- Email: [email protected]
Purification of Drosophila nucleosome remodeling factor
[44]
PURIFICATION OF NUCLEOSOME REMODELING FACTOR
757
of changes in plasmid topology, 3° of site-specific chromatin transitions on plasmids using a micrococcal nuclease assay, 31 the visualization of superhelical stress, 3e the effect of remodeling machines on transcription initiation, 33 and on promoter clearing of a nucleosome-inhibited polymeraseY Acknowledgment Patrick Varga-Weisz was supported by a grant from the Deutsche Forschungsgemeinschaft and is currently supported by the Marie Curie Cancer Care, Great Britain. 3o H. Kwon, A. Imbalzano N., P. A. Khavari, R. E. Kingston, and M. R. Green, Nature 370, 477 (1994). 31 T. Tsukiyama, P. B. Becket, and C. and Wu, Nature 367, 525 (1994). 32j. Quinn, A. M. Fyrberg, R. W. Ganster, M. C. Schmidt, and C. L. Peterson, Nature 379~ 844 (1996). 33T. Ito, M. Bulger, M. J. Pazin, R. Kobayashi, and J. T. Kodonaga, Cell 145, 1 (1997); G. Mizuguchi, T. Tsukiyama, J. Wisniewski, and C. Wu, Mol. Cell 141, (1998). 34 S. A. Brown, A. N. Imbalzano, and R. E. Kingston, Genes Dev. 10, 1479 (1996).
[44] P u r i f i c a t i o n o f Drosophila N u c l e o s o m e Remodeling Factor B y RAPHAEL SANDALTZOPOULOS, VINCENT OSSIPOW, DAVID a . GDULA, TOSHIO TSUKIYAMA, a n d CARL W U
Introduction Vital biochemical processes in the cell, such as DNA replication, transcription, repair, and recombination, take place in the environment of chromatin. The efficiency of these biochemical processes, which require access to DNA by very large molecular machines, is not compromised by the compaction of DNA in nucleosomes and higher order chromatin structures. Recent advances indicate a substantial contribution toward the regulation of chromatin accessibility by energy-consuming multisubunit protein complexes that modify, remodel, and rearrange chromatin, rendering it dynamic and accessible. The identification and purification of enzymatic complexes that regulate chromatin dynamics, such as the yeast SWI/ SNF 1 and RSC 2 complexes and the Drosophila NURF, 3 ACF, 4 and a B. R. Cairns, Y. J. Kim, M. H. Sayre, B. C. Laurent, and R. D. Kornberg, Proc. Natl. Acad. Sci. U.S.A. 91, 1950 (1994). 2 B. R. Cairns, Y. Lorch, Y. Li, M. Zhang, L. Lacomis, H. Erdjument-Bromage, P. Tempst, J. Du, B. Laurent, and R. D. Kornberg, Cell 87, 1249 (1996). 3 T. Tsukiyama and C. Wu, Cell 83, 1011 (1995). 4 T. Ito, M. Bulger, M. J. Pazin, R. Kobayashi, and J. T. Kadonaga, Cell 90, 145 (1997).
METHODS IN ENZYMOLOGY,VOL.304
Copyright© 1999by AcademicPress All rightsof reproductionin any formreserved, 0076-6879/99 $30.00
758
CHROMATIN REMODELING COMPLEXES
[44]
CHRAC 5 complexes, have modified our view of chromatin. The availability of these complexes in a pure form is fundamental for deciphering the mechanism of their functions. This article describes a detailed protocol for the purification of the Drosophila nucleosome remodeling factor (NURF). NURF is a sarkosyl-sensitive, four-subunit complex that remodels nucleosomes in an ATP-dependent manner. Under standard conditions, nucleosome remodeling takes place only in the presence of a sequencespecific DNA-binding factor and is restricted to the nucleosomes in its vicinity.3 NURF facilitates a step preceding RNA polymerase II preinitiation complex assembly resulting in transcriptional activation of a chromatin template reconstituted in vitro, 6 although the detailed mechanism of function and the nature of the remodeling that takes place remain to be elucidated. The stimulation of the ATPase activity of NURF by nucleosomes rather than free DNA or histones suggests that NURF has a special ability to recognize nucleosome structure, important elements of which include the flexible histone tails. 7 Several of the NURF subunits have been identified and cloned. NURF140 (140 kDa) is the ISWI ATPase 8 that was cloned on the basis of homology to the ATPase domain of yeast SWI2 protein. NURF-55 (55 kDa) is a WD repeat protein 9 known to be involved in histone metabolism. NURF-38 (38 kDa) has been cloned and expressed as a recombinant polypeptide. 1° The cloning and characterization of the largest subunit, NURF-215 (215 kDa), is currently in progress. Once cDNAs for all subunits become available it will be of interest to reconstitute the NURF complex with recombinant proteins and rigorously compare its activities with native NURF purified from Drosophila. Outline of Purification Scheme The purification of NURF from Drosophila nuclear extracts is a procedure of mild chromatographic steps followed by glycerol gradient centrifugation. The protocol is reliable and reproducible, and the yield of highly pure NURF complex is quite sufficient for biochemical analysis. NURF can be traced along the stages of purification either by testing fractions for 5 p. D. Varga-Weisz, M. Wilm, E. Bonte, K. Dumas, M. Mann, and P. B. Becker, Nature 388, 598 (1997). 6 G. Mizuguchi, T. Tsukiyama, J. Wisniewski, and C. Wu, Mol. Cell 1, 141 (1997). 7 p. T. Georgel, T. Tsukiyama, and C. Wu, E M B O J. 16, 4717 (1997). 8 T. Tsukiyama, C. Daniel, J. Tamkun, and C. Wu, Cell 83, 1021 (1995). 9 M. A. Martinez-Balbas, T. Tsukiyama, D. Gdula, and C. Wu, Proc. Natl. Acad. Sci. U.S.A. 95, 132 (1998). lo D. A. Gdula, R. Sandaltzopoulos, T. Tsukiyama, V. Ossipow, and C. Wu, Genes Dev. 12, 3206 (1998).
