- Email: [email protected]
Purification and Transcription Repression by Negative Cofactor 2
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[40] Purification and Transcription Repression by Negative Cofactor 2 By S. Gilfillan, G. Stelzer, E. Kremmer, and M. Meisterernst Negative cofactor 2 (NC2 also termed Dr1/DRAP) binds to promoters and inhibits transcription by RNA polymerase II by preventing formation of a bona fide preinitiation complex.1,2 Biochemical and genetic data suggest that NC2 binds directly to TBP and to DNA and competes with the general transcription factors TFIIA and TFIIB.3,4 The molecular details have been resolved at high resolution through determination of the crystal structure of the NC2–TBP–DNA complex.5 NC2 dimerizes through histone fold domains of the H2A-H2B type. As shown in Fig. 1, NC2 is situated underneath the DNA surface contacting both DNA and TBP. The inhibitory complex contacts TBP from two sides via contacts with both subunits. Loop region 1 of NC2 in the helix 1–loop 1–helix 2–loop 2–helix 3 arrangement of the histone fold and helix 5 within the carboxy-terminal region of NC2 interact directly with TBP. The structure satisfactorily explains repression by the dimeric NC2 complex through sterical occlusion of both TFIIA and TFIIB.1,2,4 It also provides a molecular model for a stimulatory function of NC2 through the histone fold in that it can, in principle, support binding of TBP to promoters. NC2 is thought to be involved in the regulation of many genes. It is possible that it attenuates transcription in a global manner. However, evidence also shows that NC2 promoter occupancy is controlled by upstream regulatory pathways. This article focuses on the human NC2 complex, provides methods for its isolation from HeLa cells, its expression in Escherichia coli, and its analysis in DNA–TBP binding and in transcription, as well as its detection on endogenous genes through chromatin immunoprecipitation (ChIP). While there is no evidence for a related factor in prokaryotes, NC2 is conserved from yeast to humans. Thus, the methods are applicable to other mammalian cell lines and, at least in part, to other eukaryotic organisms. The ChIP techniques described here will allow tracking and characterization of NC2 effects on specific genes in vivo. They will further allow determination of the relative amount of NC2 on genes and its control by regulatory factors. 1
M. Meisterernst and R. G. Roeder, Cell 67, 557 (1991). J. A. Inostroza, F. H. Mermelstein, H. Ilho, W. S. Lane, and D. Reinberg, Cell 70, 477 (1992). 3 Y. Cang, D. T. Auble, and G. Prelich, EMBO J. 18, 6662 (1999). 4 J. Xie, M. Collart, G. Stelzer, M. Lemaire, and M. Meisterernst, EMBO J. 19, 672 (2000). 2
METHODS IN ENZYMOLOGY, VOL. 370
Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00
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Fig. 1. NC2 dimerizes through histone fold domains and binds underneath the bent DNA–TBP complex.5
Purification of NC2 from HeLa Nuclear Extract
NC2 was originally defined as a factor that forms a stable complex with TBP in gel mobility shift experiments.1 While NC2 is sometimes difficult to detect in nuclear extracts of mammalian cells, the complex is readily detectable upon fractionation on phosphocellulose (see Fig. 2). Both NC2 and NC2 subunits cofractionate on P11 and subsequent columns.6 It has also been reported that Dr1 (NC2) binds DNA–TBP in the absence of NC2 (DRAP).2 This is usually not detected in crude fractions where carrier DNA is included in the gel shift experiments. Materials Phosphocellulose (P11, Whatman) DE52 (Whatman) DEAE-Sephadex (A 25, Amersham Pharmacia Biotech) Mono Q (Amersham Pharmacia Biotech) Solutions
BC buffer: 20 mM Tris (pH 7.3 at 25 ), 20% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM dithiothreitol (DTT), 1 mM EDTA, pH 8.0, 100 mM KCl (BC100), 200 mM KCl (BC200), etc. 5
K. Kamada, F. Shu, H. Chen, S. Malik, G. Stelzer, R. G. Roeder, M. Meisterernst, and S. K. Burley, Cell 106, 71 (2001). 6 A. Goppelt, G. Stelzer, F. Lottspeich, and M. Meisterernst, EMBO J. 15, 3105 (1996).
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Fig. 2. (A) Purification chart for NC2 isolated from HeLa nuclear extracts and fractionated on phosphocellulose (P11, Whatman). (B) EMSA using a 35-bp adenovirus major late promoter fragment assaying HeLa extracts fractionated on P11 fractions where NC2 elutes exclusively in the 0.5 M KCl fraction.
Procedure 1. Prepare HeLa nuclear extract from 8 1010 cells.7 2. Dialyze the nuclear extract (280 ml: approximately 10 mg/ml) against BC100 buffer and load onto a preequilibrated P11 column (capacity 10 mg/ml column volume) with a flow rate of 1 column volume per hour. Approximately 50% of the protein will be in the flow-through fraction. 3. Wash column with BC100 (10 column volumes overnight). 4. Elute the column stepwise with BC300 (approximately 30% of total protein amount), BC500 (approximately 5% of total protein amount), and BC850 (approximately 2.5% of total protein amount). 5. NC2 elutes in the 0.5 M KCl fraction. Dialyze against BC100 and load on a DE52 column (2 mg/ml resin). Flow rate is approximately 1 column volume per hour. Wash with BC100 and elute with a linear KCl gradient (0.1 to 0.5 M KCl in BC buffer). 7
M. Meisterernst, A. L. Roy, H. M. Lieu, and R. G. Roeder, Cell 66, 981 (1991).
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6. NC2 elutes at 0.18 to 0.29 M KCl; pool fractions and dialyze against BC100. Load onto a DEAE-Sephadex (A25) column and elute with a linear gradient from 0.1 to 0.5 M KCl in BC buffer. 7. Pool the fractions in the range of 0.17 to 0.28 M KCl; dialyze against BC200 and load on a Mono Q column. Wash the column with 10 column volumes of BC200 and elute directly with BC400. 8. Analyze eluted fractions by SDS–PAGE. Analysis of NC2 in the Electrophoretic Mobility Shift Assay (EMSA)
Annealed oligonucleotides or DNA fragments isolated from a vector can be used to analyze NC2–TBP and NC2 alone in EMSA. NC2 is a nonspecific DNA-binding protein and will thus also bind DNA in the absence of TBP.6 The addition of TBP shifts the complex to a lower mobility form that is more stable and more resistant to the addition of carrier DNA such as p[dGdC]. However, the amount of carrier must be determined carefully through titration. NC2 does not bind single-stranded DNA and RNA. Solutions/Reagents Annealing buffer: 200 mM NaCl, 10 mM Tris, pH 7.3, 1 mM MgCl2 MicroSpin G-25 columns (Amersham Pharmacia) Bovine serum albumin (BSA) (New England Biolabs) Klenow fragment (MBI Fermentas) [-32P]dNTP (3000 Ci/mmol, Amersham Pharmacia) TGE: 25 mM Tris, pH 7.3, 248 mM glycine, 1 mM EDTA, pH 8.0 Annealing Oligonucleotides 1. Measure the concentration of oligonucleotides by A260. 2. Mix oligonucleotides in annealing buffer (100 pmol of each oligonucleotide in a total volume of 20 l) and transfer to a heat block at 95 . Turn the heat block off after 3 min and let the sample cool gradually to room temperature. 3. The DNA can be stored at 4 and labeled by Klenow fill-in by standard procedures. Procedure for EMSA The binding reactions for EMSA are 4 mM MgCl2, 25 mM HEPES– KOH, pH 8.2, 0.4 mg/ml BSA, 5 mM DTT, 0.5 mM PMSF, 1–5 fmol/l labeled DNA. The final concentration of glycerol is kept between 6 and 10%, and the final concentration of KCl is typically 70 mM. The binding reaction is carried out in a final volume of 20 l.
