- Email: [email protected]
Assay of an Intrinsic Acetyltransferase Activity of the Transcriptional Coactivator CIITA
378
[33]
polymerase associated factors
the broadening of the average image method has lower time resolution and does not reveal either the actual length or the distribution of tether lengths but is immune to blurring. Accuracy and precision of this TPM method have been published9 and can be implemented using routines available under a general public license software agreement (www.bio. brandeis.edu/gelles/software/index.html).
[33] Assay of an Intrinsic Acetyltransferase Activity of the Transcriptional Coactivator CIITA By Jocelyn D. Weissman, Aparna Raval, and Dinah S. Singer Posttranslational modification of proteins is increasingly being recognized as an important regulatory mechanism in transcription. The best characterized of the posttranslational modifications are the phosphorylations of proteins involved in regulating signal transduction pathways and glycosylation of membrane proteins. However, studies have demonstrated that acetylation and methylation play pivotal roles in transcriptional regulation.1 Acetylation of nucleosomal histones is associated with transcriptionally active chromatin, whereas methylation correlates primarily with silent regions of chromatin. A number of nonhistone proteins have been demonstrated to undergo acetylation, although the function of this modification is not understood. Among the proteins shown to be acetylated are p53, ATF2, HIV Tat, and the coactivator CIITA.2–4 A number of proteins with acetyltransferase enzymatic (AT) activity have been identified. Most of these proteins, such as CBP, p300, and PCAF, were first identified as transcriptional coactivators and later shown to acetylate histones.5 Other proteins such as TAFII250 and Tip60, which mediate transcription but do not function as coactivators, also have been shown to have AT activity.6,7 Interestingly, the substrates of these acetyltransferases have not been well characterized. Although the coactivators appear to primarily acetylate histones, some are able to acetylate 1
T. Jenuwein and C. D. Allis, Science 294, 2477 (2001). B. Cullen, FASEB J. 5, 2361 (1991). 3 V. Ogrysko, Cell. Mol. Life Sci. 58, 683 (2001). 4 A. Raval, T. K. Howcroft, J. D. Weissman, S. Krishner, X. S. Zhu, K. Yokoyama, J. Ting, and D. S. Singer, Mol. Cell. 7, 105 (2001). 5 V. Ogryzko, L. Schiltz, V. Russanova, B. Howard, and Y. Nakatani, Cell 87, 953 (1996). 6 C. Mizzen, X. Yang, T. Kokubo, J. Brownell, A. Bannister et al. Cell 87, 1261 (1996). 7 Y. Yamamoto and M. Horikoshi, J. Biol. Chem. 272, 30595 (1997). 2
METHODS IN ENZYMOLOGY, VOL. 370
0076-6879/03 $35.00
[33]
transcriptional coactivator CIITA
379
nonhistone proteins. For example, CBP and PCAF have been shown to acetylate CIITA.8,9 Similarly, the AT activity of TAFII250 is required for its function in in vitro transcription of naked DNA in the absence of nucleosomal histones.10 The transcriptional coactivator CIITA mediates the interferon- response by nucleating formation of the transcriptional enhanceosome that activates MHC class I and class II transcription.11 We have demonstrated that CIITA, like other coactivators, contains intrinsic AT activity.4 Unlike other coactivators, this AT activity is stimulated by GTP. The CIITA enzymatic activity appears to depend on protein conformation, as it is only detected in protein purified either from transfected mammalian cells or from insect Sf9 cells; bacterially derived CIITA does not contain detectable AT activity. The methods given here describe the purification of both insect- and mammalian-derived CIITA, the acetyltransferase assay using histones as a substrate, and Western blotting to confirm purification of the protein. In all of these protocols, we have used a flag-tagged CIITA. It is also important to note that the AT activity of CIITA as measured with histone substrates is considerably less than that of CBP or p300. This may reflect either an intrinsic difference in enzymatic activity or that histones are not the native substrate of CIITA. Preparation of Recombinant CIITA from Baculovirus
rCIITA purified from a baculovirus that has been harvested from insect Sf9 cells, as described later, reproducibly displays readily detectable histone acetyltransferase activity. To facilitate purification and monitoring of the CIITA, the protein is flag tagged at the 50 end of the molecule. However, purification of enzymatically active CIITA depends on the conditions in which Sf9 cells are grown and maintained and the conditions of transfection and viral infection. The following protocols describe the growth of the cells, their transfection with the CIITA gene cloned into a baculoviral vector to generate viral stocks, the isolation of viral stocks, the infection of Sf9 cells for the quantitative isolation of virus containing CIITA, and purification of the CIITA from the virus. 8
J. D. Fontes, S. Kanazawa, D. Jean, and B. M. Peterlin, Mol. Cell Biol. 19, 941 (1999). A. Kretsovali, T. Agalioti, C. Spilianakis, E. Tzortzakaki, M. Merika, and J. Papamatheakis, Mol. Cell Biol. 18, 6777 (1998). 10 J. D. Weissman, J. Brown, T. K. Howcroft, J. Hwang, A. Chawla, P. Roche, L. Schiltz, Y. Nakatani, and D. S. Singer, Proc. Natl. Acad. Sci. USA 95, 11601 (1998). 11 K. Masternak, A. Muhlethaler-Mottet, J. Villard, M. Zufferey, V. Steimle, and W. Reith, Genes Dev. 14, 11566 (2000). 9
380
polymerase associated factors
[33]