[44]
P U R I F I C A T I O N OF N U C L E O S O M E R E M O D E L I N G F A C T O R
759
nucleosome remodeling activity or by detection of N U R F subunits in the fractions by Western analysis, using a mixture of antisera recognizing p38, p55, and ISWI. The nucleosome remodeling assay is described in detail by Mizuguchi and Wu. u For all separation methods tested, the peak of N U R F activity coincides with fractions containing p38, p55, and ISWI. Because p55 and ISWI are components of other complexes, 5,9 it is important to trace the codistribution of all N U R F subunits to avoid the inadvertent purification of other complexes. The outline of the purification scheme is shown in Fig. 1. The purity of N U R F containing fractions after each purification step is shown in Fig. 2: 1.25 t~l (lanes 1, 2, and 3), 5/~1 (lanes 4 and 5), or 10/xl (lane 6) of the indicated step was analyzed by 10% SDS-PAGE. Proteins were visualized either by silver staining or by immunoblotting with a mixture of three antisera specific for the indicated NURF subunits. Only four major bands are present after the glycerol gradient. Interestingly, all four of these proteins can be immunoprecipitated by any of the three antibodies from relatively crude fractions. 9,1° The yield of highly purified N U R F is 100-200/~g per 200 g of embryos. Preparation of Starting Material The starting material for N U R F purification is a nuclear extract prepared from Drosophila embryos according to the procedure described by Wampler et al. 12with slight modifications. For a typical purification, approximately 200 g of moist, 12- to 16-hr Drosophila embryos are dechorionated and resuspended in 3 mug of buffer I (15 mM H E P E S - K O H , pH 7.6, 10 mM KC1, 5 mM MgC12, 0.1 mM EDTA, pH 8.0, 0.5 mM EGTA, pH 8.0, 350 mM sucrose) supplemented with 1 mM dithiothreitol (DT-F) and 0.2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). The embryos are homogenized with two passes through a Yamato motordriven glass/Teflon homogenizer at 1500 rpm. The homogenate is filtered through Miracloth, and nuclei are pelleted by centrifugation for 10 min in a Beckman JA 14 rotor at 8000 rpm (10,000g). All centrifugations are at 4°. The supernatant is discarded, and nuclei are resuspended again in the original volume of buffer I. While resuspending the nuclei by swirling, it is critical to avoid the much tighter yellow yolk pellet. Nuclei are then dispersed by briefly homogenizing two to three times with a loose glass pestle (Dounce) and immediately pelleted again as described previously. After the second centrifugation there should be hardly any yolk at the bottom of the nuclear pellet. All of it should be avoided as nuclei are u G. Mizuguchi and C. Wu, "Chromatin Protocol." Humana Press, Clifton, NJ, in press. i2 S. L. Wampler, C. M. Tyree, and J. T. Kadonaga, J. Biol. Chem. 265, 21223 (1990).
760
[44]
CHROMATIN REMODELING COMPLEXES
Drosophila 0-12 hr embryos (200 g) Nuclear extract (NH4)2SO 4 precipitation (1 g) DE52 DEAE-cellulose Bio-Rex 70 (56 mg)
I
I
0.22
I
0.32
1.0 M
Q Sepharose Fast Flow (10 mg)
I
I
I
0.2
0.3
0.4
I 1.0 M
Hydroxylapatite (4.7 mg)
I
I
0.2
0.3
I
0.4
I
1.0M
Cellulose phosphate P l l (1.14 mg) 0.2
I
II
I
0.6 M
d
Glycerol gradient centrifugation (200 I~g) FIG. 1. Schematic outline of the purification protocol. The amount of total protein in the N U R F containing fractions after each purification step is indicated.
resuspended by swirling in 1 ml/g of embryos of buffer II (15 mM HEPESKOH, pH 7.6, 110 mM KC1, 5 mM MgCI2, 0.1 mM EDTA, supplemented with 1 mM DTT and 0.2 mM AEBSF). Nuclei are dispersed well in a Dounce homogenizer (loose pestle). The volume of the nuclear suspension
[44]
PURIFICATION OF NUCLEOSOME REMODELING FACTOR
761
p215 ISWI
Silver staining
p55 p38 ISWI
Western
p55 p38 12 3456
FI6.2. Protein analysis of NURF containing fractions along the purification procedure.