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1. 2. 3. 4. 5.
Distribute the components for the binding reaction to each sample. Add proteins; TBP is added last. Mix gently by pipetting up and down. Incubate at 27 for 30 min. Load samples on a 5% gel (acrylamide:bisacrylamide, 50:1). The gel is prerun 1 h at 120 V in TGE, and the TGE buffer is changed after the prerun. The gel usually runs 3 h at 100 V with a 35-bp DNA. 6. Place the gel on 3MM Whatman paper and dry it on a gel dryer. 7. Expose the gel to a film (BioMax MR, Kodak), typically 1 h at 80 and/or quantify the bands using a phosphoimager.
Expression and Purification of Recombinant NC2 in E. coli
To test the influence of NC2 on transcription, the two subunits of NC2 can be expressed individually in E.coli. This usually results in low levels of NC2. In contrast, NC2 is, under certain conditions, overexpressed greatly, but the protein is insoluble and found in inclusion bodies. When coexpressed, both subunits can be purified under native conditions. We used the pET11d expression vector and NC2 subunits that both harbor a hexa-histidine tag at their N termini (T7 system from Novagen). Materials and Reagents Escherichia coli BL21(DE3) pLys S (Novagen) Isopropyl--d-thiogalactoside (IPTG, Roth) Branson digital sonifier Columns (0.5 ml, Bio-Rad) Peristaltic pump P-1 (Amersham Pharmacia Biotech) Ni2þ-NTA resin (Qiagen) Proteins assay dye reagent concentrate (Bio-Rad) Heparin Sepharose (Amersham Pharmacia Biotech) BSA (Roche Diagnostics GmbH) Solutions Isopropyl--D-thiogalactoside, 0.5 M stock solution in water, sterile filtered Imidazole hydrochloride: 1 M stock, adjust pH to 7.9 with NaOH Lysis buffer: 500 mM NaCl, 10% glycerol, 20 mM Tris–HCl, pH 7.3. Add 2-mercaptoethanol (to 5 mM), IGEPAL CA-630 (NP-40) to 0.1%, leupeptin (to 2 g/ml), pepstatin A (to 1 g/ml), benzamidine (to 20 g/ml), and PMSF (to 1 mM) freshly HEPES–KOH 1 M stock (pH 7.9 at 25 )
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Procedure 1. Transform E. coli BL21(DE3) pLysS with the vector-containing histidine-tagged NC2 and select on LB plates containing ampicillin and chloramphenicol. 2. Inoculate a 50-ml starter culture of LB/ampicillin/chloramphenicol and grow overnight. 3. Dilute from the starting culture into 500 ml LB/ampicillin/ chloramphenicol (to OD600 0.1) and grow with vigorous shaking at 37 . 4. Add IPTG to 0.5 mM final concentration when OD600 is 0.6. 5. Grow the cells for 3 h at 30 with shaking. 6. Centrifuge the cells for 5 min at 5000 g. 7. Resuspend pellet in 20 ml lysis buffer (the resuspended bacteria can be frozen and stored at 80 ). All remaining steps are performed at 4 with prechilled buffers. 8. Sonicate the sample on ice: amplitude 20% for 3 min; on 0.2 s, off 0.8 s (i.e., total elapsed time 15 min). 9. Spin for 30 min in SS34 rotor at 18,000 rpm (39,000 g). 10. Add imidazole to the supernatant to a final concentration of 5 mM and load onto a preequilibrated Ni2þ column (0.8 ml) using peristaltic pump P1 (flow rate: 5 ml/h). 11. Wash the column with 50 ml of BC400 including 10 mM imidazole (can be done overnight). 12. Elute the recombinant NC2 dimer with 8 ml 100 mM imidazole in BC400 (flow rate 4 ml/h) and collect 1-ml aliquots. 13. Analyze an aliquot from each fraction in a 1:10 dilution with a protein assay dye reagent concentrate from Bio-Rad (diluted 1:5 and filtered through 3MM Whatman paper). 14. Pool NC2-containing fractions and adjust salt concentrations to BC200 using BC0 buffer. Load with a peristaltic pump P1 onto a heparin column (0.5 ml, preequilibrated) at a flow rate of 4 ml/h. 15. Wash column with 30 ml BC200 (can be done overnight). 16. Elute NC2 with 6 ml BC600 (flow rate 4 ml/h) and collect 0.5-ml aliquots. 17. Measure protein concentration and adjust to at least 500 ng/l with BSA. Usually, this protocol yields approximately 0.3 mg of recombinant NC2 proteins. 18. Add HEPES–KOH (pH 7.9 at 25 ) to 50 mM. 19. Aliquots are frozen in liquid nitrogen and stored at 80 .
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Immunoprecipitation of NC2 Complexes
NC2 complexes are stable in solution and can be precipitated with antibodies that recognize either the or the subunit (see Fig. 3).6 Antibodies are not yet available commercially. Hence, a stably expressed version of NC2 carrying a tag may be used instead. Here we used monoclonal antibodies directed against human NC2. The antibody coimmunoprecipitates and both of recombinant and native protein complexes isolated from logarithmically growing HeLa cells. The complex is held together, at least in part, by hydrophobic interactions, which manifest in great salt resistance in the IPs (Fig. 3A) but dissociation by RIPA buffer. Coupling of Antibody to Beads All centrifugation steps are done for 4 min at 200 g. 1. Incubate 2 mg of NC2 monoclonal antibody (from rat 4G7) with 1 ml of protein G beads for 2 h at room temperature; collect beads by centrifugation.
