Efficient Binding of NC2·TATA-binding Protein to DNA in the Absence of TATA
2004; Elsevier BV; Volume: 280; Issue: 7 Linguagem: Inglês
10.1074/jbc.m406343200
ISSN1083-351X
AutoresSiv Gilfillan, Gertraud Stelzer, Elisa Piaia, Markus Hofmann, Michael Meisterernst,
Tópico(s)RNA Interference and Gene Delivery
ResumoNegative cofactor 2 (NC2) forms a stable complex with TATA-binding protein (TBP) on promoters. This prevents the assembly of transcription factor (TF) IIA and TFIIB and leads to repression of RNA polymerase II transcription. Here we have revisited the interactions of NC2·TBP with DNA. We show that NC2·TBP complexes exhibit a significantly reduced preference for TATA box sequences compared with TBP and TBP·TFIIA complexes. In chromatin immunoprecipitations, NC2 is found on a variety of human TATA-containing and TATA-less promoters. Substantial amounts of NC2 are present in a complex with TBP in bulk chromatin. A complex of NC2·TBP displays a KD for DNA of ∼2 × 10-9m for a 35-bp major late promoter oligonucleotide. While preferentially recognizing promoter-bound TBP, NC2 also accelerates TBP binding to promoters and stabilizes TBP·DNA complexes. Our data suggest that NC2 controls TBP binding and maintenance on DNA that is largely independent of a canonical TATA sequence. Negative cofactor 2 (NC2) forms a stable complex with TATA-binding protein (TBP) on promoters. This prevents the assembly of transcription factor (TF) IIA and TFIIB and leads to repression of RNA polymerase II transcription. Here we have revisited the interactions of NC2·TBP with DNA. We show that NC2·TBP complexes exhibit a significantly reduced preference for TATA box sequences compared with TBP and TBP·TFIIA complexes. In chromatin immunoprecipitations, NC2 is found on a variety of human TATA-containing and TATA-less promoters. Substantial amounts of NC2 are present in a complex with TBP in bulk chromatin. A complex of NC2·TBP displays a KD for DNA of ∼2 × 10-9m for a 35-bp major late promoter oligonucleotide. While preferentially recognizing promoter-bound TBP, NC2 also accelerates TBP binding to promoters and stabilizes TBP·DNA complexes. Our data suggest that NC2 controls TBP binding and maintenance on DNA that is largely independent of a canonical TATA sequence. TATA-binding protein (TBP) 1The abbreviations used are: TBP, TATA-binding protein; NC2, negative cofactor 2; TAF, TBP-associated factor; AdML, adenovirus major late; TF, transcription factor; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation; wt, wild-type. binds to eukaryotic promoters to nucleate initiation of transcription (1Burley S.K. Roeder R.G. Annu. Rev. Biochem. 1996; 65: 769-799Crossref PubMed Scopus (628) Google Scholar, 2Sanders S.L. Garbett K.A. Weil P.A. Mol. Cell. Biol. 2002; 22: 6000-6013Crossref PubMed Scopus (89) Google Scholar, 3Tora L. Genes Dev. 2002; 16: 673-675Crossref PubMed Scopus (194) Google Scholar). The crystal structure of the TBP·DNA complex revealed a saddle-shaped conformation of TBP in which the highly conserved carboxyl terminus forms a concave surface that interacts with the minor groove of DNA. As a consequence, the minor groove becomes dramatically widened, and the DNA is severely bent (4Nikolov D.B. Hu S.H. Lin J. 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NC2 is shown to localize to the promoter regions of many genes. Ratios of TBP to NC2 vary from one gene to another without uncovering an obvious correlation to the presence of bona fide TATA boxes. In fact, in vitro analyses revealed that NC2·TBP displays moderate sequence specificity for TATA. We further show that NC2 increases both the on-rate of TBP to DNA and the stability of TBP·DNA complexes. Expression and Purification of Recombinant Proteins—Human NC2 subunits were expressed as both two single subunits and bicistronic co-expression constructs in pET11d as described previously (46Gilfillan S. Stelzer G. Kremmer E. Meisterernst M. Methods Enzymol. 