is measured and divided equally into screw-cap tubes for the Beckman Type 35 rotor. Nuclei are lysed by adding 1/10 volume of 4 M (NH4)2804, pH 8.0, in each tube. The viscous lysate is mixed vigorously for 20 min on a fast rotating wheel and then centrifuged in a precooled ultracentrifuge for 1 hr at 35,000 rpm (90,000g). The clear supernatant is collected by plunging a pipette beneath the lipid top layer. The volume is measured again and poured into a beaker. An equal volume of 4 M (NH4)2804, pH 8.0, is added slowly (over a period of 5 rain) and the mix is stirred vigorously for another 10 min at 4°. Proteins are precipitated by centrifugation in a JA-14 rotor at 11,000 rpm (18,600g) for 30 min. The pellets may be flash frozen in liquid nitrogen and kept at - 8 0 ° until needed. The pellet is allowed to thaw and is resuspended in 0.2 ml/g of embryos of HEGN-40 [25 mM H E P E S - K O H , pH 7.6, 1 mM EDTA, 10% (v/v) glycerol, 0.02% (v/v) Nonidet P-40 (NP-40), 40 mM KC1; the indicator 40 stands for the concentration of KC1 in raM] supplemented with 1 mM DTT and 0.2 mM AEBSF by pipetting up and down. It is important to ensure thorough solubilization of the pellet at this step as severe reprecipitation may occur during subsequent dialysis. The extract is then placed in dialysis bags (Spectra/Por 2, molecular weight cutoff 12,000-14,000) and dialyzed twice for 1 hr against 1 liter of HEGN-40 (with 1 mM DTT and 0.2 mM AEBSF). The dialysis buffer is changed once more and the dialysis continues until the conductivity of the extract equals that of HEGN-150. The conductivity is monitored every 15 min. Finally the dia-
762
CHROMATIN REMODELING COMPLEXES
[44]
lyzed extract is harvested and cleared before proceeding to chromatography by centrifugation at 8000 rpm in a precooled JA-14 rotor (10,000g) for 10 rain. Ion-Exchange Chromatography All chromatographic steps are performed on an FPLC (fast protein liquid chromatography) system (Pharmacia, Piscataway, N J). Every column is packed 1 day in advance and equilibrated overnight with at least 5 bed volumes of buffer at a slow flow rate. The conductivity, as well as the pH of the flow through, is monitored to verify equilibration. The estimated volumes of buffers needed are prepared in advance and the appropriate loading superloop is mounted on the system so that they equilibrate to 4° overnight. The first ion-exchange chromatography step consists of a DE52 DEAE-cellulose and a Bio-Rex 70 column, in tandem. For each gram of extract protein (expect approximately 1 g protein per 200 g of embryos) use 20 ml of packed DE52 and 50 ml Bio-Rex 70 resins. An appropriate amount of preswollen Whatman DE52 resin is resuspended in at least 6 ml of HEG-150 (as HEGN-150 without NP-40) per gram of resin with gentle agitation on a shaking platform. The swollen resin is allowed to settle by gravity and the supernatant containing fines is aspirated away. The resin is then resuspended in fresh HEG-150 buffer and allowed to settle once more to remove ultrafine particles that need a longer time to settle. Then the resin is transferred into a XK16 column (Pharmacia) and allowed to pack. In parallel, a sufficient amount of Bio-Rex 70 resin (200-400 mesh; BioRad, Richmond, CA) is resuspended in 5 ml HEG-150 per gram of resin. The swollen resin is transferred in a XK26 (Pharmacia) column. Once packed by gravity, the columns are mounted on the FPLC system such that the bottom of the DE52 column is connected to the top of the Bio-Rex 70 column. We use HEGN-0 (containing no KC1) as buffer A of the FPLC and HEGN-1000 (containing 1 M KC1) as buffer B, both supplemented with 1 mM DTT and 0.2 mM AEBSF. The columns are equilibrated with 15% buffer B (containing 150 mM KC1) overnight (at least 5 bed volumes). The next day, the flow rate is increased to 2 ml/min for 15 mirl to ensure tight packing, then the top lid of each column is readjusted to minimize the dead volume. The extract is loaded at 1 ml/min with 15% buffer B. NURF does not bind efficiently to DE52 under these conditions but it is retained on the Bio-Rex 70. As soon as the UV monitor signal drops to the baseline, the DE52 column is disconnected. The flow rate is increased to 2 ml/min, and the Bio-Rex 70 column is washed with 22% buffer B until the UV signal
[44]
P U R I F I C A T I O N OF N U C L E O S O M E R E M O D E L I N G F A C T O R
763
drops to the baseline. Then N U R F is eluted in a single step by raising the relative concentration of buffer B to 32%. The entire peak (about 40 ml for 1 g of loaded protein) is collected. Although N U R F is eluted as a broad peak between approximately 280 and 380 mM KC1, the elution at 320 mM is a good compromise between yield and purification factor. The majority of free N U R F subunits not associated in the complex is removed (as indicated by filtration methods) and NURF activity is extremely high. This first step is highly reproducible. However, as NURF proteins bind avidly to DE52 resin at pH greater than 8.0, careful adjustment of the buffer pH is imperative. N U R F containing fractions are then dialyzed against HEGN-100 until conductivity equals that of HEGN-150. The sample is now ready for the second purification step. Sufficient Q Sepharose fast flow (Pharmacia) slurry is dispensed in a beaker to give 7-8 ml of packed resin. The resin is allowed to settle. The ethanol containing supernatant is aspirated away and the resin is resuspended in an equal volume of starting buffer (HEG-150, Nonidet P-40 is avoided again during packing). When the resin settles again, the supernatant is decanted and is replaced by fresh buffer. The slurry is degassed and poured