Fig. 3. (A) NC2 forms a stable complex in solution. (Top) IP and wash conditions. RIPA buffer contains 150 mM NaCl, 1% NP-40 (IGEPAL CA-630), 0.1% SDS, and 50 mM Tris– HCl (pH 8.0). BC buffer: 20 mM Tris–HCl (pH 7.3 at real time), 20% glycerol, 1 mM EDTA, and 150 mM KCl (BC150), 300 mM KCl (BC300), 500 mM KCl (BC500), or 850 mM KCl (BC850). (B) Chromatin IP using a monoclonal antibody against NC2 and polyclonal antibodies against TBP and NC2, respectively. The control was performed with an isotype antibody of the rat monoclonal NC2. The real-time PCR reaction is quantified by serial dilutions of input material. Radioactive PCR reaction is quantified by a phosphoimager.
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2. Wash beads twice with 10 ml phosphate-buffered saline (PBS). 3. Wash beads twice with 0.2 M sodium borate, pH 9.0. 4. Resuspend beads in 10 ml of 0.2 M sodium borate, pH 9.0, and add 52 mg dimethyl pimelidate (DMP) (final concentration of 20 mM). 5. Incubate for 30 min at room temperature with gentle mixing. 6. Block excess DMP by washing the beads once in 0.2 M ethanolamine, pH 8.0, followed by an incubation for 2 h at room temperature in 0.2 M ethanolamine, pH 8.0, with gentle mixing. 7. Wash beads twice with 10 ml PBS. 8. Resuspend the beads in PBS and add sodium azide to a final concentration of 0.02% and store at 4 . Procedure 1. Mix 20 l protein G–NC2 antibody-coupled beads, 300 ng recombinant NC2 proteins, and 100 l BC150. The BC150 buffer used in this method is supplemented with PMSF (final concentration 5 mM), DTT (final concentration 1 mM), and IGEPAL CA-630 (final concentration 0.1%). 2. Incubate for 3 h at 4 with gentle mixing. 3. Wash four times with 1 ml BC150. Recover the beads by centrifugation for 3 min at 300 g. 4. NC2 is eluted from antibody-coupled beads either with SDS buffer (4 min at 95 ) or with a peptide corresponding to the epitope. Add 40 l of the peptide (1 mg/ml in BC150) and incubate at room temperature for 1 h with shaking. 5. Analyze eluted samples by SDS–PAGE and Western blot. Detection of NC2 on Genes by Chromatin IP8,9
NC2 can be found on many eukaryotic genes.10 The amounts vary greatly depending on the microenvironment, the promoter structure, and signals (unpublished observations). However, the signals and factors that control NC2 occupancy are not fully understood. Hence, to understand the molecular processes on specific genes and their promoters, it will be interesting to monitor NC2 levels. The ChIP technique allows visualization of binding sites and levels of NC2 in vivo. As expected from the X-ray structure, NC2 is cross-linked readily to DNA and output signals are in the range of several percent of input comparable with histones. 8
V. Orlando, H. Strutt, and R. Paro, Methods 11, 205 (1997). V. Orlando, Trends Biochem. Sci. 25, 99 (2000). 10 J. V. Geisberg, F. C. Holstege, R. A. Young, and K. Struhl, Mol Cell Biol. 21, 2736 (2001). 9
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Materials 37% formaldehyde solution (Merck) Branson sonifier Glass beads (212–300 m, Sigma) Glycogen (Sigma) Protein G Sepharose 4 fast flow (Amersham Pharmacia Biotech) Bovine serum albumin (20 mg/ml, Roche Diagnostics GmbH) Proteinase K solution (15.6 mg/ml, Roche Diagnostics GmbH) Solutions PBS: 137 mM NaCl, 2.7 mM KCl, 6.5 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2–7.4 RPMI 1640 medium with l-glutamine (GIBCO) TBS: 10 mM Tris, pH 8.0, 150 mM NaCl Buffer A (sonification buffer): 50 mM HEPES–KOH, pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton, 0.1% sodium deoxycholate, 1 mM PMSF Buffer A with 500 mM NaCl Buffer B: 10 mM Tris, pH 8.0, 250 mM LiCl, 0.5% IGEPAL CA-630 (Sigma), 0.5 % sodium deoxycholate, 1 mM EDTA Buffer D (dialysis buffer): 5% glycerol, 1 mM EDTA, 10 mM Tris, pH 8.0, 1 mM PMSF Buffer E (elution buffer): 1% SDS, 0.1 M NaHCO3 10% sodium lauryl sarcosine Cells Jurkat J6 cells (human leukemic T-cell lymphoblast) Procedure 1. Suspension cultures of human J6 Jurkat cells are used to prepare cross-linked extracts. Typically 2 108 cells are harvested at a density of 5 105/ml by centrifugation at 1000 rpm (204 g) in a benchtop centrifuge. Wash and resuspend in 50 ml PBS. 2. For cross-linking, add 1.35 ml of 37% formaldehyde to a 50-ml cell suspension (final concentration 1%) and incubate at room temperature for 9 min on a roller mixer. Stop the cross-linking reaction with 0.47 g of solid glycine to 50 ml (final concentration 125 mM). 3. Spin down the cells at 1000 rpm (204 g) and wash twice with cold TBS. The pellet can be stored at 80 .
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4. Resuspend the cross-linked material in 10 ml buffer A, add glass beads, and sonicate in an ice-ethanol bath 10 times for 30 s with cooling phases of 1 min in between (duty cycle 70% and output control 3). The sample must not get warm! 5. Centrifuge for 10 min at 3500 rpm (2490 g) to remove glass beads. Collect the lysate in a new tube and adjust to 0.5% sarcosyl and 1.42 g/cm3 CsCl. 6. Separate cross-linked material from free protein, DNA, and RNA by gradient centrifugation in a Beckman SW 41 swinging bucket rotor at 38,000 rpm for 36 to 72 h at 18 . 7. After centrifugation, puncture the tube with a syringe and collect 500-l fractions dropwise. Check fractions on a 0.5 % agarose gel. Free DNA is usually in fractions 1–3, whereas cross-linked DNA (smear with a size of 2.0 to >30 kb) is found in fractions 6–12 corresponding to 1.38–1.39 g/cm3 CsCl. Take an aliquot of the fractions containing cross linked DNA, incubate for 2 h at 65 to remove cross-links, and check on a 1% agarose gel for DNA size. Pool fractions with an average size of 500 bp and dialyze overnight against buffer D. 8. Use 2.5 A260 for immunoprecipitation and adjust to buffer A in a total volume of 1 ml. As a preclearing step, 30 l of protein G-Sepharose is added and incubated for 2 h at 4 . 9. Protein G-Sepharose is removed by centrifugation at 4000 rpm (1500 g) in a microcentrifuge. Add 15 g of a monoclonal NC2 antibody to the supernatant and incubate overnight on a rotating instrument at 4 . As an ideal control, serve beads loaded with an isotype antibody. 10. Spin down at 13,000 rpm for 10 min to remove precipitates and transfer supernatant to fresh tubes. 11. Antibody-bound complexes are recovered by the addition of 30 l protein G-Sepharose, 2 g sonicated salmon sperm DNA, and 100 g BSA to the sample and by incubation for 2 h at 4 . 12. Immunocomplexes are precipitated by centrifugation at 4000 rpm (1500 g). Wash twice with buffer A, twice with buffer A adjusted to 500 mM NaCl, twice with buffer B, and twice with TE (1 ml each time). 13. For elution, incubate the beads with 400 l buffer E for 30 min at room temperature, spin down, and transfer the supernatant to a fresh tube. 14. Adjust to 500 mM NaCl and incubate for 5 h (or overnight) at 65 to reverse the cross-links. Add 6 g proteinase K and adjust to 100 mM Tris, pH 6.8, and 50 mM EDTA. Incubate at 56 for 1 h. 15. Extract once with phenol–chloroform and once with chloroform– isoamylalcohol. Precipitate with 2.5 volumes ethanol and 10 g glycogen.