Reagents SF9 culture medium: TMN-FH (Gemini Bio-Products) Baculogold transfection kit: Pharmingen kit Phosphate-buffered saline (PBS) Lysis buffer: 50 mM Tris–Cl, pH 8.0, 5 mM MgCl2, 150 mM KCl, 0.1% NP-40, 10% glycerol, freshly added 10 g/ml pepstatin, 5 g/ml leupeptin, 20 g/ml aprotinin, 1 mM phenylethylsulforyl fluoride (PMSF) Antiflag agarose beads: Sigma A1205 TBS: 50 mM Tris, pH 8.0, 150 mM NaCl 0.1 M glycine, pH 3.5 Flag peptide stock solution: 5 mg/ml in TBS Buffer D: 20 mM HEPES, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol (DTT), 20% glycerol(for Hi-Trap columns) Growth of Sf9 Cells Sf9 insect cells can be grown as either a monolayer in a tissue culture flask or in suspension culture in spinner flasks. In either case, care should be taken while growing Sf9 cells, as they are extremely sensitive to fluctuations in temperature, cell density, and agitation. Spinner SF9 cultures have to be maintained at 27 at a density of 5 5 7 10 to 2 10 cells/ml and under gentle shaking. Cells are harvested by centrifugation at 500 rpm or less for 10 min with no brake. Monolayer cultures should be plated at a density of 107 cells in 30 ml medium in a T100 plate. Cells should be transferred to fresh medium every 2–3 days by harvesting with gentle agitation and dilution at about 1:3 into a clean plate to reestablish the original seeding density. (The cells are extremely fragile and may be broken by scraping with a rubber policeman.) Transfection of Sf9 Cells to Generate Viral Seed Culture The following protocol describes transfection into SF9 cells of the baculovirus expression vector, PVL 1393 with the inserted CIITA cDNA, recovery of the recombinant virus, and expansion of the virus. Then, expression of the CIITA recombinant protein is analyzed by Western blotting of infected SF9 cell extracts. Day 2: Transfer Sf9 cells to fresh medium before transfection to ensure that cells are growing in an exponential phase. Day 1: Harvest cells and plate at 3 million cells/10 ml in a T25 flask. Plate an additional flask for a nontransfected control. Close and set it aside for 1 h to let cells adhere. Check under a microscope for adherence before
[33]
transcriptional coactivator CIITA
381
proceeding. Bring buffers A and B (from Pharmingen Baculovirus Gold kit) to room temperature. Combine 0.5 g of Baculovirus Gold DNA from the kit with 4 g of CIITA expression vector PVL 1323 (instead of the 2 g suggested by the kit protocol) in an Eppendorf tube. Leave at room temperature for 5 min. While waiting, aspirate the medium from Sf9 cells in the T25 flask. Add 1.25 ml of buffer A. After the 5-min incubation, add 1.25 ml buffer B to the DNA mixture and mix well. Add this mix dropwise to the flask containing the cells. Leave the flask at 27 for 4 h. At this point, the supernatant should look cloudy. Remove the supernatant by aspiration and add 4 ml fresh SF9 medium. Incubate for 48 h at 27 . Day 3: Examine the cells in the flask under a microscope. Transfected cells should appear larger than those in the nontransfected control flask. Replace 2 ml of the old culture medium with 2 ml fresh SF9 medium. Day 5: At this point, most of the remaining cells should be floating, not adherent, although a few may remain attached to the flask. This is an indication of a successful transfection. Using sterile technique, remove the medium with the floating cells in a sterile 15-ml tube and centrifuge at 1200 rmp for 10 min to clear debris. Store the cleared supernatant at 4 . If large numbers of cells remain attached to the flask, add an additional 4 ml of fresh medium and continue to incubate the cells for 2 more days. Day 7: Remove the medium, spin, and store the supernatant at 4 . Do not combine with first supernatant. Viral Assay To assay for the presence of viral particles in the culture supernatants, seed 3 million Sf9 cells in a small T25 flask, allowing them to adhere at 27 for 1 h. Remove medium. Sterilely mix 600 l of supernatant from day 5 with 600 l of fresh medium and add to flask. Incubate for 1 h. Every 10–15 min rotate the flask gently to make sure that the entire surface of the flask is covered. After 1 h add 3 ml medium and incubate at 27 for 48 h. After 48 h, harvest the cells and make lysate (see later). Run SDS–PAGE and Western to determine if protein has been made. Because there is considerable variation in the rate and the stability of the produced recombinant proteins, a time course should be run to determine the optimal time for harvesting of the supernatants after transfection. Preparing a Large-Scale Viral Stock If the transfection has been successful, as measured by the production of CIITA protein, a viral stock is generated. Take 30 million cells in a large T flask and let them adhere for 1 h. Add 2 ml of the viral seed culture (supernatant from day 5 or 7) and 8 ml fresh
382
polymerase associated factors
[33]
medium. Incubate at 27 for 1 h, rotating every 10–15 min. After 1 h, add 30 ml fresh medium. Harvest cells on day 5 in a 50-ml sterile tube and spin at 1200 rpm for 10 min and store the supernatant at 4 . This is the viral stock. Protein Production and Purification Place 200 million cells (1–1.5 million cells/ml) in a spinner flask and add 10 ml of viral stock. (At some point, you will need to make Sf9 cell extract from a nontransfected culture to use as control.) Incubate at 27 for 48 h. Harvest cells and spin at room temperature, 500 rpm with no brake. Remove the supernatant and wash the pellet once with room temperature PBS. Immediately freeze the pellet on dry ice. In general, the pellet should be processed immediately, although some proteins may be left at 70 for a few days. Thaw the pellet at 37 for 5 min. Add 3–4 ml lysis buffer and incubate on ice for 20 min. Break open