into an H R 10/10 column (Pharmacia). HEGN-0 is used as buffer A and HEGN-1000 as buffer B. The column is equilibrated with 5 volumes of HEGN-150 (with 1 mM DTT and 0.2 mM AEBSF freshly added). The flow rate is adjusted at 2 ml/min for 5 min. The sample is centrifuged in a JA-20 rotor for 15 min at 12,000 rpm (17,400g). The supernatant is filtered through a 0.22-/zm Millex-GV low protein binding filter (Millipore, Bedford, MA) and loaded onto the column at 1 ml/min in HEGN-150. When the UV signal drops to baseline, NURF is eluted in steps with 20, 30, 40, and 100% buffer B. Three-milliliter fractions are collected. NURF elutes in the 0.3 M peak (30% of buffer B). N U R F containing fractions are pooled and dialyzed against PPEGN100 [0.1 mM EDTA, 10% (v/v) glycerol, 0.02% (v/v) Nonidet P-40, 100 mM K2 HPO4/KH2PO4, pH 7.6] for 2.5 hr. Hydroxylapatite and Cellulose Phosphate Chromatography Hydroxylapatite resin (Bio-Rad) is resuspended in PPEGN-100. The slurry is poured into a H R 10/10 (Pharmacia) column and allowed to pack by gravity. A sufficient amount of slurry to yield 4 ml of packed bed volume is added. PPEGN-0 (as PPEGN-100 without K2HPO4/KH2PO4) is used as buffer A and PPEGN-800 (contains 0.8 M K2HPO4/KH2PO4, pH 7.6) as buffer B. The column is equilibrated with at least 5 bed volumes of PPEGN100 (i.e., 12.5% buffer B). The sample is filtered through a 0.22-/zm MillexGV filter and loaded at 0.5 ml/min. When the UV signal drops to baseline,
764
CHROMATIN REMODELING COMPLEXES
[44]
proteins are eluted with steps of PPEGN-200, PPEGN-300, PPEGN-400, and PPEGN-800 collecting 1.5-ml fractions. NURF elutes mainly in the PPEGN-200 peak. Fractions containing N U R F are pooled (expect about 12 ml total volume of pooled N U R F containing fractions). Although some N U R F is also present in the PPEGN-300 peak, these fractions are not pooled because they contain far more impurities. In order to minimize losses due to nonspecific protein sticking to the dialysis bag, pure pH neutralized insulin (Boehringer) is added to a final concentration of 0.2 mg/ml and dialyzed against HEGN-150 until the conductivity is equal to HEGN-200 (about 2 hr). The last chromatographic step is cellulose phosphate P l l (Whatman). P l l resin comes in a fibrous form that needs precycling before use (see manufacturer's specifications). P l l can be stored at 4° in 0.5 M phosphate buffer at pH 7.0. Precycled cellulose phosphate P l l is transfered into 10-20 volumes of HEGN-200. The resin is mixed without vigorous stirring to avoid the generation of fines and is allowed to settle. The supernatant is discarded and the resin is resuspended in 3-5 volumes of HEGN-200. The resin is degassed and the equivalent of 1 ml resin volume is poured into a H R 5/5 column. After the resin has settled by gravity, the top is refilled with resin (to compensate for the reduction of volume after close packing) and the column is closed. HEGN-0 and HEGN-1000 buffers are used as buffers A and B respectively. The column is equilibrated in HEGN-200 with 20 bed volumes at 0.2 ml/min. The flow rate is increased to 0.4 ml/ min for 3 min. Then the sample is loaded at 0.4 ml/min for 3 min. Then the sample is loaded at 0.4 ml/min in HEGN-200. When the UV signal drops back to baseline, the column is developed with a shallow linear salt gradient (HEGN-200 to HEGN-600 in 20 column volumes). Four hundredmicroliter fractions are collected. NURF elutes as a very broad peak, spanning from HEGN-250 to HEGN-450, probably because P11 is a bifunctional cation exchanger containing both strong and weak acid groups. It is essential not to pool the N U R F containing fractions; each one is processed further separately. Glycerol Gradient Centrifugation The ultimate purification step is a glycerol gradient centrifugation. Glycerol gradients are formed in SW50Ti tubes (swinging bucket rotor tubes; Beckman) by carefully applying 450-~1 aliquots of each of 10 HEGN-150 solutions (supplemented with a cocktail of protein inhibitors; the 100× concentrated inhibitor mix contains 100 mM phenylmethylsulfonyl fluoride, 200 b~M pepstatin A, 60 ~M leupeptin, 200 mM benzamidine, and 200 ~g/ml chymostatin in ethanol) containing 35 to 17% (v/v) glycerol in inere-
[44]
PURIFICATION OF NUCLEOSOME REMODELING FACTOR
765
ments of 2%, i.e., the first layer (bottom) is 35% (v/v) and the last layer (top) is 17% (v/v) glycerol. All buffers as well as the rotor and the centrifugation chamber must be precooled to 4° because the viscosity of the gradient is a function of the temperature. Four hundred microliters of the NURF containing P l l fractions is applied on top. The gradients are centrifuged at 49,000 rpm (160,000g) for 20 hr at 4 ° (no brake). Five hundred-microliter fractions are collected from the bottom of each tube. NURF is always found in the third fraction. Glycerol gradient centrifugation is a very effective and reproducible purification step that conditions the purified NURF fractions for prolonged storage. Early N U R F containing fractions of the P l l column yield very pure NURF preparations when applied to glycerol gradient centrifugation. After this step, only four major protein bands corresponding to the four NURF subunits are visible on a silver-stained S D S - P A G E gel (Fig. 2, lane 6). Late N U R F containing P l l fractions usually contain impurities that are not completely removed after glycerol centrifugation. Acknowledgments R.S. was supported by an E M B O postdoctoral fellowship. V.O. was a recipient of a long-term fellowship from the Human Frontier Science Program Organization. D.A.G. is a Leukemia Society of America postdoctoral fellow.