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16. Dissolve the pellet in 30 l TE and use 1.5 l per polymerase chain reaction (PCR). Ten percent of input material is processed like eluted samples (reverse cross-link and phenol/chloroform extract) and used as a standard in the PCR reaction. PCR reactions are performed either with Taq polymerase in the presence of 1 Ci [-32P]dCTP (10 Ci/l, 3000 Ci/mmol) and separated on an polyacrylamide gel or by real time PCR according to the manufacturer’s instructions. A serial dilution of input DNA serves as a standard. Repression of Transcription by NC2
NC2 is a potent repressor of basal transcription in reconstituted in vitro systems. Efficient repression is dependent on the presence of both subunits and especially effective in reconstituted systems that contain TBP and lack TFIIA (Fig. 4) together with other recombinant and native partially purified basal factors (TFIIB, TFIIE, TFIIF and TFIIH, and RNA polymerase II, respectively). Repression is also seen, although to a lesser extent in the presence of TFIID (TBP and TAFs). In crude systems, excess NC2 also represses many (if not all) RNA polymerase II promoters. However, (and this discrepancy is not fully understood), stimulatory effects of NC2 were also reported for yeast and drosophila NC2 complexes.10–14
Fig. 4. Repression of transcription by NC2. Individual subunits and NC2 complex (approximately 50 ng) were added to a purified RNA polymerase II transcription system as indicated.
11
H. Ge, E. Martinez, C.-M. Chiang, and R. G. Roeder, Methods Enzymol. 274, 57 (1996). G. Prelich, Mol Cell Biol. 17, 2057 (1997). 13 M. Lemaire, J. Xie, M. Meisterernst, and M. A. Collart, Mol Microbiol. 36, 163 (2000). 14 P. J. Willy, R. Kobayashi, and J. T. Kadonaga, Science 290, 982 (2000). 12
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Materials 20 mg/ml bovine serum albumin (Roche Diagnostics GmbH) 40 U/l RNase block (Stratagene) Ultrapure NTP set (Amersham Pharmacia Biotech) 30 -O-Methylguanosine 50 -triphosphate (omG, Amersham Pharmacia) Plasmids6,11 pML53, carrying a downstream G-less cassette pMRG5, hybrid core promoter composed of HIV TATA box, the AdML initiator region, and five Gal4-binding sites upstream of the promoter upstream of G-less cassette Solutions Transcription stop solution: 7 M urea, 10 mM Tris, pH 7.8, 10 mM EDTA, 0.5% SDS, 100 mM LiCl, 100 g/ml tRNA, 300 mM sodium acetate Nucleotide mix: 10 mM ATP, 10 mM TTP, 0.5 mM CTP, 10 mM GTP, or 2 mM omG Loading dye: 97% deionized formamide, 20 mM Tris, pH 7.3, 10 mM EDTA, 0.05% bromphenol blue For reconstitution of basal transcription with a partially purified system, see Fig. 4. The following transcription reactions are based on factors purified as described elsewhere.6,11 Procedure Prepare a DNA mix, per reaction add: 50 ng pMRG5, 50 ng pML53, 0.8 l MgCl2 (100 mM stock solution), 0.5 l HEPES–KOH, pH 8.2 (1 M stock solution), 0.1 l DTT (1 M stock solution), 0.05 l PMSF (0.2 M stock solution), 0.2 l omG mix, and 0.8 l [-32P]CTP (10 Ci/l, 3000 Ci/mmol); adjust the DNA mixture to a volume of 10 l with H2O and BC buffer. The final salt concentration should be 60 mM KCl in a total reaction volume of 20 l. 1. In a separate microcentrifuge tube, mix 2 l of a NC2-phosphocellulose fraction (P11 0.5; 2 mg/ml) dialyzed to BC100, 10 ng recombinant TBP, 20 g BSA, and 0.5 l RNAse block to a total volume of 5 l. 2. Mix 14 l DNA mix and 5 l protein mixture and add 1 l either NC2 (40 ng/l) or NC2 (100 ng/l) or 1 l of a premixed NC2
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3. 4. 5. 6.
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(40 ng and 100 ng /l) in BC100 buffer. Alternatively, the coexpressed NC2 complex can be added at similar concentrations. Incubate at 28 for 60 min. Stop with 400 l stop solution, extract with phenol/chloroform, and precipitate with isopropanol for 1 h at 20 . Spin down for 30 min at 13,500 rpm in a microfuge, wash with 80% ethanol, and dissolve the dried pellet in 10 l loading dye. Analyze the transcripts on a 5 % gel (acrylamide:bisacrylamide 19:1) containing 8 M urea.
Reconstitution of Transcription in Nuclear Extract 1. Prepare DNA mix as described for the purified system but without adding the DNA template. For runon transcription, use linear RNA polymerase II transcription templates (20 to 200 ng), appropriate nucleotide mix and 50 to 200 ng of purified recombinant NC2 complex. RNA polymerase III transcription is readily measurable, i.e., from the adenovirus VAI promoter, but is not decreased under these conditions. 2. Incubate DNA mix with NC2 premixed in HeLa nuclear extracts (25–50 g of total protein) for 60 min at 28 and stop and process the reaction as described previously. Acknowledgments We thank Gregor Gilfillan and the members of the Meisterernst laboratory for critical reading of the manuscript. This work has been supported by grants of the DFG, the BMBF, and the EC to M.M.