the cells with passage through a 23-gauge needle twice. Spin lysate at 40,000 rpm at 4 for 20 min to remove debris. Collect the supernatant, which can be stored at 70 . Repeated freezing and thawing may lead to protein degradation. Antiflag Bead Activation The CIITA protein used in our studies has an N-terminal flag tag that is used as the basis of the protein purification. During ultracentrifugation of the lysate described earlier activate the antiflag beads. Wash 500 l of antiflag agarose beads three times with TBS as follows: place beads in a 1.5-ml Eppendorf tube, add 1 ml of TBS, and invert several times. Spin at 2000 rpm for 2–3 min. Discard the supernatant and repeat the washing step. Then wash three times with 0.1 M glycine, pH 3.5, to activate the beads. Wash again with TBS. Finally wash once in lysis buffer with protease inhibitors. Resuspend in 1 ml lysis buffer (i.e., 2X original bead volume). Notes. The beads should be activated fresh everytime, before the immunoprecipitation. The beads should not be left in the glycine buffer more than 20 min. Protein Immunoprecipitation Add 1 ml of the activated antiflag agarose bead slurry to the 3–4 ml of cleared viral lysate prepared in earlier. (A nontransfected control lysate should be processed in parallel.) Rotate the mixture at 4 for 4 h and then spin down the bead at 2000 rpm for 2–3 min. Wash the beads five times with 10 ml of lysis buffer (with protease inhibitors). Elute the bound
[33]
transcriptional coactivator CIITA
383
protein with 500 l of the flag peptide solution, freshly diluted to 200 g/ml in TBS. Elute four times by adding 500 l each time of this 200-g/ml solution (before eluting, check pH of flag peptides, which should not be acidic). After each 500-l addition, spin in a horizontal rotor at 2000 rpm for 15 min at 4 . Collect the supernatant in a fresh tube. The first two elutions have most of the proteins. Therefore, combine the first and second elutions separately from the third and fourth. This results in two aliquots of protein of 1 ml each (four elutions of 500 l each). Dialyze each aliquot against three changes of buffer of interest for 4 h to remove flag peptides. If flag peptides remain in the protein preparation after the dialysis, they can be removed by gel chromatography or Hi-Trap columns. For Hi-Trap column chromatography, the Hi-Trap column is washed with buffer D and elution is also done in buffer D. After elution, concentrate the eluate in a 30 or 50K cutoff Microcon or Centricon and then add fresh protease inhibi tors, pepstatin, leupeptin, and aprotinin. Make aliquots and store at 70 . To prevent loss of protein due to denaturation, the entire process should be completed in 1 day. Preparation of CIITA from Transfected HeLa Cells
CIITA is prepared from HeLa cells, transfected by CaPO4 precipitation, or any other method of choice. For the assays described here, 10 150-mm plates seeded with HeLa cells are each transfected with 10 g of the CIITA mammalian expression vector DNA. Transfection is for 48 h. After transfection, the cell lysate is prepared and the flag-tagged CIITA is immunoprecipitated using antiflag agarose beads, as described later. Immunoprecipitation is done in two parts: an aliquot of the lysate is used for Western immunoblotting and the remainder is used in the acetyltransferase assay. Immunoprecipitation of CIITA from Transfected Hela Cells Reagents Lysis buffer: 50 mM Tris–Cl, pH 8.0, 5 mM MgCl2, 150 mM KCl, 0.1% NP-40, 10% glycerol, freshly added 10 g/ml pepstatin, 5 g/ml leupeptin, 20 g/ml aprotinin, 1 mM PMSF Agarose beads: Antiflag (Sigma, A1205) Bead wash buffer: Cold TBS (50 mM Tris, pH 7.4, and 150 mM NaCl) 0.1 M glycine, pH 3.5 Antibody: Monoclonal Ab (M2, antiflag Sigma F3165), antimouse HRP conjugate (Santa Cruz).
384
polymerase associated factors
[33]
After 48 h of transfection, adherent cells are rinsed once with cold PBS and then harvested in 2 ml cold PBS by scraping with a rubber police man. Cells are pooled and centrifuged at 1200 rpm at 4 for 5 min. The cell pellet is resuspended at a volume equal to two times the packed cell volume in cold lysis buffer. Cells are swollen on ice for 20 min and then lysed by passage twice through a 23-gauge needle. The lysate is centrifuged at 40,000 rpm for 20 min at 4 . The supernatant is collected and stored at 70 . For Western immunoblotting, 25 l of the cleared cell lysate is combined with 10–15 l of activated antiflag agarose beads (activated as described previously) for assay of acetyltransferase activity. Two hundred microliters of cleared cell lysate is combined with 60 l of activated beads. In both cases, the slurries are incubated overnight at 4 on a vertical rotating wheel. After overnight incubation, the sample is centrifuged for 10 min at 2000 rpm. The supernatant can be checked for residual, unbound protein and then discarded. The beads should be washed three more times with 1 ml of cold lysis buffer. The bulk of the protein should be attached to the beads, which are processed further. Western Immunoblot Analysis of CIITA Reagents 4 stacking buffer: 60.6 g Tris, 40 ml 10% SDS, pH 6.8, per liter 4 sample buffer: 250 l 4 stacking buffer, 10 l bromphenol blue (saturated solution), 90 l H2O, 50 l glycerol. Immediately before use, combine 80 l of 4 sample buffer with 20 l 2-mercaptoethanol Transfer buffer: for 1 liter, combine 5.92 g Tris, 2.93 g glycine, 10% SDS, and 200 ml methanol TBS: 10 mM Tris, pH 8.0, 150 mM NaCl TBST: 10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween Blocking buffer: 5.0% nonfat dried milk in TBST or TBS Primary antibody: Monoclonal antibody antiflag M2 (antiflag Sigma F3165), 10 g/ml secondary antibody: antimouse HRP conjugate (Santa Cruz), 1:2000 dilution Substrate: Pierce Supersignal Chemiluminescent Substrate Agarose beads as described earlier, with the captured CIITA protein, are resuspended in 30 l of lysis buffer and 10 l of 4 sample buffer and heated at 55 for 20 min. After centrifugation, the immunoprecipitated proteins are separated on a 10% SDS–PAGE gel. Following electrophoretic separation, proteins are transferred to a nitrocellulose membrane (Schleicher and Schuell, BA-S85, 0.45 m). The membrane should be wet once in water and then in transfer buffer. Transfer