PURIFICATION OF NUCLEOSOME REMODELING FACTOR
757
of changes in plasmid topology, 3° of site-specific chromatin transitions on plasmids using a micrococcal nuclease assay, 31 the visualization of superhelical stress, 3e the effect of remodeling machines on transcription initiation, 33 and on promoter clearing of a nucleosome-inhibited polymeraseY Acknowledgment Patrick Varga-Weisz was supported by a grant from the Deutsche Forschungsgemeinschaft and is currently supported by the Marie Curie Cancer Care, Great Britain. 3o H. Kwon, A. Imbalzano N., P. A. Khavari, R. E. Kingston, and M. R. Green, Nature 370, 477 (1994). 31 T. Tsukiyama, P. B. Becket, and C. and Wu, Nature 367, 525 (1994). 32j. Quinn, A. M. Fyrberg, R. W. Ganster, M. C. Schmidt, and C. L. Peterson, Nature 379~ 844 (1996). 33T. Ito, M. Bulger, M. J. Pazin, R. Kobayashi, and J. T. Kodonaga, Cell 145, 1 (1997); G. Mizuguchi, T. Tsukiyama, J. Wisniewski, and C. Wu, Mol. Cell 141, (1998). 34 S. A. Brown, A. N. Imbalzano, and R. E. Kingston, Genes Dev. 10, 1479 (1996).
[44] P u r i f i c a t i o n o f Drosophila N u c l e o s o m e Remodeling Factor B y RAPHAEL SANDALTZOPOULOS, VINCENT OSSIPOW, DAVID a . GDULA, TOSHIO TSUKIYAMA, a n d CARL W U
Introduction Vital biochemical processes in the cell, such as DNA replication, transcription, repair, and recombination, take place in the environment of chromatin. The efficiency of these biochemical processes, which require access to DNA by very large molecular machines, is not compromised by the compaction of DNA in nucleosomes and higher order chromatin structures. Recent advances indicate a substantial contribution toward the regulation of chromatin accessibility by energy-consuming multisubunit protein complexes that modify, remodel, and rearrange chromatin, rendering it dynamic and accessible. The identification and purification of enzymatic complexes that regulate chromatin dynamics, such as the yeast SWI/ SNF 1 and RSC 2 complexes and the Drosophila NURF, 3 ACF, 4 and a B. R. Cairns, Y. J. Kim, M. H. Sayre, B. C. Laurent, and R. D. Kornberg, Proc. Natl. Acad. Sci. U.S.A. 91, 1950 (1994). 2 B. R. Cairns, Y. Lorch, Y. Li, M. Zhang, L. Lacomis, H. Erdjument-Bromage, P. Tempst, J. Du, B. Laurent, and R. D. Kornberg, Cell 87, 1249 (1996). 3 T. Tsukiyama and C. Wu, Cell 83, 1011 (1995). 4 T. Ito, M. Bulger, M. J. Pazin, R. Kobayashi, and J. T. Kadonaga, Cell 90, 145 (1997).
METHODS IN ENZYMOLOGY,VOL.304
Copyright© 1999by AcademicPress All rightsof reproductionin any formreserved, 0076-6879/99 $30.00
758
CHROMATIN REMODELING COMPLEXES
[44]
CHRAC 5 complexes, have modified our view of chromatin. The availability of these complexes in a pure form is fundamental for deciphering the mechanism of their functions. This article describes a detailed protocol for the purification of the Drosophila nucleosome remodeling factor (NURF). NURF is a sarkosyl-sensitive, four-subunit complex that remodels nucleosomes in an ATP-dependent manner. Under standard conditions, nucleosome remodeling takes place only in the presence of a sequencespecific DNA-binding factor and is restricted to the nucleosomes in its vicinity.3 NURF facilitates a step preceding RNA polymerase II preinitiation complex assembly resulting in transcriptional activation of a chromatin template reconstituted in vitro, 6 although the detailed mechanism of function and the nature of the remodeling that takes place remain to be elucidated. The stimulation of the ATPase activity of NURF by nucleosomes rather than free DNA or histones suggests that NURF has a special ability to recognize nucleosome structure, important elements of which include the flexible histone tails. 7 Several of the NURF subunits have been identified and cloned. NURF140 (140 kDa) is the ISWI ATPase 8 that was cloned on the basis of homology to the ATPase domain of yeast SWI2 protein. NURF-55 (55 kDa) is a WD repeat protein 9 known to be involved in histone metabolism. NURF-38 (38 kDa) has been cloned and expressed as a recombinant polypeptide. 1° The cloning and characterization of the largest subunit, NURF-215 (215 kDa), is currently in progress. Once cDNAs for all subunits become available it will be of interest to reconstitute the NURF complex with recombinant proteins and rigorously compare its activities with native NURF purified from Drosophila. Outline of Purification Scheme The purification of NURF from Drosophila nuclear extracts is a procedure of mild chromatographic steps followed by glycerol gradient centrifugation. The protocol is reliable and reproducible, and the yield of highly pure NURF complex is quite sufficient for biochemical analysis. NURF can be traced along the stages of purification either by testing fractions for 5 p. D. Varga-Weisz, M. Wilm, E. Bonte, K. Dumas, M. Mann, and P. B. Becker, Nature 388, 598 (1997). 6 G. Mizuguchi, T. Tsukiyama, J. Wisniewski, and C. Wu, Mol. Cell 1, 141 (1997). 7 p. T. Georgel, T. Tsukiyama, and C. Wu, E M B O J. 16, 4717 (1997). 8 T. Tsukiyama, C. Daniel, J. Tamkun, and C. Wu, Cell 83, 1021 (1995). 9 M. A. Martinez-Balbas, T. Tsukiyama, D. Gdula, and C. Wu, Proc. Natl. Acad. Sci. U.S.A. 95, 132 (1998). lo D. A. Gdula, R. Sandaltzopoulos, T. Tsukiyama, V. Ossipow, and C. Wu, Genes Dev. 12, 3206 (1998).