2003; 370: 467-479Crossref PubMed Scopus (2) Google Scholar). Truncation mutants were constructed using PCR primers harboring a stop codon at the desired position. All vectors were confirmed by sequencing. The human TBP, also in pET11d, was expressed and purified as described previously (47Stelzer G. Goppelt A. Lottspeich F. Meisterernst M. Mol. Cell. Biol. 1994; 14: 4712-4721Crossref PubMed Scopus (55) Google Scholar, 48Kretzschmar M. Stelzer G. Roeder R.G. Meisterernst M. Mol. Cell. Biol. 1994; 14: 3927-3937Crossref PubMed Scopus (55) Google Scholar). Human TFIIA was expressed and purified as described elsewhere (31Xie J. Collart M. Lemaire M. Stelzer G. Meisterernst M. EMBO J. 2000; 19: 672-682Crossref PubMed Scopus (46) Google Scholar). Electrophoretic Mobility Shift Assay—The binding reactions were carried out in a buffer containing 4 mm MgCl2,25 mm HEPES-KOH, pH 8.2, 0.4 mg/ml bovine serum albumin, 5 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, glycerol (7–10%), and 70–90 mm KCl. Protein and DNA amounts are given in the figure legends. Reactions were incubated for 30 min at 27 °C, loaded on a 5% acrylamide gel (acrylamide:bisacrylamide, 50:1), and run at 120 V in Tris-glycine-EDTA buffer (25 mm Tris-HCl, pH 7.3, 248 mm glycine, and 1 mm EDTA, pH 8.0). Gels were usually run at room temperature, but when TFIIA was included, the gels were run at 4 °C. Band intensity was quantified using a phosphorimager (Packard Instantimager). DNA Templates Used in EMSA—Oligonucleotides were annealed in buffer (200 mm NaCl, 10 mm Tris-HCl, pH 7.3, and 1 mm MgCl2) by heating to 95 °C for 3 min and cooling gradually to room temperature. The annealed oligonucleotides (designed to contain 4-bp 5′ overhangs) were stored at 4 °C and labeled with [α-32P]dCTP (Amersham Biosciences) by Klenow fill-in using standard procedures. The 35-bp sequences used were as follows (the TATA sequence is underlined): H2B promoter, CTGAAGCGATTCTATATAAAAGCGCCTTGTCATAC; Vβ promoter, CCAGGATGCATTCTGTGGGGATAAAATGTCACAAA; HLAA promoter, TCGCGGTCGCTGTTCTAAAGTCCGCACGCACCCAC; interleukin-2 promoter, CCAGAATTAACAGTATAAATTGCATCTCTTGTTCA; and adenovirus major late promoter (AdML-TATA and AdML-wt), CCTGAAGGGGGGCTATAAAAGGGGGTGGGGGCGCG. Mutations in the TATA sequence were as follows (mutations are underlined): TGCTGGG, GATAAAA, CATAAAA, GAGAAAA, CTCGAGA, or TGTAAAA. Longer DNA fragments used for EMSA (217 bp) were isolated from pB2-MLP carrying a 99-bp EcoRI/SmaI fragment of the AdML promoter (49Kim J. Zwieb C. Wu C. Adhya S. Gene (Amst.). 1989; 85: 15-23Crossref PubMed Scopus (321) Google Scholar). A TATA to TGTA mutation in this vector (pSO18) was produced with the QuikChange site-directed mutagenesis kit (Stratagene). EcoRI fragments of both vectors were purified and labeled with [α-32P]dATP. Cell Culture—Suspension cultures of human Jurkat (J6 human leukemia T cell) or HeLa cells were grown in RPMI 1640 medium (Invitrogen) enriched with 5% fetal calf serum (Invitrogen). Cell density was kept between 2 and 9 × 105 cells/ml. Cell lysate from Jurkat cells was prepared by washing cells once in phosphate-buffered saline and suspending them in radioimmune precipitation assay buffer (50 mm HEPES-KOH, pH 7.9, 140 mm NaCl, 1 mm EDTA, pH 8.0, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mm phenylmethylsulfonyl fluoride, 5 mm dithiothreitol, and 0.2 mm benzamidine) using a Dounce homogenizer. The cell lysate was centrifuged (7000 × g, 30 min), and the pellet was discarded. Chromatin Immunoprecipitation—ChIP was performed with a rat monoclonal NC2α antibody (4G7) and polyclonal TBP antibody on formaldehyde cross-linked Jurkat cells. In brief, cells were fixed in 1% formaldehyde for 9 min at room temperature, sonicated, and run on a CsCl gradient as described previously (46Gilfillan S. Stelzer G. Kremmer E. Meisterernst M. Methods Enzymol. 