[33]
transcriptional coactivator CIITA
385
is done using Transblot SD, semidry transfer cells from Bio-Rad at 15 V for 60 min. After the transfer, the membrane must be blocked for nonspecific proteins. Blocking can be done at room temperature for 4 h in 5% milk/TBST. Alternatively, block overnight at 4 in 5% milk/TBS only, followed by a 1-h incubation at room temperature 5% milk/TBST in the morning. Incubate the membrane with the antiflag M2 antibody (10 g/ml) in 5% milk/TBST for 60 min at room temperature on a rocker. Drain away antibody and wash four times for 10 min each with TBST. The membrane is next incubated with the antimouse HRP conjugate at 1:2000 dilution in TBST for 30 min at room temperature, followed by four washes for 5 min min each in TBST and one wash in TBS. The HRP tag of the second antibody is developed using the Pierce Supersignal Westpico Chemiluminescent Substrate. Mix equal volumes of buffers A and B (as directed by manufacturer) and add to the membrane. Use enough to cover entirely. Rock for 5 min on a platform. Drain. Place the membrane on 3MM Whatman paper, cover with Saran wrap, and expose to X-ray film. Various times of exposure should be tested to yield maximum detection. Histone Acetyltransferase Assay Reagents 10 HAT buffer: 500 mM Tris, pH 8.0, 10 mM DTT, 1 mM EDTA 10 butyric acid: 100 mM in water 3 H Acetyl-CoA (10 Ci/pmol) (Perkin Elmer #290, 50 Ci) 14 C Acetyl-Co A (60 mCi/pmol) (from Amersham, CFA729, 10 Ci) Histones H3/H4: 1 mg/ml (Boehringer Mannheim) To assay the acetyltransferase activity of recombinant CIITA, prepare the master mix, as follows, per reaction: 3.0 l 10 HAT buffer 3.0 l 10 butyric acid 3.0 l glycerol 0.7 l 3H acetyl-CoA 0.3 l 14C acetyl-CoA 5.0 l lysis buffer (freshly added protease inhibitor) 1.25 l (50 ng) of recombinant protein CIITA 28 l total volume with water To this mix add 2.0 l of histone mixture [mix 1 l of H3 (1 mg/ml) þ 1 l H4 (1 mg/ml)]. Incubate at 30 for 30 min and stop the reaction by adding 10 l of 4 SDS sample buffer (without glycerol). Heat at 55 for 20 min.
386
[34]
polymerase associated factors
Load on to a SDS–PAGE minigel (Bio-Rad), 16% resolving gel, and 5% stacking gel. Run at 150 V. Stain the gel in 35% methanol, 10% acetic acid with 0.25 mg Coomassie R250/100 ml for 1 h at room temperature. Destain the gel in 35% methanol and 10% acetic acid O/N. Dry gel under slow temperature rise settings to prevent cracking. Expose to a tritium enhancing screen. Visualize and quantitate on a Molecular Dynamics Storm phosphorimager or equivalent. To assay the AT activity of transfected CIITA isolated from HeLa cells, combine 60 l of activated antiflag agarose beads with 200–300 l of the HeLa cell lysate and immunoprecipitate as described earlier. After the last wash, add 28 l of the HAT assay master mix to the beads in each tube. Add 2.0 l of histones (H3 þ H4). Continue as described for recombinant CIITA.
[34] Purification and Assay of Saccharomyces cerevisiae Phosphatase That Acts on the C-Terminal Domain of the Largest Subunit of RNA Polymerase II By Susanne Hoheisel, Michael S. Kobor, Erik Pierstorff, Jack Greenblatt, and Caroline M. Kane The largest subunit of RNA polymerase II contains a heptad repeat with the consensus sequence YSPTSPS at its C terminus, referred to as the CTD. The heptad is repeated multiple times, with 26 repeats in Saccharomyces cerevisiae and 52 repeats in rodents and humans. The phosphorylation along this repeated sequence varies during transcription, during the cell cycle, and with changes in cellular metabolism.1,2 Several kinases have been shown to participate in the differential phosphorylation of this repeat, but only one phosphatase been isolated that is specific for the polymerase (Fcp1p). Another phosphatase, PP1 in mammals, has been shown capable of participating in dephosphorylation of RNA polymerase II as well.3 However, it is the phosphatase encoded by FCP1 in S. cerevisiae that is the topic of this article. Several assays used to quantitate the phosphatase activity are presented, as are several methods for purification of the phosphatase itself, from the more traditional biochemical method to 1
M. S. Kobor and J. Greenblatt, Biochim. Biophys. Acta. 1577, 261 (2002). P. S. Lin, N. F. Marshall, M. E. Dahmus, Prog. Nucleic Acid Res. Mol. Biol. 72, 333 (2002). 3 K. Washington, T. Ammosova, M. Beullens, M. Jerebtsova, A. Kumar, M. Bollen, S. Nekhai, J. Biol. Chem. 277, 40442 (2000). 2
METHODS IN ENZYMOLOGY, VOL. 370
Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00
[33]
polymerase associated factors
the broadening of the average image method has lower time resolution and does not reveal either the actual length or the distribution of tether lengths but is immune to blurring. Accuracy and precision of this TPM method have been published9 and can be implemented using routines available under a general public license software agreement (www.bio. brandeis.edu/gelles/software/index.html).