[44]
P U R I F I C A T I O N OF N U C L E O S O M E R E M O D E L I N G F A C T O R
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nucleosome remodeling activity or by detection of N U R F subunits in the fractions by Western analysis, using a mixture of antisera recognizing p38, p55, and ISWI. The nucleosome remodeling assay is described in detail by Mizuguchi and Wu. u For all separation methods tested, the peak of N U R F activity coincides with fractions containing p38, p55, and ISWI. Because p55 and ISWI are components of other complexes, 5,9 it is important to trace the codistribution of all N U R F subunits to avoid the inadvertent purification of other complexes. The outline of the purification scheme is shown in Fig. 1. The purity of N U R F containing fractions after each purification step is shown in Fig. 2: 1.25 t~l (lanes 1, 2, and 3), 5/~1 (lanes 4 and 5), or 10/xl (lane 6) of the indicated step was analyzed by 10% SDS-PAGE. Proteins were visualized either by silver staining or by immunoblotting with a mixture of three antisera specific for the indicated NURF subunits. Only four major bands are present after the glycerol gradient. Interestingly, all four of these proteins can be immunoprecipitated by any of the three antibodies from relatively crude fractions. 9,1° The yield of highly purified N U R F is 100-200/~g per 200 g of embryos. Preparation of Starting Material The starting material for N U R F purification is a nuclear extract prepared from Drosophila embryos according to the procedure described by Wampler et al. 12with slight modifications. For a typical purification, approximately 200 g of moist, 12- to 16-hr Drosophila embryos are dechorionated and resuspended in 3 mug of buffer I (15 mM H E P E S - K O H , pH 7.6, 10 mM KC1, 5 mM MgC12, 0.1 mM EDTA, pH 8.0, 0.5 mM EGTA, pH 8.0, 350 mM sucrose) supplemented with 1 mM dithiothreitol (DT-F) and 0.2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). The embryos are homogenized with two passes through a Yamato motordriven glass/Teflon homogenizer at 1500 rpm. The homogenate is filtered through Miracloth, and nuclei are pelleted by centrifugation for 10 min in a Beckman JA 14 rotor at 8000 rpm (10,000g). All centrifugations are at 4°. The supernatant is discarded, and nuclei are resuspended again in the original volume of buffer I. While resuspending the nuclei by swirling, it is critical to avoid the much tighter yellow yolk pellet. Nuclei are then dispersed by briefly homogenizing two to three times with a loose glass pestle (Dounce) and immediately pelleted again as described previously. After the second centrifugation there should be hardly any yolk at the bottom of the nuclear pellet. All of it should be avoided as nuclei are u G. Mizuguchi and C. Wu, "Chromatin Protocol." Humana Press, Clifton, NJ, in press. i2 S. L. Wampler, C. M. Tyree, and J. T. Kadonaga, J. Biol. Chem. 265, 21223 (1990).
760
[44]
CHROMATIN REMODELING COMPLEXES
Drosophila 0-12 hr embryos (200 g) Nuclear extract (NH4)2SO 4 precipitation (1 g) DE52 DEAE-cellulose Bio-Rex 70 (56 mg)
I
I
0.22
I
0.32
1.0 M
Q Sepharose Fast Flow (10 mg)
I
I
I
0.2
0.3
0.4
I 1.0 M
Hydroxylapatite (4.7 mg)
I
I
0.2
0.3
I
0.4
I
1.0M
Cellulose phosphate P l l (1.14 mg) 0.2
I
II
I
0.6 M
d
Glycerol gradient centrifugation (200 I~g) FIG. 1. Schematic outline of the purification protocol. The amount of total protein in the N U R F containing fractions after each purification step is indicated.
resuspended by swirling in 1 ml/g of embryos of buffer II (15 mM HEPESKOH, pH 7.6, 110 mM KC1, 5 mM MgCI2, 0.1 mM EDTA, supplemented with 1 mM DTT and 0.2 mM AEBSF). Nuclei are dispersed well in a Dounce homogenizer (loose pestle). The volume of the nuclear suspension
[44]
PURIFICATION OF NUCLEOSOME REMODELING FACTOR
761
p215 ISWI
Silver staining
p55 p38 ISWI
Western
p55 p38 12 3456
FI6.2. Protein analysis of NURF containing fractions along the purification procedure.
