Immunoprecipitation of chromatin

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IMMUNOPRECIPITATION OF CHROMATIN

[ 1 5] I m m u n o p r e c i p i t a t i o n By

LAURA P. O'NEILL

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of Chromatin

and BRYAN M. TURNER

A valuable approach for analyzing the association of specific proteins with DNA, in the context of chromatin, has been to produce specific antibodies and use them in immunoprecipitation experiments. Various techniques have been developed that incorporate adaptations to increase the efficiency of the precipitation, dependent on both the antibody specificity and the starting chromatin population. A standard procedure is outlined in Fig. 1. This chapter offers three standard protocols, all of which have been used successfully to analyze the role of specific proteins within defined regions of the eukaryotic genome. It is becoming increasingly important to analyze both the D N A and the protein after immunoprecipitation. This requires a careful examination of the protocol chosen as not all are amenable to protein analysis. If firm conclusions regarding the role of a particular protein are to be drawn, then the researcher must show convincingly that the target protein has indeed been selectively precipitated in the experiment. This relies on the specificity of the antibody, which should be tested not only by enzyme-linked immunosorbent assay (ELISA) and Western blotting, but also against chromatin in the form in which it is to be used for immunoprecipitation. The conformation of protein antigens is likely to vary depending on whether they are in their native state in solution, formaldehyde-fixed, bound to nitrocellulose filters, or fixed to plastic microtiter plates. Nonspecific binding of the antibody to chromatin should also be carefully controlled. Antibodies may be added in excess if complete depletion of the target protein is required. This may exacerbate problems of nonspecific binding that should be carefully monitored by using both preimmune sera and no-antibody controls. Affinity purification of antisera also minimizes background and, if possible, should be routinely carried out. The starting chromatin population should also be carefully considered. If the precise positioning of proteins on a stretch of DNA is of interest, then the exchange or sliding of these proteins during chromatin isolation must be minimized. In such cases formaldehyde fixation 1,~ or UV-induced cross-linking 3-5 have been successfully employed and are discussed in detail 1 V. Jackson, Cell 15, 945 (1978). 2 j. Solomon and A. Varshavsky, Proc. Natl. Acad, Sci. U.S.A. 82, 6470 (1985). 3 S. Gilmor and J. T, Lis, Mol. Cell Biol. 5, 2009 (1985). 4 R. T. K a m a k a k a and J. O. T h o m a s , E M B O J. 9, 3997 (1990). 5 A. D. Mirzabekov, Gene 135, 111 (1993).

METHODS IN ENZYMOLOGY.VOL. 274

Copyright © 1996by AcademicPress, Inc. All rights of reproduction in any form reserved.

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CHROMATIN AS TEMPLATES FOR TRANSCRIPTION

Antibody

+

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Chromatin

Incubate O/N at 4°

Add Protein A- Sepharose

Incubate for 1-4 hr at R.T.

Centrifuge Supernatant = UNBOUND

Pellet = BOUND

Wash pellet with NaCl

Elute bound material

Isolate DNA and PROTEIN

FIG. 1. Schematic representation of a typical immunoprecipitation experiment.

later. The choice of immunoabsorbent is also important. Heat-killed formalin-fixed Staphylococcus aureus has been commonly used, but commercially available pure protein A coupled to Sepharose CL-4B, although more expensive, generally gives cleaner results, with nonspecific binding of chromatin being kept to a minimum. Washing of the bound material on the immunoabsorbent is also critical and, in some cases, it has been found that large volume washes reduce nonspecific binding considerably. The stringency of the washing should be based on the avidity with which the antibody binds to the target protein. Up to 0.5 M NaC1 has been used as a final wash, but in these cases any information about linker histones and other salt-extractable proteins is lost. 6,7 6 C. A. Perry, C. A. Dadd, C. D. Allis, and A. T. Annunziato, Biochemistry 32, 13605 (1993). 7C. A. Perry, C. D. Allis, and A. T. Annunziato, Biochemistry 32, 13615 (1993).