[33] Assay of an Intrinsic Acetyltransferase Activity of the Transcriptional Coactivator CIITA By Jocelyn D. Weissman, Aparna Raval, and Dinah S. Singer Posttranslational modification of proteins is increasingly being recognized as an important regulatory mechanism in transcription. The best characterized of the posttranslational modifications are the phosphorylations of proteins involved in regulating signal transduction pathways and glycosylation of membrane proteins. However, studies have demonstrated that acetylation and methylation play pivotal roles in transcriptional regulation.1 Acetylation of nucleosomal histones is associated with transcriptionally active chromatin, whereas methylation correlates primarily with silent regions of chromatin. A number of nonhistone proteins have been demonstrated to undergo acetylation, although the function of this modification is not understood. Among the proteins shown to be acetylated are p53, ATF2, HIV Tat, and the coactivator CIITA.2–4 A number of proteins with acetyltransferase enzymatic (AT) activity have been identified. Most of these proteins, such as CBP, p300, and PCAF, were first identified as transcriptional coactivators and later shown to acetylate histones.5 Other proteins such as TAFII250 and Tip60, which mediate transcription but do not function as coactivators, also have been shown to have AT activity.6,7 Interestingly, the substrates of these acetyltransferases have not been well characterized. Although the coactivators appear to primarily acetylate histones, some are able to acetylate 1
T. Jenuwein and C. D. Allis, Science 294, 2477 (2001). B. Cullen, FASEB J. 5, 2361 (1991). 3 V. Ogrysko, Cell. Mol. Life Sci. 58, 683 (2001). 4 A. Raval, T. K. Howcroft, J. D. Weissman, S. Krishner, X. S. Zhu, K. Yokoyama, J. Ting, and D. S. Singer, Mol. Cell. 7, 105 (2001). 5 V. Ogryzko, L. Schiltz, V. Russanova, B. Howard, and Y. Nakatani, Cell 87, 953 (1996). 6 C. Mizzen, X. Yang, T. Kokubo, J. Brownell, A. Bannister et al. Cell 87, 1261 (1996). 7 Y. Yamamoto and M. Horikoshi, J. Biol. Chem. 272, 30595 (1997). 2
METHODS IN ENZYMOLOGY, VOL. 370
0076-6879/03 $35.00
[33]
transcriptional coactivator CIITA
379
nonhistone proteins. For example, CBP and PCAF have been shown to acetylate CIITA.8,9 Similarly, the AT activity of TAFII250 is required for its function in in vitro transcription of naked DNA in the absence of nucleosomal histones.10 The transcriptional coactivator CIITA mediates the interferon- response by nucleating formation of the transcriptional enhanceosome that activates MHC class I and class II transcription.11 We have demonstrated that CIITA, like other coactivators, contains intrinsic AT activity.4 Unlike other coactivators, this AT activity is stimulated by GTP. The CIITA enzymatic activity appears to depend on protein conformation, as it is only detected in protein purified either from transfected mammalian cells or from insect Sf9 cells; bacterially derived CIITA does not contain detectable AT activity. The methods given here describe the purification of both insect- and mammalian-derived CIITA, the acetyltransferase assay using histones as a substrate, and Western blotting to confirm purification of the protein. In all of these protocols, we have used a flag-tagged CIITA. It is also important to note that the AT activity of CIITA as measured with histone substrates is considerably less than that of CBP or p300. This may reflect either an intrinsic difference in enzymatic activity or that histones are not the native substrate of CIITA. Preparation of Recombinant CIITA from Baculovirus
rCIITA purified from a baculovirus that has been harvested from insect Sf9 cells, as described later, reproducibly displays readily detectable histone acetyltransferase activity. To facilitate purification and monitoring of the CIITA, the protein is flag tagged at the 50 end of the molecule. However, purification of enzymatically active CIITA depends on the conditions in which Sf9 cells are grown and maintained and the conditions of transfection and viral infection. The following protocols describe the growth of the cells, their transfection with the CIITA gene cloned into a baculoviral vector to generate viral stocks, the isolation of viral stocks, the infection of Sf9 cells for the quantitative isolation of virus containing CIITA, and purification of the CIITA from the virus. 8
J. D. Fontes, S. Kanazawa, D. Jean, and B. M. Peterlin, Mol. Cell Biol. 19, 941 (1999). A. Kretsovali, T. Agalioti, C. Spilianakis, E. Tzortzakaki, M. Merika, and J. Papamatheakis, Mol. Cell Biol. 18, 6777 (1998). 10 J. D. Weissman, J. Brown, T. K. Howcroft, J. Hwang, A. Chawla, P. Roche, L. Schiltz, Y. Nakatani, and D. S. Singer, Proc. Natl. Acad. Sci. USA 95, 11601 (1998). 11 K. Masternak, A. Muhlethaler-Mottet, J. Villard, M. Zufferey, V. Steimle, and W. Reith, Genes Dev. 14, 11566 (2000). 9
380
polymerase associated factors
[33]