is measured and divided equally into screw-cap tubes for the Beckman Type 35 rotor. Nuclei are lysed by adding 1/10 volume of 4 M (NH4)2804, pH 8.0, in each tube. The viscous lysate is mixed vigorously for 20 min on a fast rotating wheel and then centrifuged in a precooled ultracentrifuge for 1 hr at 35,000 rpm (90,000g). The clear supernatant is collected by plunging a pipette beneath the lipid top layer. The volume is measured again and poured into a beaker. An equal volume of 4 M (NH4)2804, pH 8.0, is added slowly (over a period of 5 rain) and the mix is stirred vigorously for another 10 min at 4°. Proteins are precipitated by centrifugation in a JA-14 rotor at 11,000 rpm (18,600g) for 30 min. The pellets may be flash frozen in liquid nitrogen and kept at - 8 0 ° until needed. The pellet is allowed to thaw and is resuspended in 0.2 ml/g of embryos of HEGN-40 [25 mM H E P E S - K O H , pH 7.6, 1 mM EDTA, 10% (v/v) glycerol, 0.02% (v/v) Nonidet P-40 (NP-40), 40 mM KC1; the indicator 40 stands for the concentration of KC1 in raM] supplemented with 1 mM DTT and 0.2 mM AEBSF by pipetting up and down. It is important to ensure thorough solubilization of the pellet at this step as severe reprecipitation may occur during subsequent dialysis. The extract is then placed in dialysis bags (Spectra/Por 2, molecular weight cutoff 12,000-14,000) and dialyzed twice for 1 hr against 1 liter of HEGN-40 (with 1 mM DTT and 0.2 mM AEBSF). The dialysis buffer is changed once more and the dialysis continues until the conductivity of the extract equals that of HEGN-150. The conductivity is monitored every 15 min. Finally the dia-
762
CHROMATIN REMODELING COMPLEXES
[44]
lyzed extract is harvested and cleared before proceeding to chromatography by centrifugation at 8000 rpm in a precooled JA-14 rotor (10,000g) for 10 rain. Ion-Exchange Chromatography All chromatographic steps are performed on an FPLC (fast protein liquid chromatography) system (Pharmacia, Piscataway, N J). Every column is packed 1 day in advance and equilibrated overnight with at least 5 bed volumes of buffer at a slow flow rate. The conductivity, as well as the pH of the flow through, is monitored to verify equilibration. The estimated volumes of buffers needed are prepared in advance and the appropriate loading superloop is mounted on the system so that they equilibrate to 4° overnight. The first ion-exchange chromatography step consists of a DE52 DEAE-cellulose and a Bio-Rex 70 column, in tandem. For each gram of extract protein (expect approximately 1 g protein per 200 g of embryos) use 20 ml of packed DE52 and 50 ml Bio-Rex 70 resins. An appropriate amount of preswollen Whatman DE52 resin is resuspended in at least 6 ml of HEG-150 (as HEGN-150 without NP-40) per gram of resin with gentle agitation on a shaking platform. The swollen resin is allowed to settle by gravity and the supernatant containing fines is aspirated away. The resin is then resuspended in fresh HEG-150 buffer and allowed to settle once more to remove ultrafine particles that need a longer time to settle. Then the resin is transferred into a XK16 column (Pharmacia) and allowed to pack. In parallel, a sufficient amount of Bio-Rex 70 resin (200-400 mesh; BioRad, Richmond, CA) is resuspended in 5 ml HEG-150 per gram of resin. The swollen resin is transferred in a XK26 (Pharmacia) column. Once packed by gravity, the columns are mounted on the FPLC system such that the bottom of the DE52 column is connected to the top of the Bio-Rex 70 column. We use HEGN-0 (containing no KC1) as buffer A of the FPLC and HEGN-1000 (containing 1 M KC1) as buffer B, both supplemented with 1 mM DTT and 0.2 mM AEBSF. The columns are equilibrated with 15% buffer B (containing 150 mM KC1) overnight (at least 5 bed volumes). The next day, the flow rate is increased to 2 ml/min for 15 mirl to ensure tight packing, then the top lid of each column is readjusted to minimize the dead volume. The extract is loaded at 1 ml/min with 15% buffer B. NURF does not bind efficiently to DE52 under these conditions but it is retained on the Bio-Rex 70. As soon as the UV monitor signal drops to the baseline, the DE52 column is disconnected. The flow rate is increased to 2 ml/min, and the Bio-Rex 70 column is washed with 22% buffer B until the UV signal
[44]
P U R I F I C A T I O N OF N U C L E O S O M E R E M O D E L I N G F A C T O R
763
drops to the baseline. Then N U R F is eluted in a single step by raising the relative concentration of buffer B to 32%. The entire peak (about 40 ml for 1 g of loaded protein) is collected. Although N U R F is eluted as a broad peak between approximately 280 and 380 mM KC1, the elution at 320 mM is a good compromise between yield and purification factor. The majority of free N U R F subunits not associated in the complex is removed (as indicated by filtration methods) and NURF activity is extremely high. This first step is highly reproducible. However, as NURF proteins bind avidly to DE52 resin at pH greater than 8.0, careful adjustment of the buffer pH is imperative. N U R F containing fractions are then dialyzed against HEGN-100 until conductivity equals that of HEGN-150. The sample is now ready for the second purification step. Sufficient Q Sepharose fast flow (Pharmacia) slurry is dispensed in a beaker to give 7-8 ml of packed resin. The resin is allowed to settle. The ethanol containing supernatant is aspirated away and the resin is resuspended in an equal volume of starting buffer (HEG-150, Nonidet P-40 is avoided again during packing). When the resin settles again, the supernatant is decanted and is replaced by fresh buffer. The slurry is degassed and