[41] Hap1p Photofootprinting as an In Vivo Assay of Repression Mechanism in Saccharomyces cerevisiae By Mitsuhiro Shimizu and Aaron P. Mitchell Gene expression levels result from the dynamic interplay of activators and repressors. These factors may influence the basal transcriptional machinery directly or indirectly, through interactions that govern each other’s activity or access to DNA. One fundamental question in analysis of a repressor is whether it acts by preventing transcriptional activators from binding to DNA target sites (i.e., UASs or enhancers). We have used a
METHODS IN ENZYMOLOGY, VOL. 370
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[40] Purification and Transcription Repression by Negative Cofactor 2 By S. Gilfillan, G. Stelzer, E. Kremmer, and M. Meisterernst Negative cofactor 2 (NC2 also termed Dr1/DRAP) binds to promoters and inhibits transcription by RNA polymerase II by preventing formation of a bona fide preinitiation complex.1,2 Biochemical and genetic data suggest that NC2 binds directly to TBP and to DNA and competes with the general transcription factors TFIIA and TFIIB.3,4 The molecular details have been resolved at high resolution through determination of the crystal structure of the NC2–TBP–DNA complex.5 NC2 dimerizes through histone fold domains of the H2A-H2B type. As shown in Fig. 1, NC2 is situated underneath the DNA surface contacting both DNA and TBP. The inhibitory complex contacts TBP from two sides via contacts with both subunits. Loop region 1 of NC2 in the helix 1–loop 1–helix 2–loop 2–helix 3 arrangement of the histone fold and helix 5 within the carboxy-terminal region of NC2 interact directly with TBP. The structure satisfactorily explains repression by the dimeric NC2 complex through sterical occlusion of both TFIIA and TFIIB.1,2,4 It also provides a molecular model for a stimulatory function of NC2 through the histone fold in that it can, in principle, support binding of TBP to promoters. NC2 is thought to be involved in the regulation of many genes. It is possible that it attenuates transcription in a global manner. However, evidence also shows that NC2 promoter occupancy is controlled by upstream regulatory pathways. This article focuses on the human NC2 complex, provides methods for its isolation from HeLa cells, its expression in Escherichia coli, and its analysis in DNA–TBP binding and in transcription, as well as its detection on endogenous genes through chromatin immunoprecipitation (ChIP). While there is no evidence for a related factor in prokaryotes, NC2 is conserved from yeast to humans. Thus, the methods are applicable to other mammalian cell lines and, at least in part, to other eukaryotic organisms. The ChIP techniques described here will allow tracking and characterization of NC2 effects on specific genes in vivo. They will further allow determination of the relative amount of NC2 on genes and its control by regulatory factors. 1
M. Meisterernst and R. G. Roeder, Cell 67, 557 (1991). J. A. Inostroza, F. H. Mermelstein, H. Ilho, W. S. Lane, and D. Reinberg, Cell 70, 477 (1992). 3 Y. Cang, D. T. Auble, and G. Prelich, EMBO J. 18, 6662 (1999). 4 J. Xie, M. Collart, G. Stelzer, M. Lemaire, and M. Meisterernst, EMBO J. 19, 672 (2000). 2
METHODS IN ENZYMOLOGY, VOL. 370
Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00
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Fig. 1. NC2 dimerizes through histone fold domains and binds underneath the bent DNA–TBP complex.5
Purification of NC2 from HeLa Nuclear Extract
NC2 was originally defined as a factor that forms a stable complex with TBP in gel mobility shift experiments.1 While NC2 is sometimes difficult to detect in nuclear extracts of mammalian cells, the complex is readily detectable upon fractionation on phosphocellulose (see Fig. 2). Both NC2 and NC2 subunits cofractionate on P11 and subsequent columns.6 It has also been reported that Dr1 (NC2) binds DNA–TBP in the absence of NC2 (DRAP).2 This is usually not detected in crude fractions where carrier DNA is included in the gel shift experiments. Materials Phosphocellulose (P11, Whatman) DE52 (Whatman) DEAE-Sephadex (A 25, Amersham Pharmacia Biotech) Mono Q (Amersham Pharmacia Biotech) Solutions
BC buffer: 20 mM Tris (pH 7.3 at 25 ), 20% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM dithiothreitol (DTT), 1 mM EDTA, pH 8.0, 100 mM KCl (BC100), 200 mM KCl (BC200), etc. 5
K. Kamada, F. Shu, H. Chen, S. Malik, G. Stelzer, R. G. Roeder, M. Meisterernst, and S. K. Burley, Cell 106, 71 (2001). 6 A. Goppelt, G. Stelzer, F. Lottspeich, and M. Meisterernst, EMBO J. 15, 3105 (1996).
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Fig. 2. (A) Purification chart for NC2 isolated from HeLa nuclear extracts and fractionated on phosphocellulose (P11, Whatman). (B) EMSA using a 35-bp adenovirus major late promoter fragment assaying HeLa extracts fractionated on P11 fractions where NC2 elutes exclusively in the 0.5 M KCl fraction.
Procedure 1. Prepare HeLa nuclear extract from 8 1010 cells.7 2. Dialyze the nuclear extract (280 ml: approximately 10 mg/ml) against BC100 buffer and load onto a preequilibrated P11 column (capacity 10 mg/ml column volume) with a flow rate of 1 column volume per hour. Approximately 50% of the protein will be in the flow-through fraction. 3. Wash column with BC100 (10 column volumes overnight). 4. Elute the column stepwise with BC300 (approximately 30% of total protein amount), BC500 (approximately 5% of total protein amount), and BC850 (approximately 2.5% of total protein amount). 5. NC2 elutes in the 0.5 M KCl fraction. Dialyze against BC100 and load on a DE52 column (2 mg/ml resin). Flow rate is approximately 1 column volume per hour. Wash with BC100 and elute with a linear KCl gradient (0.1 to 0.5 M KCl in BC buffer). 7
M. Meisterernst, A. L. Roy, H. M. Lieu, and R. G. Roeder, Cell 66, 981 (1991).
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6. NC2 elutes at 0.18 to 0.29 M KCl; pool fractions and dialyze against BC100. Load onto a DEAE-Sephadex (A25) column and elute with a linear gradient from 0.1 to 0.5 M KCl in BC buffer. 7. Pool the fractions in the range of 0.17 to 0.28 M KCl; dialyze against BC200 and load on a Mono Q column. Wash the column with 10 column volumes of BC200 and elute directly with BC400. 8. Analyze eluted fractions by SDS–PAGE. Analysis of NC2 in the Electrophoretic Mobility Shift Assay (EMSA)
Annealed oligonucleotides or DNA fragments isolated from a vector can be used to analyze NC2–TBP and NC2 alone in EMSA. NC2 is a nonspecific DNA-binding protein and will thus also bind DNA in the absence of TBP.6 The addition of TBP shifts the complex to a lower mobility form that is more stable and more resistant to the addition of carrier DNA such as p[dGdC]. However, the amount of carrier must be determined carefully through titration. NC2 does not bind single-stranded DNA and RNA. Solutions/Reagents Annealing buffer: 200 mM NaCl, 10 mM Tris, pH 7.3, 1 mM MgCl2 MicroSpin G-25 columns (Amersham Pharmacia) Bovine serum albumin (BSA) (New England Biolabs) Klenow fragment (MBI Fermentas) [-32P]dNTP (3000 Ci/mmol, Amersham Pharmacia) TGE: 25 mM Tris, pH 7.3, 248 mM glycine, 1 mM EDTA, pH 8.0 Annealing Oligonucleotides 1. Measure the concentration of oligonucleotides by A260. 2. Mix oligonucleotides in annealing buffer (100 pmol of each oligonucleotide in a total volume of 20 l) and transfer to a heat block at 95 . Turn the heat block off after 3 min and let the sample cool gradually to room temperature. 3. The DNA can be stored at 4 and labeled by Klenow fill-in by standard procedures. Procedure for EMSA The binding reactions for EMSA are 4 mM MgCl2, 25 mM HEPES– KOH, pH 8.2, 0.4 mg/ml BSA, 5 mM DTT, 0.5 mM PMSF, 1–5 fmol/l labeled DNA. The final concentration of glycerol is kept between 6 and 10%, and the final concentration of KCl is typically 70 mM. The binding reaction is carried out in a final volume of 20 l.