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Immunoprecipitation of Unfixed Chromatin Chromatin can be isolated from tissue culture cells or whole tissues following standard procedures previously detailed in this series s employing micrococcal nuclease or DNase I digestion. This procedure generates fragments ranging in size from mononucleosomes up to 10- to 12-mers depending on the time of digestion. The use of freshly prepared chromatin is recommended to minimize proteolysis, especially of chromatin isolated from tissues. Selective precipitation of larger chromatin fragments has been observed by several workers 9-11 and, if problematic, can be avoided by isolating pure mononucleosomes from sucrose gradients. 12 In the experiments cited, the conclusions drawn were similar irrespective of the starting nucleosome population. However, in some cases when precise localization of a protein is being examined, the isolation of mononucleosomes may be required. The following immunoprecitation procedure 11 was developed in our own laboratory based on previous protocols. 4'1° We have found that it is essential to use siliconized Eppendorf tubes and pipette tips to ensure maximum recovery of DNA. Procedure

1. Add 100-200/zl affinity-purified antibody (50-100/zg Ig) to 100-200 /zg unfixed chromatin (based on A260 readings-input) and add incubation buffer [50 mM NaC1, 20 mM Tris-HCl, pH 7.5, 20 mM sodium butyrate, 5 mM Na2EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF)] to a final volume of 1 ml. For experiments with cultured cells, DNA is routinely labeled by growth of the cells overnight in [3H]thymidine (1/xCi/ml) prior to chromatin preparation. 2. After overnight incubation (on a slowly rotating platform) at 4 °, add 200 tzl preswollen protein A-Sepharose [50% (v/v) slurry] and continue the incubation for an additional 3 hr at room temperature. 3. Centrifuge the antibody-chromatin mixture at 11,600 g at 4° for 10 min. Carefully remove the supernatant and keep on ice. This is designated the unbound fraction. Resuspend the protein A-Sepharose pellet in 1 ml buffer A (50 mM Tris-HC1, pH 7.5, 10 mM EDTA, 5 mM sodium butyrate) containing 50 mM NaCI and layer onto 9 ml of the same buffer. After centrifugation at 600 g for 10 min at 4°, remove the supernatant by aspiration 8 R. D. Kornberg, J. W. LaPointe, and Y. Lorch, Methods Enzymol. 170, 1 (1989). 9 E. Mendelson, D. Landsman, S. Druckmann, and M. Bustin, Eur. J. Biochem. 160, 253 (1986). 10 T. R. Hebbes, A. W. Thorne, A. L. Clayton, and C. Crane-Robinson, Nucleic Acids Res. 20, 1017 (1988). 11 L. P. O'Neill and B. M. Turner, E M B O J. 14, 3946 (1995). 12 M. Noll and R. D. Kornberg, J. Mol. Biol. 109, 393 (1977).

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CHROMATINAS TEMPLATESFOR TRANSCRIPTION

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and wash the pellet in 10 ml buffer A containing 100 m M NaC1 and in 10 ml of buffer A containing 150 m M NaC1. 4. Elute the bound material from the protein A-Sepharose by the addition of 125/~1 1% sodium dodecyl sulfate (SDS) in incubation buffer and incubate for 15 min at room temperature with constant inversion. After centrifugation at 11,600 g at 4 ° for 10 min, remove the supernatant and store on ice. Repeat this step once more and combine the two supernatants. Add an equal volume of incubation buffer to reduce the concentration of SDS to 0.5%. This is designated as the bound fraction. 5. Isolation of DNA: Add one-third volume of phenol : chloroform to the input, unbound, and bound fractions. Vortex and centrifuge at 600 g for 10 min at 4 ° to separate the phases. Remove the supernatant and add an equal volume of phenol:chloroform. Repeat the centrifugation step. Add an equal volume of chloroform : isoamyl alcohol, centrifuge as before, and transfer the supernatant to a 6-ml centrifuge tube. Finally precipitate the D N A at - 2 0 ° using two volumes of ice-cold ethanol in the presence of 0.1 M LiC1. 6. Isolation of proteins: Precipitate the proteins from the first phenol:chloroform phase 13 by the addition of 5 /zg bovine serum albumin (BSA, carrier), 1/100th volume 10 M H2804, and 12 volumes of acetone. After overnight precipitation at - 2 0 °, wash the protein pellets once in acidified acetone (1 : 6 (v/v) 100 m M H2804 : acetone) and three times in dry acetone. Notes: Step 1. Sodium butyrate is added as an inhibitor of histone deacetylase and is not essential for immunoprecipitations where histone acetylation is not of primary interest. The amount of antibody added is dependent on the titer, concentration of Ig, and amount of target protein present in the chromatin. Step 2. Protein A-Sepharose is available commercially as a freeze-dried powder (Pharmacia, Piscataway, N J) and should be preswelled in 50 m M Tris-HCl, 5 m M Na EDTA, 50 m M NaC1. The concentration of NaC1 can be increased in order to reduce nonspecific binding, although it may also release the antibody from the target protein or associated proteins from chromatin. Preliminary experiments should be employed to determine the optimum washing conditions for the antibody used. Step 3. The concentration of the NaC1 can be increased to reduce nonspecific binding. See step 2. We have found that large volume washes, carried out in 15-ml siliconized centrifuge tubes, greatly reduce nonspecific binding. Other workers, however, have found with their antibodies that increasing the number of washes in a smaller volume is satisfactory.6,1° 13C. G. P. Matthews, G. H. Goodwin, and E. W. Johns, Nucleic Acids Res. 6, 167 (1979).