poured into an H R 10/10 column (Pharmacia). HEGN-0 is used as buffer A and HEGN-1000 as buffer B. The column is equilibrated with 5 volumes of HEGN-150 (with 1 mM DTT and 0.2 mM AEBSF freshly added). The flow rate is adjusted at 2 ml/min for 5 min. The sample is centrifuged in a JA-20 rotor for 15 min at 12,000 rpm (17,400g). The supernatant is filtered through a 0.22-/zm Millex-GV low protein binding filter (Millipore, Bedford, MA) and loaded onto the column at 1 ml/min in HEGN-150. When the UV signal drops to baseline, NURF is eluted in steps with 20, 30, 40, and 100% buffer B. Three-milliliter fractions are collected. NURF elutes in the 0.3 M peak (30% of buffer B). N U R F containing fractions are pooled and dialyzed against PPEGN100 [0.1 mM EDTA, 10% (v/v) glycerol, 0.02% (v/v) Nonidet P-40, 100 mM K2 HPO4/KH2PO4, pH 7.6] for 2.5 hr. Hydroxylapatite and Cellulose Phosphate Chromatography Hydroxylapatite resin (Bio-Rad) is resuspended in PPEGN-100. The slurry is poured into a H R 10/10 (Pharmacia) column and allowed to pack by gravity. A sufficient amount of slurry to yield 4 ml of packed bed volume is added. PPEGN-0 (as PPEGN-100 without K2HPO4/KH2PO4) is used as buffer A and PPEGN-800 (contains 0.8 M K2HPO4/KH2PO4, pH 7.6) as buffer B. The column is equilibrated with at least 5 bed volumes of PPEGN100 (i.e., 12.5% buffer B). The sample is filtered through a 0.22-/zm MillexGV filter and loaded at 0.5 ml/min. When the UV signal drops to baseline,
764
CHROMATIN REMODELING COMPLEXES
[44]
proteins are eluted with steps of PPEGN-200, PPEGN-300, PPEGN-400, and PPEGN-800 collecting 1.5-ml fractions. NURF elutes mainly in the PPEGN-200 peak. Fractions containing N U R F are pooled (expect about 12 ml total volume of pooled N U R F containing fractions). Although some N U R F is also present in the PPEGN-300 peak, these fractions are not pooled because they contain far more impurities. In order to minimize losses due to nonspecific protein sticking to the dialysis bag, pure pH neutralized insulin (Boehringer) is added to a final concentration of 0.2 mg/ml and dialyzed against HEGN-150 until the conductivity is equal to HEGN-200 (about 2 hr). The last chromatographic step is cellulose phosphate P l l (Whatman). P l l resin comes in a fibrous form that needs precycling before use (see manufacturer's specifications). P l l can be stored at 4° in 0.5 M phosphate buffer at pH 7.0. Precycled cellulose phosphate P l l is transfered into 10-20 volumes of HEGN-200. The resin is mixed without vigorous stirring to avoid the generation of fines and is allowed to settle. The supernatant is discarded and the resin is resuspended in 3-5 volumes of HEGN-200. The resin is degassed and the equivalent of 1 ml resin volume is poured into a H R 5/5 column. After the resin has settled by gravity, the top is refilled with resin (to compensate for the reduction of volume after close packing) and the column is closed. HEGN-0 and HEGN-1000 buffers are used as buffers A and B respectively. The column is equilibrated in HEGN-200 with 20 bed volumes at 0.2 ml/min. The flow rate is increased to 0.4 ml/ min for 3 min. Then the sample is loaded at 0.4 ml/min for 3 min. Then the sample is loaded at 0.4 ml/min in HEGN-200. When the UV signal drops back to baseline, the column is developed with a shallow linear salt gradient (HEGN-200 to HEGN-600 in 20 column volumes). Four hundredmicroliter fractions are collected. NURF elutes as a very broad peak, spanning from HEGN-250 to HEGN-450, probably because P11 is a bifunctional cation exchanger containing both strong and weak acid groups. It is essential not to pool the N U R F containing fractions; each one is processed further separately. Glycerol Gradient Centrifugation The ultimate purification step is a glycerol gradient centrifugation. Glycerol gradients are formed in SW50Ti tubes (swinging bucket rotor tubes; Beckman) by carefully applying 450-~1 aliquots of each of 10 HEGN-150 solutions (supplemented with a cocktail of protein inhibitors; the 100× concentrated inhibitor mix contains 100 mM phenylmethylsulfonyl fluoride, 200 b~M pepstatin A, 60 ~M leupeptin, 200 mM benzamidine, and 200 ~g/ml chymostatin in ethanol) containing 35 to 17% (v/v) glycerol in inere-
[44]
PURIFICATION OF NUCLEOSOME REMODELING FACTOR
765
ments of 2%, i.e., the first layer (bottom) is 35% (v/v) and the last layer (top) is 17% (v/v) glycerol. All buffers as well as the rotor and the centrifugation chamber must be precooled to 4° because the viscosity of the gradient is a function of the temperature. Four hundred microliters of the NURF containing P l l fractions is applied on top. The gradients are centrifuged at 49,000 rpm (160,000g) for 20 hr at 4 ° (no brake). Five hundred-microliter fractions are collected from the bottom of each tube. NURF is always found in the third fraction. Glycerol gradient centrifugation is a very effective and reproducible purification step that conditions the purified NURF fractions for prolonged storage. Early N U R F containing fractions of the P l l column yield very pure NURF preparations when applied to glycerol gradient centrifugation. After this step, only four major protein bands corresponding to the four NURF subunits are visible on a silver-stained S D S - P A G E gel (Fig. 2, lane 6). Late N U R F containing P l l fractions usually contain impurities that are not completely removed after glycerol centrifugation. Acknowledgments R.S. was supported by an E M B O postdoctoral fellowship. V.O. was a recipient of a long-term fellowship from the Human Frontier Science Program Organization. D.A.G. is a Leukemia Society of America postdoctoral fellow.