2003; 370: 467-479Crossref PubMed Scopus (2) Google Scholar). Antibody and extract were incubated overnight, followed by incubation with a mixture of protein A- and G-Sepharose beads (Amersham Biosciences). After extensive washing, complexes were eluted from the beads, cross-links were reversed, and DNA was purified by phenol-chloroform extraction and precipitated. Quantitative PCR was performed (28 cycles; sequences of primers are available upon request). Mononucleosome Preparation—The chromatin pellet of extracted HeLa nuclei (36Meisterernst M. Roy A.L. Lieu H.M. Roeder R.G. Cell. 1991; 66: 981-993Abstract Full Text PDF PubMed Scopus (225) Google Scholar) was resuspended using a Dounce homogenizer in buffer (15 mm HEPES-KOH, pH 7.4, 15 mm NaCl, 60 mm KCl, 2 mm MgCl2, 0.2 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 2 μg/ml pepstatin, and 3 μg/ml aprotinin). The pellet was collected by centrifugation (200 × g, 5 min) and washed twice with the same buffer. CaCl2 was added to a final concentration of 0.23 mm. 200 units of micrococcal nuclease (N-5386; Sigma) were added and incubated at 37 °C for 3–20 min. The reaction was stopped by the addition of EDTA to a final concentration of 5 mm, incubated on ice for 5 min, and centrifuged at 200 × g for 15 min. The presence of mononucleosomes in the supernatant was confirmed by sizing columns, protein gels, and digestion with proteinase K and subsequent analysis of the DNA on agarose gels. Immunoprecipitation—Protein G beads and NC2α monoclonal antibody (4G7) were incubated for at least 4 h. Mononucleosome fraction or nuclear extract was added; adjusted to BC150 supplemented with phenylmethylsulfonyl fluoride (final concentration, 5 mm), dithiothreitol (final concentration, 1 mm), and IGEPAL CA-630 (final concentration, 0.1%); and incubated for 3 h at 4 °C with gentle mixing, followed by four rounds of washes with 1 ml of BC150 each. Beads were recovered by centrifugation for 3 min at 300 × g. NC2 was eluted with SDS loading buffer (4 min at 95 °C). Calculation of Active Protein Concentration—Either NC2 or TBP was kept at limiting concentration while the other protein was in excess, and DNA was titrated until no further increase in complex formation was observed. This was defined as the active concentration, which is always referred to in the text. The active amount of TBP was ∼65% in the specific preparation and ∼60% for NC2 relative to total protein. Calculation of the Equilibrium Dissociation Constant KD—The equation used to determine the binding constant derives from the equilibrium binding constant: 1/KD = KB = [c]/([TBP·NC2] × [DNA]), where [c] is the concentration of NC2·TBP·DNA complex at equilibrium, and [protein] and [DNA] are the equilibrium concentrations of TBP and DNA, respectively. To simplify the equation, TBP·NC2 was treated as a single protein entity. NC2·TBP Is Localized on TATA and TATA-less Promoters in Vivo—ChIP experiments were performed to monitor NC2 and TBP on the promoters of endogenous human genes in Jurkat cells. NC2 was found on all promoters tested. NC2 and TBP levels are high and the NC2/TBP ratio is low at the highly active H2A/H2B locus. The HLAA locus carrying a cryptic TATA displayed the highest level of NC2. The TATA-less eIF4E promoter also binds NC2, whereas on the promoter of interleukin-2, TBP and NC2 levels are low, although it harbors a reasonable TATA box (Fig. 1). The limited set of data does not allow for a statistical correlation of NC2 occupancy relative to the core promoter structure. Presently, the data suggest that NC2 binds to many promoters in human cells. This binding does not require the presence of a TATA box. Finally, NC2/TBP ratios vary significantly from one promoter to another. NC2 and TBP Are Present in Bulk Chromatin—Localization of NC2 on many genes may indicate that the factor resides in chromatin together with TBP. To test this hypothesis, mononucleosomes were prepared from chromatin extracts of HeLa cells and tested for the presence of NC2. Approximately 10–20% of total NC2 was found in the mononucleosome fraction. NC2α was precipitated using a monoclonal antibody described previously (46Gilfillan S. Stelzer G. Kremmer E. Meisterernst M. Methods Enzymol. 