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1. 2. 3. 4. 5.
Distribute the components for the binding reaction to each sample. Add proteins; TBP is added last. Mix gently by pipetting up and down. Incubate at 27 for 30 min. Load samples on a 5% gel (acrylamide:bisacrylamide, 50:1). The gel is prerun 1 h at 120 V in TGE, and the TGE buffer is changed after the prerun. The gel usually runs 3 h at 100 V with a 35-bp DNA. 6. Place the gel on 3MM Whatman paper and dry it on a gel dryer. 7. Expose the gel to a film (BioMax MR, Kodak), typically 1 h at 80 and/or quantify the bands using a phosphoimager.
Expression and Purification of Recombinant NC2 in E. coli
To test the influence of NC2 on transcription, the two subunits of NC2 can be expressed individually in E.coli. This usually results in low levels of NC2. In contrast, NC2 is, under certain conditions, overexpressed greatly, but the protein is insoluble and found in inclusion bodies. When coexpressed, both subunits can be purified under native conditions. We used the pET11d expression vector and NC2 subunits that both harbor a hexa-histidine tag at their N termini (T7 system from Novagen). Materials and Reagents Escherichia coli BL21(DE3) pLys S (Novagen) Isopropyl--d-thiogalactoside (IPTG, Roth) Branson digital sonifier Columns (0.5 ml, Bio-Rad) Peristaltic pump P-1 (Amersham Pharmacia Biotech) Ni2þ-NTA resin (Qiagen) Proteins assay dye reagent concentrate (Bio-Rad) Heparin Sepharose (Amersham Pharmacia Biotech) BSA (Roche Diagnostics GmbH) Solutions Isopropyl--D-thiogalactoside, 0.5 M stock solution in water, sterile filtered Imidazole hydrochloride: 1 M stock, adjust pH to 7.9 with NaOH Lysis buffer: 500 mM NaCl, 10% glycerol, 20 mM Tris–HCl, pH 7.3. Add 2-mercaptoethanol (to 5 mM), IGEPAL CA-630 (NP-40) to 0.1%, leupeptin (to 2 g/ml), pepstatin A (to 1 g/ml), benzamidine (to 20 g/ml), and PMSF (to 1 mM) freshly HEPES–KOH 1 M stock (pH 7.9 at 25 )
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Procedure 1. Transform E. coli BL21(DE3) pLysS with the vector-containing histidine-tagged NC2 and select on LB plates containing ampicillin and chloramphenicol. 2. Inoculate a 50-ml starter culture of LB/ampicillin/chloramphenicol and grow overnight. 3. Dilute from the starting culture into 500 ml LB/ampicillin/ chloramphenicol (to OD600 0.1) and grow with vigorous shaking at 37 . 4. Add IPTG to 0.5 mM final concentration when OD600 is 0.6. 5. Grow the cells for 3 h at 30 with shaking. 6. Centrifuge the cells for 5 min at 5000 g. 7. Resuspend pellet in 20 ml lysis buffer (the resuspended bacteria can be frozen and stored at 80 ). All remaining steps are performed at 4 with prechilled buffers. 8. Sonicate the sample on ice: amplitude 20% for 3 min; on 0.2 s, off 0.8 s (i.e., total elapsed time 15 min). 9. Spin for 30 min in SS34 rotor at 18,000 rpm (39,000 g). 10. Add imidazole to the supernatant to a final concentration of 5 mM and load onto a preequilibrated Ni2þ column (0.8 ml) using peristaltic pump P1 (flow rate: 5 ml/h). 11. Wash the column with 50 ml of BC400 including 10 mM imidazole (can be done overnight). 12. Elute the recombinant NC2 dimer with 8 ml 100 mM imidazole in BC400 (flow rate 4 ml/h) and collect 1-ml aliquots. 13. Analyze an aliquot from each fraction in a 1:10 dilution with a protein assay dye reagent concentrate from Bio-Rad (diluted 1:5 and filtered through 3MM Whatman paper). 14. Pool NC2-containing fractions and adjust salt concentrations to BC200 using BC0 buffer. Load with a peristaltic pump P1 onto a heparin column (0.5 ml, preequilibrated) at a flow rate of 4 ml/h. 15. Wash column with 30 ml BC200 (can be done overnight). 16. Elute NC2 with 6 ml BC600 (flow rate 4 ml/h) and collect 0.5-ml aliquots. 17. Measure protein concentration and adjust to at least 500 ng/l with BSA. Usually, this protocol yields approximately 0.3 mg of recombinant NC2 proteins. 18. Add HEPES–KOH (pH 7.9 at 25 ) to 50 mM. 19. Aliquots are frozen in liquid nitrogen and stored at 80 .
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Immunoprecipitation of NC2 Complexes
NC2 complexes are stable in solution and can be precipitated with antibodies that recognize either the or the subunit (see Fig. 3).6 Antibodies are not yet available commercially. Hence, a stably expressed version of NC2 carrying a tag may be used instead. Here we used monoclonal antibodies directed against human NC2. The antibody coimmunoprecipitates and both of recombinant and native protein complexes isolated from logarithmically growing HeLa cells. The complex is held together, at least in part, by hydrophobic interactions, which manifest in great salt resistance in the IPs (Fig. 3A) but dissociation by RIPA buffer. Coupling of Antibody to Beads All centrifugation steps are done for 4 min at 200 g. 1. Incubate 2 mg of NC2 monoclonal antibody (from rat 4G7) with 1 ml of protein G beads for 2 h at room temperature; collect beads by centrifugation.