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Step 4. In the given procedure we used SDS to elute the bound fraction from protein A-Sepharose. Guanidine hydrochloride (2 M) also gives efficient elution of the bound material following the same steps for subsequent protein isolation. Readers should also refer to an earlier volume of this series for alternative elution buffersJ 4 Step 5. We routinely add glycogen (5/xg) as a carrier to maximize the precipitation of DNA from the bound fraction. Ethanol used in the precipitation should be Analar grade and precooled to - 2 0 ° to ensure rapid precipitation. Keep the first phenol : chloroform phase for subsequent protein isolation. We routinely analyze the DNA samples by electrophoresis on 1.2% agarose gels and staining with ethidium bromide. DNA content in each sample is followed by measurement of [3H]thymidine by scintillation counting. Step 6. We routinely analyze the proteins by electrophoresis in SDS-polyacrylamide gels. as Proteins can also be analyzed on acid/urea/Triton gels, 16 in which case the protein pellet must be washed, diluted, and reconcentrated to remove SDS that interferes with these gels. Resuspend protein pellets initially in 500 /zl distilled H20 and centrifuge using microconcentrators (Amicon) for 30 min at 13,000 rpm. Repeat this step before adding 2 volumes of AUT-loading buffer (8 M urea, 5% (v/v) 2-mercaptoethanol, 1 M glacial acetic acid, plus a few drops of tracking dye pyronin Y) to the final concentrated sample. Western blotting, immunostaining, and detection by enhanced chemiluminescence (ECL, Amersham) or iodine-125 conjugated antibody should be performed following standard proceduresJ 7

Immunoprecipitation of Formaldehyde-Fixed Chromatin Some studies requiring the precise positioning of DNA-binding proteins are hampered by the exchange, sliding, or artificial movements of proteins during chromatin isolation. Treatment with formaldehyde,1 dimethyl sulfate/borohydride] 8 or UV irradiation 4,18 efficiently cross-links proteins to DNA. Methods for formaldehyde cross-linking of chromatin in nuclei have been described in detail by Jackson. 1 Formaldehyde produces reversible cross-links between protein-DNA, protein-RNA, and proteinproteinJ ,2 This method has been used to map protein-DNA interactions

14j. Anderson and G. Blobel, Methods EnzymoL 96, 111 (1983). 15 U. K. Laemmli, Nature 277, 680 (1970). 16W. M. Bonner, M. H. P. West, and J. D. Stedman, Eur. ,L Biochem. 109, 17 (1980). 17 B. M. Turner, L. P. O'Neill, and I. M. Allan, FEBS Lett. 253, 141 (1989). 18 A, D. Mirzabekov, V. V. Shick, A. V. Belavsky, and S. G. Bavykin, Proc. Natl. Acad. Sci. U.S.A. 75, 4184 (1978).

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CHROMATIN AS TEMPLATES FOR TRANSCRIPTION

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along the heat shock protein 70 (hsp 70) g e n e , 19"2° the yeast silent mating loci, 21 and the polycomb proteins within the bithorax domain. 22 Although clean DNA results were obtained from all of these papers, analysis of the proteins was not carried out. Although the DNA-protein bonds are easily reversed by heating in the presence of SDS, 1 very rarely are both protein and D N A analyzed from the same immunoprecipitate. This is possibly due to the small amount of material available following precipitation. Comprehensive methodology using a simpler three-step approach has now been developed. 23'24 In these experiments the time of formaldehyde fixation and NaCI concentrations are well characterized. The only other major difference between these protocols is that Dedon et al. 23'24 do not use a CsC1 gradient to remove contaminating, nonchromatin-bound cross-linked proteins and free DNA and RNA. Depending on the degree of nonspecific binding acceptable, this step may have to be included. The following method outlined is that developed by Solomon et al. 19 although readers should also refer to the simpler method of Dedon et aL 23 which gives excellent details regarding the pitfalls associated with the immunoprecipitation of chromatin. P r o ced u r e A