2003; 370: 467-479Crossref PubMed Scopus (2) Google Scholar). The precipitated material was analyzed for the presence of NC2β, TBP, TAFs, and BTAF1 (formerly called TAF170/172) using polyclonal antibodies. TBP, but not TAFs or BTAF1 (50Timmers H.T. Sharp P.A. Genes Dev. 1991; 5: 1946-1956Crossref PubMed Scopus (94) Google Scholar), co-precipitated with NC2 (Fig. 2A). We also could not detect histones in the precipitated material (data not shown). Given the stringency of the immunoprecipitation, this does not exclude interactions of NC2 with BTAF1 and the TFIID complex. Nevertheless, the data argue for a strong preference of NC2 for free TBP. Notably, antibodies did not co-precipitate detectable amounts of TBP with NC2 in HeLa nuclear extracts. We reasoned that co-precipitation from chromatin extracts might be facilitated by DNA (Fig. 2B). To test this hypothesis, the immunoprecipitated material was treated with DNase I. This resulted in a complete loss of TBP in the NC2 immunoprecipitation (Fig. 2C), arguing for interaction of TBP and NC2 through DNA. It is somewhat puzzling that promoter and coding regions of the H2A/H2B locus seemed underrepresented in NC2 immunoprecipitations compared with the input as analyzed by PCR (data not shown). This could be explained if TBP·NC2 localizes to open chromatin, in which the DNA is readily accessed by micrococcus nuclease. Although the average length of micrococcus nuclease-treated DNA was chosen to be in the range of kilobases in our analysis, hypersensitivity could perhaps lead to trimming of the genomic DNA flanking the TBP·NC2 binding sites, which could render them inaccessible to PCR analysis. This hypothetical model and the ChIP analysis support the notion that NC2 localizes to active promoters. However, at present, the exact nature of the preferred binding sites of chromatin-bound TBP·NC2 remains elusive. Even if these were in part not promoter regions, formation of a TBP·NC2 could make sense in that it could prevent transcription from non-promoter regions. This primarily requires an excess of NC2 over TBP. Western analysis of Jurkat whole cell extracts revealed that NC2 is indeed present in moderate excess over TBP (with TBP comprising ∼0.11 ng/μg and NC2 comprising ∼0.18 ng/μg of the total protein mass; Fig. 2D). In addition, part of TBP is probably engaged in complexes, for example, with the class I and class III TAFs. Another major part is thought to be in a complex with BTAF1. In summary, NC2 is probably present in significant excess over TBP in the extracts of human cells. The NC2·TBP Complex Shows Moderate Preference for TATA—EMSA was used to compare the binding of NC2·TBP to oligonucleotides comprising core regions of the selected promoters studied above in ChIP. Consistent with the in vivo localization, NC2·TBP bound to all promoters tested, irrespective of the presence of a consensus TATA motif. A moderate preference for promoters harboring a consensus TATA sequence (H2B and interleukin-2) as compared with promoters with a poor TATA sequence (HLAA and Vβ; Fig. 3A) was seen. In contrast to NC2·TBP, and as expected from the published data, TFIIA·TBP exhibits a clear preference for TATA (Figs. 3C and 4B). Specific TFIIA·TBP binding to TATA is seen even at high concentrations where NC2·TBP complexes do not show priority for TATA (Fig. 3, B and C). To document the specific influence of TATA, point mutations were analyzed in the context of the AdML promoter. Again, NC2·TBP complexes did not discriminate between wt and mutant oligonucleotides at nanomolar concentrations (Fig. 4A). However, a moderate preference for TATA (∼3-fold) was seen if limiting concentrations of TBP and NC2 were