Reagents SF9 culture medium: TMN-FH (Gemini Bio-Products) Baculogold transfection kit: Pharmingen kit Phosphate-buffered saline (PBS) Lysis buffer: 50 mM Tris–Cl, pH 8.0, 5 mM MgCl2, 150 mM KCl, 0.1% NP-40, 10% glycerol, freshly added 10 g/ml pepstatin, 5 g/ml leupeptin, 20 g/ml aprotinin, 1 mM phenylethylsulforyl fluoride (PMSF) Antiflag agarose beads: Sigma A1205 TBS: 50 mM Tris, pH 8.0, 150 mM NaCl 0.1 M glycine, pH 3.5 Flag peptide stock solution: 5 mg/ml in TBS Buffer D: 20 mM HEPES, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol (DTT), 20% glycerol(for Hi-Trap columns) Growth of Sf9 Cells Sf9 insect cells can be grown as either a monolayer in a tissue culture flask or in suspension culture in spinner flasks. In either case, care should be taken while growing Sf9 cells, as they are extremely sensitive to fluctuations in temperature, cell density, and agitation. Spinner SF9 cultures have to be maintained at 27 at a density of 5 5 7 10 to 2 10 cells/ml and under gentle shaking. Cells are harvested by centrifugation at 500 rpm or less for 10 min with no brake. Monolayer cultures should be plated at a density of 107 cells in 30 ml medium in a T100 plate. Cells should be transferred to fresh medium every 2–3 days by harvesting with gentle agitation and dilution at about 1:3 into a clean plate to reestablish the original seeding density. (The cells are extremely fragile and may be broken by scraping with a rubber policeman.) Transfection of Sf9 Cells to Generate Viral Seed Culture The following protocol describes transfection into SF9 cells of the baculovirus expression vector, PVL 1393 with the inserted CIITA cDNA, recovery of the recombinant virus, and expansion of the virus. Then, expression of the CIITA recombinant protein is analyzed by Western blotting of infected SF9 cell extracts. Day 2: Transfer Sf9 cells to fresh medium before transfection to ensure that cells are growing in an exponential phase. Day 1: Harvest cells and plate at 3 million cells/10 ml in a T25 flask. Plate an additional flask for a nontransfected control. Close and set it aside for 1 h to let cells adhere. Check under a microscope for adherence before
[33]
transcriptional coactivator CIITA
381
proceeding. Bring buffers A and B (from Pharmingen Baculovirus Gold kit) to room temperature. Combine 0.5 g of Baculovirus Gold DNA from the kit with 4 g of CIITA expression vector PVL 1323 (instead of the 2 g suggested by the kit protocol) in an Eppendorf tube. Leave at room temperature for 5 min. While waiting, aspirate the medium from Sf9 cells in the T25 flask. Add 1.25 ml of buffer A. After the 5-min incubation, add 1.25 ml buffer B to the DNA mixture and mix well. Add this mix dropwise to the flask containing the cells. Leave the flask at 27 for 4 h. At this point, the supernatant should look cloudy. Remove the supernatant by aspiration and add 4 ml fresh SF9 medium. Incubate for 48 h at 27 . Day 3: Examine the cells in the flask under a microscope. Transfected cells should appear larger than those in the nontransfected control flask. Replace 2 ml of the old culture medium with 2 ml fresh SF9 medium. Day 5: At this point, most of the remaining cells should be floating, not adherent, although a few may remain attached to the flask. This is an indication of a successful transfection. Using sterile technique, remove the medium with the floating cells in a sterile 15-ml tube and centrifuge at 1200 rmp for 10 min to clear debris. Store the cleared supernatant at 4 . If large numbers of cells remain attached to the flask, add an additional 4 ml of fresh medium and continue to incubate the cells for 2 more days. Day 7: Remove the medium, spin, and store the supernatant at 4 . Do not combine with first supernatant. Viral Assay To assay for the presence of viral particles in the culture supernatants, seed 3 million Sf9 cells in a small T25 flask, allowing them to adhere at 27 for 1 h. Remove medium. Sterilely mix 600 l of supernatant from day 5 with 600 l of fresh medium and add to flask. Incubate for 1 h. Every 10–15 min rotate the flask gently to make sure that the entire surface of the flask is covered. After 1 h add 3 ml medium and incubate at 27 for 48 h. After 48 h, harvest the cells and make lysate (see later). Run SDS–PAGE and Western to determine if protein has been made. Because there is considerable variation in the rate and the stability of the produced recombinant proteins, a time course should be run to determine the optimal time for harvesting of the supernatants after transfection. Preparing a Large-Scale Viral Stock If the transfection has been successful, as measured by the production of CIITA protein, a viral stock is generated. Take 30 million cells in a large T flask and let them adhere for 1 h. Add 2 ml of the viral seed culture (supernatant from day 5 or 7) and 8 ml fresh
382
polymerase associated factors
[33]
medium. Incubate at 27 for 1 h, rotating every 10–15 min. After 1 h, add 30 ml fresh medium. Harvest cells on day 5 in a 50-ml sterile tube and spin at 1200 rpm for 10 min and store the supernatant at 4 . This is the viral stock. Protein Production and Purification Place 200 million cells (1–1.5 million cells/ml) in a spinner flask and add 10 ml of viral stock. (At some point, you will need to make Sf9 cell extract from a nontransfected culture to use as control.) Incubate at 27 for 48 h. Harvest cells and spin at room temperature, 500 rpm with no brake. Remove the supernatant and wash the pellet once with room temperature PBS. Immediately freeze the pellet on dry ice. In general, the pellet should be processed immediately, although some proteins may be left at 70 for a few days. Thaw the pellet at 37 for 5 min. Add 3–4 ml lysis buffer and incubate on ice for 20 min. Break open the cells with passage through a 23-gauge needle twice. Spin