). However, the complex becomes increasingly stable with increased DNA levels (lanes 2–7). When the DNA is in excess the complex remains stable and competition is observed (see Figs. 2B and 3A). The observation that the TAF4b·TAF12-DNA complex becomes more efficient and stable at a high DNA:protein ratio (compare lanes 4 to 5, Fig. 1B) raises the possibility that there are several points of contacts between TAF4b·TAF12 and the DNA. In a large excess of TAF4b·TAF12 some of these contacts are competed out and the complex becomes less stable.FIGURE 3The optimal length of DNA for TAF4b·TAF12 binding is half the size of nucleosomal DNA. A, the TAF4b·TAF12 complex was incubated with a radiolabeled oligonucleotide containing a 70-bp AdML promoter (WT) in the absence (lane 2) or the presence (lanes 3–10) of cold DNA competitors as indicated at the top of the lanes. The sequences of the DNA used as competitors are shown below. B, EMSA experiment as in A using equivalent molar amounts of different length DNAs whose sequences are shown in A. The relative amount of bound DNA is indicated at the bottom. C, competition experiment as in A. Sequences of competitor DNA are shown at the bottom. Mutated nucleotides are in lowercase letters.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We determined the apparent dissociation constant (Kd) of the TAF4b·TAF12·DNA complex, in which the concentration was calculated according to its heterodimer composition, to be ∼30 nm (Fig. 1C), which is within the physiological range. To examine whether TAF4b·TAF12 binds DNA in a sequence-specific manner we examined the binding of the purified recombinant TAF4b·TAF12 complex to four different DNA fragments of 74- to 105-bp length (Fig. 2A). The first fragment, A20, is the core promoter of a gene regulated by TAF4b (25Yamit-Hezi A. Nir S. Wolstein O. Dikstein R. J. Biol. Chem. 2000; 275: 18180-18187Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Two others are the AdML and the IκB gene core promoters, and the last is a control DNA derived from a plasmid with no promoter sequences. We tested the binding of the TAF4b·TAF12 complex to these four DNA fragments with EMSA. Using labeled AdML DNA we examined the affinity of TAF4b·TAF12 to each of the DNA fragments by competition with an excess of unlabeled fragments (Fig. 2B). The experiment revealed differences in the ability of the fragments to compete with AdML suggesting that TAF4b·TAF12 discriminates between the different sequences. The AdML promoter had the highest affinity to the complex followed by IκB. The results were verified by reciprocal experiments in which either the A20 (Fig. 2C) or the IκB (data not shown) promoters were labeled. Enzymatic and chemical footprinting assays of the AdML promoter failed to reveal a specific DNA sequence bound by the complex indicating that the sequence preference is weak. The observation that the affinity for the A20 core promoter derived from a gene regulated by TAF4b was the lowest is surprising. In activating A20 transcription TAF4b serves as coactivator for NF-κB, an activity that requires direct interaction between TAF4b and NF-κB (25Yamit-Hezi A. Nir S. Wolstein O. Dikstein R. J. Biol. Chem. 2000; 275: 18180-18187Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 26Silkov A. Wolstein O. Shachar I. Dikstein R. J. Biol. Chem. 2002; 277: 17821-17829Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Thus, it seems that the DNA binding and coactivation functions of TAF4b are independent of each other. We extended the characterization of TAF4b·TAF12 binding to the AdML promoter with a competition experiment between the AdML core promoter probe and an excess of cold DNAs, either the wild-type AdML core promoter or mutants in which the upstream or downstream segments or both were deleted (Fig. 3A). In this experiment we observed differences between the mutants for competition with the labeled 70-bp AdML promoter. The central 40-bp region lacking both the upstream and the downstream ends was the least effective competitor (compare lanes 3 and 4 to lanes 9 and 10), whereas mutants lacking either the upstream or the downstream region competed more efficiently than the central 40-bp but less than the full-length 70-bp AdML promoter (compare lanes 3 and 4 to lanes 5–8). The results indicate that areas both upstream and downstream to the central 40 bp area are important for binding by TAF4b·TAF12. To gain support for this finding, the full-length 70-bp AdML promoter, the mutants lacking either the upstream (60 bp) or the downstream (50 bp) or both (40 bp) and an extended promoter (98 bp) were each labeled and used for binding to the TAF4b·TAF12 complex by EMSA using equimolar amounts. The results show that the level and the stability of the TAF4b·TAF12·DNA complex increase gradually with the addition of the upstream and downstream sequences (Fig. 3B) up to 70 bp. Beyond this length the binding efficiency is similar. To examine further whether DNA length or the sequence surrounding the central AdML promoter contribute to increased DNA binding, the nucleotide sequence of the upstream and downstream regions were changed in the context of the full-length 70-bp AdML promoter and used for competition assay. As shown in Fig. 3C these substitutions did not reduce the binding affinity of TAF4b·TAF12, suggesting that DNA sequence may be less important than its length. To examine further the length requirement for binding we performed similar binding assays with the PEL98 promoter, which is distinct in its sequence from the AdML promoter (supplemental Fig. 2A). This promoter was chosen because of its dependency on TAF4 DNA binding (see below). With this sequence we observed a clear DNA length preference as was found for the AdML promoter as the affinity to 70 bp > 55 bp > 40 bp. In this promoter context we also examined whether the spacing between the TATA-box and the Initiator is important for high affinity binding. The 20 nucleotides between TATA and Initiator were either relocated to the 5′-end or deleted shortening the DNA to 50 bp (supplemental Fig. 2B). The results revealed that changing the spacing but retaining the length does not significantly affect binding affinity, whereas binding is reduced with the shorter 50-bp fragment. To test further the sequence preference we compared the binding between the PEL98 and the non-target promoter PMM2 and found that the complex binds preferentially the PEL98 DNA (supplemental Fig. 2C). These findings together confirm that high affinity DNA binding by TAF4b·TAF12 is achieved through contacts with DNA spanning 70 bp combined with weak sequence preference. A possible explanation that integrates these findings is that TAF4b·TAF12 DNA binding has several points of contact spanning the length of the DNA, some specific and some non-specific, that all contribute to stable binding. The TAF4b" @default.
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