employed (Fig. 4C). Competition experiments further confirmed the limited specificity for TATA (Fig. 4D). To analyze the specificity of the NC2·TBP complex to DNA under more physiological conditions, the binding reaction was conducted at 140 mm potassium glutamate. Also, under these stringent conditions, TBP·NC2 binds DNA with very limited specificity for TATA (Fig. 4E).Fig. 4Specificity of binding by NC2·TBP and TFIIA·TBP to the AdML promoter, depending on different TATA mutations.A, EMSA of NC2·TBP with AdML promoter wt (TATAAAA) and mutants GATAAAA, CATAAAA, GAGAAAA, TGTAAAA, or TGCTGGG, as indicated. All lanes contained 21 nm DNA template and increasing TBP·NC2 with 6.5, 32, and 161 nm TBP and 9.6, 19.3, and 96.3 nm NC2 as indicated (96.3 nm NC2 in lane 2 and161 nm TBP in lane 3 and the last lane). B, TFIIA·TBP·DNA formation with 1.2, 11.7, and 23.3 nm TFIIA and 6.5, 32, and 161 nm TBP as indicated. Lane 2 contained 23.3 nm TFIIA; lane 3 contained 161 nm TBP. DNA was as described in A. C, relative binding of NC2·TBP to AdML (TND) wt versus mutated sequences. 0.5 nm DNA, 0.55 nm NC2, and 1.5 nm TBP were used. D, TBP·NC2·DNA complex formation on the AdML promoter in the presence of 10-fold unlabeled competitor DNA with the indicated sequences. 0.5 nm DNA, 0.55 nm NC2, and 1.5 nm TBP were used (n = 3; results are the mean ± S.E.). E, EMSA analysis of TBP·NC2 specificity at low and high salt. Reactions contained 0.25 nm AdML-TATAAA or AdML-CTCGAG oligonucleotides in 60 mm KCl or 2.5 nm AdML-TATAAA or AdML-CTCGAG oligonucleotides in 140 mm potassium glutamate, limiting amounts (0.5 nm) of yeast TBP at four different concentrations of NC2 (0.2, 0.8, 2.5, and 10 nm). F, complex formation on a 217-bp fragment containing a wt AdML TATA versus a mutation to TGTA. Concentrations of protein were as follows: 1.5, 5.3, and 15 nm NC2; 0.7, 2.4, and 7.2 nm TFIIA; and 7.6, 15, and 32 nm TBP. Each lane contained 7.5 nm DNA. The first and last lanes contained 32 nm TBP.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The reduced specificity of TBP binding in the presence of NC2 was also seen with a 217-bp fragment comprising 80 bp of AdML promoter flanked by polylinker sequences and containing either a wt TATA sequence or a single point mutation (TGTAAAA). The mutation did not influence binding of the (fast) monomeric TBP·NC2 complexes (Fig. 4F). Under comparable conditions, the TFIIA·TBP complex that is seen on the wt fragment was not formed on a TGTA mutant. Further consistent with low specificity for TATA, NC2·TBP complexes usually form a ladder of monomeric, dimeric, and trimeric complexes (and so forth) on longer DNA fragments that contain both TBP and NC2 (data not shown). 2G. Stelzer, E. Piaia, and M. Meisterernst, manuscript in preparation. The appearance of higher order complexes in the case of TBP·TFIIA probably results from binding to TATA-like sequences present in the polylinker. This is not seen if limiting concentrations of TBP are used and, contrary to TBP-NC2, is not seen on other DNA fragments. Efficient Recognition of DNA-bound TBP by NC2—Next we wished to determine the binding constant of NC2·TBP·DNA complexes. Initially, the limiting factor for complex formation was identified via titration of TBP and NC2 at constant and limiting (0.1 nm) DNA concentrations (Fig. 5A). At 0.5 nm TBP and high (20 nm) NC2, templates were not yet saturated (note that TBP was present in excess over DNA). If TBP levels are raised to 3 nm, low concentrations of NC2 (0.15 nm) shift more than 50% of the promoter. We conclude that TBP, but not NC2, is limiting for complex formation. Similar results were obtained at somewhat higher (0.5 nm) DNA concentrations (Fig. 5B). This set of experiments allows for a rough estimation of the affinity of NC2 for TBP·DNA (KD 1–2 × 10-10m). As demonstrated below and consistent with the notion that TBP is t
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