lysate at 40,000 rpm at 4 for 20 min to remove debris. Collect the supernatant, which can be stored at 70 . Repeated freezing and thawing may lead to protein degradation. Antiflag Bead Activation The CIITA protein used in our studies has an N-terminal flag tag that is used as the basis of the protein purification. During ultracentrifugation of the lysate described earlier activate the antiflag beads. Wash 500 l of antiflag agarose beads three times with TBS as follows: place beads in a 1.5-ml Eppendorf tube, add 1 ml of TBS, and invert several times. Spin at 2000 rpm for 2–3 min. Discard the supernatant and repeat the washing step. Then wash three times with 0.1 M glycine, pH 3.5, to activate the beads. Wash again with TBS. Finally wash once in lysis buffer with protease inhibitors. Resuspend in 1 ml lysis buffer (i.e., 2X original bead volume). Notes. The beads should be activated fresh everytime, before the immunoprecipitation. The beads should not be left in the glycine buffer more than 20 min. Protein Immunoprecipitation Add 1 ml of the activated antiflag agarose bead slurry to the 3–4 ml of cleared viral lysate prepared in earlier. (A nontransfected control lysate should be processed in parallel.) Rotate the mixture at 4 for 4 h and then spin down the bead at 2000 rpm for 2–3 min. Wash the beads five times with 10 ml of lysis buffer (with protease inhibitors). Elute the bound
[33]
transcriptional coactivator CIITA
383
protein with 500 l of the flag peptide solution, freshly diluted to 200 g/ml in TBS. Elute four times by adding 500 l each time of this 200-g/ml solution (before eluting, check pH of flag peptides, which should not be acidic). After each 500-l addition, spin in a horizontal rotor at 2000 rpm for 15 min at 4 . Collect the supernatant in a fresh tube. The first two elutions have most of the proteins. Therefore, combine the first and second elutions separately from the third and fourth. This results in two aliquots of protein of 1 ml each (four elutions of 500 l each). Dialyze each aliquot against three changes of buffer of interest for 4 h to remove flag peptides. If flag peptides remain in the protein preparation after the dialysis, they can be removed by gel chromatography or Hi-Trap columns. For Hi-Trap column chromatography, the Hi-Trap column is washed with buffer D and elution is also done in buffer D. After elution, concentrate the eluate in a 30 or 50K cutoff Microcon or Centricon and then add fresh protease inhibi tors, pepstatin, leupeptin, and aprotinin. Make aliquots and store at 70 . To prevent loss of protein due to denaturation, the entire process should be completed in 1 day. Preparation of CIITA from Transfected HeLa Cells
CIITA is prepared from HeLa cells, transfected by CaPO4 precipitation, or any other method of choice. For the assays described here, 10 150-mm plates seeded with HeLa cells are each transfected with 10 g of the CIITA mammalian expression vector DNA. Transfection is for 48 h. After transfection, the cell lysate is prepared and the flag-tagged CIITA is immunoprecipitated using antiflag agarose beads, as described later. Immunoprecipitation is done in two parts: an aliquot of the lysate is used for Western immunoblotting and the remainder is used in the acetyltransferase assay. Immunoprecipitation of CIITA from Transfected Hela Cells Reagents Lysis buffer: 50 mM Tris–Cl, pH 8.0, 5 mM MgCl2, 150 mM KCl, 0.1% NP-40, 10% glycerol, freshly added 10 g/ml pepstatin, 5 g/ml leupeptin, 20 g/ml aprotinin, 1 mM PMSF Agarose beads: Antiflag (Sigma, A1205) Bead wash buffer: Cold TBS (50 mM Tris, pH 7.4, and 150 mM NaCl) 0.1 M glycine, pH 3.5 Antibody: Monoclonal Ab (M2, antiflag Sigma F3165), antimouse HRP conjugate (Santa Cruz).
384
polymerase associated factors
[33]
After 48 h of transfection, adherent cells are rinsed once with cold PBS and then harvested in 2 ml cold PBS by scraping with a rubber police man. Cells are pooled and centrifuged at 1200 rpm at 4 for 5 min. The cell pellet is resuspended at a volume equal to two times the packed cell volume in cold lysis buffer. Cells are swollen on ice for 20 min and then lysed by passage twice through a 23-gauge needle. The lysate is centrifuged at 40,000 rpm for 20 min at 4 . The supernatant is collected and stored at 70 . For Western immunoblotting, 25 l of the cleared cell lysate is combined with 10–15 l of activated antiflag agarose beads (activated as described previously) for assay of acetyltransferase activity. Two hundred microliters of cleared cell lysate is combined with 60 l of activated beads. In both cases, the slurries are incubated overnight at 4 on a vertical rotating wheel. After overnight incubation, the sample is centrifuged for 10 min at 2000 rpm. The supernatant can be checked for residual, unbound protein and then discarded. The beads should be washed three more times with 1 ml of cold lysis buffer. The bulk of the protein should be attached to the beads, which are processed further. Western Immunoblot Analysis of CIITA Reagents 4 stacking buffer: 60.6 g Tris, 40 ml 10% SDS, pH 6.8, per liter 4 sample buffer: 250 l 4 stacking buffer, 10 l bromphenol blue (saturated solution), 90 l H2O, 50 l glycerol. Immediately before use, combine 80 l of 4 sample buffer with 20 l 2-mercaptoethanol Transfer buffer: for 1 liter, combine 5.92 g Tris, 2.93 g glycine, 10% SDS, and 200 ml methanol TBS: 