Fig. 3. (A) NC2 forms a stable complex in solution. (Top) IP and wash conditions. RIPA buffer contains 150 mM NaCl, 1% NP-40 (IGEPAL CA-630), 0.1% SDS, and 50 mM Tris– HCl (pH 8.0). BC buffer: 20 mM Tris–HCl (pH 7.3 at real time), 20% glycerol, 1 mM EDTA, and 150 mM KCl (BC150), 300 mM KCl (BC300), 500 mM KCl (BC500), or 850 mM KCl (BC850). (B) Chromatin IP using a monoclonal antibody against NC2 and polyclonal antibodies against TBP and NC2, respectively. The control was performed with an isotype antibody of the rat monoclonal NC2. The real-time PCR reaction is quantified by serial dilutions of input material. Radioactive PCR reaction is quantified by a phosphoimager.
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2. Wash beads twice with 10 ml phosphate-buffered saline (PBS). 3. Wash beads twice with 0.2 M sodium borate, pH 9.0. 4. Resuspend beads in 10 ml of 0.2 M sodium borate, pH 9.0, and add 52 mg dimethyl pimelidate (DMP) (final concentration of 20 mM). 5. Incubate for 30 min at room temperature with gentle mixing. 6. Block excess DMP by washing the beads once in 0.2 M ethanolamine, pH 8.0, followed by an incubation for 2 h at room temperature in 0.2 M ethanolamine, pH 8.0, with gentle mixing. 7. Wash beads twice with 10 ml PBS. 8. Resuspend the beads in PBS and add sodium azide to a final concentration of 0.02% and store at 4 . Procedure 1. Mix 20 l protein G–NC2 antibody-coupled beads, 300 ng recombinant NC2 proteins, and 100 l BC150. The BC150 buffer used in this method is supplemented with PMSF (final concentration 5 mM), DTT (final concentration 1 mM), and IGEPAL CA-630 (final concentration 0.1%). 2. Incubate for 3 h at 4 with gentle mixing. 3. Wash four times with 1 ml BC150. Recover the beads by centrifugation for 3 min at 300 g. 4. NC2 is eluted from antibody-coupled beads either with SDS buffer (4 min at 95 ) or with a peptide corresponding to the epitope. Add 40 l of the peptide (1 mg/ml in BC150) and incubate at room temperature for 1 h with shaking. 5. Analyze eluted samples by SDS–PAGE and Western blot. Detection of NC2 on Genes by Chromatin IP8,9
NC2 can be found on many eukaryotic genes.10 The amounts vary greatly depending on the microenvironment, the promoter structure, and signals (unpublished observations). However, the signals and factors that control NC2 occupancy are not fully understood. Hence, to understand the molecular processes on specific genes and their promoters, it will be interesting to monitor NC2 levels. The ChIP technique allows visualization of binding sites and levels of NC2 in vivo. As expected from the X-ray structure, NC2 is cross-linked readily to DNA and output signals are in the range of several percent of input comparable with histones. 8
V. Orlando, H. Strutt, and R. Paro, Methods 11, 205 (1997). V. Orlando, Trends Biochem. Sci. 25, 99 (2000). 10 J. V. Geisberg, F. C. Holstege, R. A. Young, and K. Struhl, Mol Cell Biol. 21, 2736 (2001). 9
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Materials 37% formaldehyde solution (Merck) Branson sonifier Glass beads (212–300 m, Sigma) Glycogen (Sigma) Protein G Sepharose 4 fast flow (Amersham Pharmacia Biotech) Bovine serum albumin (20 mg/ml, Roche Diagnostics GmbH) Proteinase K solution (15.6 mg/ml, Roche Diagnostics GmbH) Solutions PBS: 137 mM NaCl, 2.7 mM KCl, 6.5 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2–7.4 RPMI 1640 medium with l-glutamine (GIBCO) TBS: 10 mM Tris, pH 8.0, 150 mM NaCl Buffer A (sonification buffer): 50 mM HEPES–KOH, pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton, 0.1% sodium deoxycholate, 1 mM PMSF Buffer A with 500 mM NaCl Buffer B: 10 mM Tris, pH 8.0, 250 mM LiCl, 0.5% IGEPAL CA-630 (Sigma), 0.5 % sodium deoxycholate, 1 mM EDTA Buffer D (dialysis buffer): 5% glycerol, 1 mM EDTA, 10 mM Tris, pH 8.0, 1 mM PMSF Buffer E (elution buffer): 1% SDS, 0.1 M NaHCO3 10% sodium lauryl sarcosine Cells Jurkat J6 cells (human leukemic T-cell lymphoblast) Procedure 1. Suspension cultures of human J6 Jurkat cells are used to prepare cross-linked extracts. Typically 2 108 cells are harvested at a density of 5 105/ml by centrifugation at 1000 rpm (204 g) in a benchtop centrifuge. Wash and resuspend in 50 ml PBS. 2. For cross-linking, add 1.35 ml of 37% formaldehyde to a 50-ml cell suspension (final concentration 1%) and incubate at room temperature for 9 min on a roller mixer. Stop the cross-linking reaction with 0.47 g of solid glycine to 50 ml (final concentration 125 mM). 3. Spin down the cells at 1000 rpm (204 g) and wash twice with cold TBS. The pellet can be stored at 80 .
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4. Resuspend the cross-linked material in 10 ml buffer A, add glass beads, and sonicate in an ice-ethanol bath 10 times for 30 s with cooling phases of 1 min in between (duty cycle 70% and output control 3). The sample must not get warm! 5. Centrifuge for 10 min at 3500 rpm (2490 g) to remove glass beads. Collect the lysate in a new tube and adjust to 0.5% sarcosyl and 1.42 g/cm3 CsCl. 6. Separate cross-linked material from free protein, DNA, and RNA by gradient centrifugation in a Beckman SW 41 swinging bucket rotor at 38,000 rpm for 36 to 72 h at 18 . 7. After centrifugation, puncture the tube with a syringe and collect 500-l fractions dropwise. Check fractions on a 0.5 % agarose gel. Free DNA is usually in fractions 1–3, whereas cross-linked DNA (smear with a size of 2.0 to >30 kb) is found in fractions 6–12 corresponding to 1.38–1.39 g/cm3 CsCl. Take an aliquot of the fractions containing cross linked DNA, incubate for 2 h at 65 to remove cross-links, and check on a 1% agarose gel for DNA size. Pool fractions with an average size of 500 bp and dialyze overnight against buffer D. 8. Use 2.5 A260 for immunoprecipitation and adjust to buffer A in a total volume of 1 ml. As a preclearing step, 30 l of protein G-Sepharose is added and incubated for 2 h at 4 . 9. Protein G-Sepharose is removed by centrifugation at 4000 rpm (1500 g) in a microcentrifuge. Add 15 g of a monoclonal NC2 antibody to the supernatant and incubate overnight on a rotating instrument at 4 . As an ideal control, serve beads loaded with an isotype antibody. 10. Spin down at 13,000 rpm for 10 min to remove precipitates and transfer supernatant to fresh tubes. 11. Antibody-bound complexes are recovered by the addition of 30 l protein G-Sepharose, 2 g sonicated salmon sperm DNA, and 100 g BSA to the sample and by incubation for 2 h at 4 . 12. Immunocomplexes are precipitated by centrifugation at 4000 rpm (1500 g). Wash twice with buffer A, twice with buffer A adjusted to 500 mM NaCl, twice with buffer B, and twice with TE (1 ml each time). 13. For elution, incubate the beads with 400 l buffer E for 30 min at room temperature, spin down, and transfer the supernatant to a fresh tube. 14. Adjust to 500 mM NaCl and incubate for 5 h (or overnight) at 65 to reverse the cross-links. Add 6 g proteinase K and adjust to 100 mM Tris, pH 6.8, and 50 mM EDTA. Incubate at 56 for 1 h. 15. Extract once with phenol–chloroform and once with chloroform– isoamylalcohol. Precipitate with 2.5 volumes ethanol and 10 g glycogen.