10 mM Tris, pH 8.0, 150 mM NaCl TBST: 10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween Blocking buffer: 5.0% nonfat dried milk in TBST or TBS Primary antibody: Monoclonal antibody antiflag M2 (antiflag Sigma F3165), 10 g/ml secondary antibody: antimouse HRP conjugate (Santa Cruz), 1:2000 dilution Substrate: Pierce Supersignal Chemiluminescent Substrate Agarose beads as described earlier, with the captured CIITA protein, are resuspended in 30 l of lysis buffer and 10 l of 4 sample buffer and heated at 55 for 20 min. After centrifugation, the immunoprecipitated proteins are separated on a 10% SDS–PAGE gel. Following electrophoretic separation, proteins are transferred to a nitrocellulose membrane (Schleicher and Schuell, BA-S85, 0.45 m). The membrane should be wet once in water and then in transfer buffer. Transfer
[33]
transcriptional coactivator CIITA
385
is done using Transblot SD, semidry transfer cells from Bio-Rad at 15 V for 60 min. After the transfer, the membrane must be blocked for nonspecific proteins. Blocking can be done at room temperature for 4 h in 5% milk/TBST. Alternatively, block overnight at 4 in 5% milk/TBS only, followed by a 1-h incubation at room temperature 5% milk/TBST in the morning. Incubate the membrane with the antiflag M2 antibody (10 g/ml) in 5% milk/TBST for 60 min at room temperature on a rocker. Drain away antibody and wash four times for 10 min each with TBST. The membrane is next incubated with the antimouse HRP conjugate at 1:2000 dilution in TBST for 30 min at room temperature, followed by four washes for 5 min min each in TBST and one wash in TBS. The HRP tag of the second antibody is developed using the Pierce Supersignal Westpico Chemiluminescent Substrate. Mix equal volumes of buffers A and B (as directed by manufacturer) and add to the membrane. Use enough to cover entirely. Rock for 5 min on a platform. Drain. Place the membrane on 3MM Whatman paper, cover with Saran wrap, and expose to X-ray film. Various times of exposure should be tested to yield maximum detection. Histone Acetyltransferase Assay Reagents 10 HAT buffer: 500 mM Tris, pH 8.0, 10 mM DTT, 1 mM EDTA 10 butyric acid: 100 mM in water 3 H Acetyl-CoA (10 Ci/pmol) (Perkin Elmer #290, 50 Ci) 14 C Acetyl-Co A (60 mCi/pmol) (from Amersham, CFA729, 10 Ci) Histones H3/H4: 1 mg/ml (Boehringer Mannheim) To assay the acetyltransferase activity of recombinant CIITA, prepare the master mix, as follows, per reaction: 3.0 l 10 HAT buffer 3.0 l 10 butyric acid 3.0 l glycerol 0.7 l 3H acetyl-CoA 0.3 l 14C acetyl-CoA 5.0 l lysis buffer (freshly added protease inhibitor) 1.25 l (50 ng) of recombinant protein CIITA 28 l total volume with water To this mix add 2.0 l of histone mixture [mix 1 l of H3 (1 mg/ml) þ 1 l H4 (1 mg/ml)]. Incubate at 30 for 30 min and stop the reaction by adding 10 l of 4 SDS sample buffer (without glycerol). Heat at 55 for 20 min.
386
[34]
polymerase associated factors
Load on to a SDS–PAGE minigel (Bio-Rad), 16% resolving gel, and 5% stacking gel. Run at 150 V. Stain the gel in 35% methanol, 10% acetic acid with 0.25 mg Coomassie R250/100 ml for 1 h at room temperature. Destain the gel in 35% methanol and 10% acetic acid O/N. Dry gel under slow temperature rise settings to prevent cracking. Expose to a tritium enhancing screen. Visualize and quantitate on a Molecular Dynamics Storm phosphorimager or equivalent. To assay the AT activity of transfected CIITA isolated from HeLa cells, combine 60 l of activated antiflag agarose beads with 200–300 l of the HeLa cell lysate and immunoprecipitate as described earlier. After the last wash, add 28 l of the HAT assay master mix to the beads in each tube. Add 2.0 l of histones (H3 þ H4). Continue as described for recombinant CIITA.
[34] Purification and Assay of Saccharomyces cerevisiae Phosphatase That Acts on the C-Terminal Domain of the Largest Subunit of RNA Polymerase II By Susanne Hoheisel, Michael S. Kobor, Erik Pierstorff, Jack Greenblatt, and Caroline M. Kane The largest subunit of RNA polymerase II contains a heptad repeat with the consensus sequence YSPTSPS at its C terminus, referred to as the CTD. The heptad is repeated multiple times, with 26 repeats in Saccharomyces cerevisiae and 52 repeats in rodents and humans. The phosphorylation along this repeated sequence varies during transcription, during the cell cycle, and with changes in cellular metabolism.1,2 Several kinases have been shown to participate in the differential phosphorylation of this repeat, but only one phosphatase been isolated that is specific for the polymerase (Fcp1p). Another phosphatase, PP1 in mammals, has been shown capable of participating in dephosphorylation of RNA polymerase II as well.3 However, it is the phosphatase encoded by FCP1 in S. cerevisiae that is the topic of this article. Several assays used to quantitate the phosphatase activity are presented, as are several methods for purification of the phosphatase itself, from the more traditional biochemical method to 1
M. S. Kobor and J. Greenblatt, Biochim. Biophys. Acta. 1577, 261 (2002). P. S. Lin, N. F. Marshall, M. E. Dahmus, Prog. Nucleic Acid Res. Mol. Biol. 72, 333 (2002). 3 K. Washington, T. Ammosova, M. Beullens, M. Jerebtsova, A. Kumar, M. Bollen, S. Nekhai, J. Biol. Chem. 277, 40442 (2000). 2
METHODS IN ENZYMOLOGY, VOL. 370
Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00