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16. Dissolve the pellet in 30 l TE and use 1.5 l per polymerase chain reaction (PCR). Ten percent of input material is processed like eluted samples (reverse cross-link and phenol/chloroform extract) and used as a standard in the PCR reaction. PCR reactions are performed either with Taq polymerase in the presence of 1 Ci [-32P]dCTP (10 Ci/l, 3000 Ci/mmol) and separated on an polyacrylamide gel or by real time PCR according to the manufacturer’s instructions. A serial dilution of input DNA serves as a standard. Repression of Transcription by NC2
NC2 is a potent repressor of basal transcription in reconstituted in vitro systems. Efficient repression is dependent on the presence of both subunits and especially effective in reconstituted systems that contain TBP and lack TFIIA (Fig. 4) together with other recombinant and native partially purified basal factors (TFIIB, TFIIE, TFIIF and TFIIH, and RNA polymerase II, respectively). Repression is also seen, although to a lesser extent in the presence of TFIID (TBP and TAFs). In crude systems, excess NC2 also represses many (if not all) RNA polymerase II promoters. However, (and this discrepancy is not fully understood), stimulatory effects of NC2 were also reported for yeast and drosophila NC2 complexes.10–14
Fig. 4. Repression of transcription by NC2. Individual subunits and NC2 complex (approximately 50 ng) were added to a purified RNA polymerase II transcription system as indicated.
11
H. Ge, E. Martinez, C.-M. Chiang, and R. G. Roeder, Methods Enzymol. 274, 57 (1996). G. Prelich, Mol Cell Biol. 17, 2057 (1997). 13 M. Lemaire, J. Xie, M. Meisterernst, and M. A. Collart, Mol Microbiol. 36, 163 (2000). 14 P. J. Willy, R. Kobayashi, and J. T. Kadonaga, Science 290, 982 (2000). 12
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Materials 20 mg/ml bovine serum albumin (Roche Diagnostics GmbH) 40 U/l RNase block (Stratagene) Ultrapure NTP set (Amersham Pharmacia Biotech) 30 -O-Methylguanosine 50 -triphosphate (omG, Amersham Pharmacia) Plasmids6,11 pML53, carrying a downstream G-less cassette pMRG5, hybrid core promoter composed of HIV TATA box, the AdML initiator region, and five Gal4-binding sites upstream of the promoter upstream of G-less cassette Solutions Transcription stop solution: 7 M urea, 10 mM Tris, pH 7.8, 10 mM EDTA, 0.5% SDS, 100 mM LiCl, 100 g/ml tRNA, 300 mM sodium acetate Nucleotide mix: 10 mM ATP, 10 mM TTP, 0.5 mM CTP, 10 mM GTP, or 2 mM omG Loading dye: 97% deionized formamide, 20 mM Tris, pH 7.3, 10 mM EDTA, 0.05% bromphenol blue For reconstitution of basal transcription with a partially purified system, see Fig. 4. The following transcription reactions are based on factors purified as described elsewhere.6,11 Procedure Prepare a DNA mix, per reaction add: 50 ng pMRG5, 50 ng pML53, 0.8 l MgCl2 (100 mM stock solution), 0.5 l HEPES–KOH, pH 8.2 (1 M stock solution), 0.1 l DTT (1 M stock solution), 0.05 l PMSF (0.2 M stock solution), 0.2 l omG mix, and 0.8 l [-32P]CTP (10 Ci/l, 3000 Ci/mmol); adjust the DNA mixture to a volume of 10 l with H2O and BC buffer. The final salt concentration should be 60 mM KCl in a total reaction volume of 20 l. 1. In a separate microcentrifuge tube, mix 2 l of a NC2-phosphocellulose fraction (P11 0.5; 2 mg/ml) dialyzed to BC100, 10 ng recombinant TBP, 20 g BSA, and 0.5 l RNAse block to a total volume of 5 l. 2. Mix 14 l DNA mix and 5 l protein mixture and add 1 l either NC2 (40 ng/l) or NC2 (100 ng/l) or 1 l of a premixed NC2
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3. 4. 5. 6.
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(40 ng and 100 ng /l) in BC100 buffer. Alternatively, the coexpressed NC2 complex can be added at similar concentrations. Incubate at 28 for 60 min. Stop with 400 l stop solution, extract with phenol/chloroform, and precipitate with isopropanol for 1 h at 20 . Spin down for 30 min at 13,500 rpm in a microfuge, wash with 80% ethanol, and dissolve the dried pellet in 10 l loading dye. Analyze the transcripts on a 5 % gel (acrylamide:bisacrylamide 19:1) containing 8 M urea.
Reconstitution of Transcription in Nuclear Extract 1. Prepare DNA mix as described for the purified system but without adding the DNA template. For runon transcription, use linear RNA polymerase II transcription templates (20 to 200 ng), appropriate nucleotide mix and 50 to 200 ng of purified recombinant NC2 complex. RNA polymerase III transcription is readily measurable, i.e., from the adenovirus VAI promoter, but is not decreased under these conditions. 2. Incubate DNA mix with NC2 premixed in HeLa nuclear extracts (25–50 g of total protein) for 60 min at 28 and stop and process the reaction as described previously. Acknowledgments We thank Gregor Gilfillan and the members of the Meisterernst laboratory for critical reading of the manuscript. This work has been supported by grants of the DFG, the BMBF, and the EC to M.M.
[41] Hap1p Photofootprinting as an In Vivo Assay of Repression Mechanism in Saccharomyces cerevisiae By Mitsuhiro Shimizu and Aaron P. Mitchell Gene expression levels result from the dynamic interplay of activators and repressors. These factors may influence the basal transcriptional machinery directly or indirectly, through interactions that govern each other’s activity or access to DNA. One fundamental question in analysis of a repressor is whether it acts by preventing transcriptional activators from binding to DNA target sites (i.e., UASs or enhancers). We have used a
METHODS IN ENZYMOLOGY, VOL. 370
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