1. Dilute 0.2-0.5 /zg D N A (maximum volume 180/zl) in 0.1% SDS, 1 mM Na2EDTA, 10 mM Tris-HC1 (pH 8) and incubate for 5 min at room temperature. Add sodium deoxycholate and Triton X-100 to 0.1 and 1% final concentration (RIPA buffer). 2. Incubate for 10 rain at room temperature before adding 30-90/xg lgG and continue the incubation overnight at 4 °. 3. Add a volume of 50% protein A-Sepharose slurry equivalent to the volume of IgG added and continue incubation for a further 3 hr at room temperature. 4. Centrifuge the immunocomplexes at 12,000 g at 4 ° for 15 sec. 5. Wash the pellet four times (10 min per wash) in 0.4 ml of 0.25 M LiC1, 0.5% (v/v) NP-40, 0.5% (w/v) sodium-deoxycholate, 1 mM NazEDTA, 10 mM Tris-HC1 and twice in 0.2 ml 1 mM Na2EDTA, 10 mM Tris-HC1 (pH 8). i9 M. J. Solomon, P. L. Larsen, and A. Varshavsky, Cell 53, 937 (1985). 20 G. A. Nacheva, D. Y. Guschin, O. V. Preobrazhenskaya, V. L. Karpov, K. K. Ebralidse, and A. D. Mirzabekov, Cell 58, 27 (1988). 21 M. Braunstein, A. B. Rose, S. G. Holmes, C. D. Allis, and J. R. Broach, Genes Dev. 7, 592 (1993). 2z V. Orlando and R. Paro, Cell 75, 1187 (1994). 23 p. C. Dedon, J. A. Soults, C. D. Allis, and M. A. Gorovsky, Anal. Biochem. 197, 83 (1991). 24 p. C. Dedon, J. A. Soults, C. D. Allis, and M. A. Gorovsky, MoL Cell. Biol. U , 1729 (1991).

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6. Resuspend the pellet in 0.2 ml of 1 mM Na2EDTA, 10 mM Tris-HC1, add RNase A at 50 txg/ml, and incubate at 25 ° for 10 rain. Add pronase and SDS to 0.1 mg/ml and 0.15% final concentration and continue the incubation for a further 2 hr at 37°. 7. Reverse pronase-resistant peptide-DNA cross-links for DNA samples to be analyzed on Southern blots by heating samples to 65° followed by ethanol precipitation. 8. DNA samples analyzed by slot hybridization are incubated at 95° for 30 rain followed by ethanol precipitation. Notes: This procedure does not allow the analysis of both protein and DNA from the same sample given the addition of RNase A and pronase in steps 7 and 8. This method has recently been used to map the polycomb proteins along the bithorax domain and includes a ligated mediated polymerase chain reaction step to amplify the immunoprecipitated DNA. 22This step is potentially very useful if only small amounts of DNA have been obtained and should allow extensive analysis of the precipitated material.

Procedure B This procedure has slight variations in the buffer concentrations used compared to those outlined in Procedure A. However, this method does include analysis of the unbound proteins following the reversal of the protein-DNA cross-links. 1. Dilute 0.1-0.4 /xg DNA (based on [3H]thymidine counts) in 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaC1, 20 mM TrisHC1, pH 8.1 (TSE), in a final volume of 0.25-0.75 ml. 2. Add preimmune or immune sera together with protease inhibitors (1 mM PMSF, 0.8/xg/ml pepstatin, 0.6/xg/ml leupeptin) and incubate for 12-16 hr at 4°. 3. Add a 50% (w/v) protein A-Sepharose slurry equivalent to six times the antibody volume added in step 2 and continue the incubation for another hour at 4°. 4. Centrifuge the immunocomplex at 16,000 g, 4°, for 15 sec and wash with gentle agitation for 5-10 min in 1 ml of TSE followed by TSE containing 0.5 M NaC1, 0.25 M LiC1, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris, pH 8.1, and finally with two washes in TE.

Analysis of Proteins from Unbound Fraction 5. Label cultures with [3H]lysine (15 /zCi/ml) for 16 hr. Combine the supernatant designated unbound and the first wash and reverse the protein-DNA cross-links by boiling the solution for 30 rain.

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6. Add a carrier protein (e.g., 5 t~g BSA) and precipitate the sample using 30% (v/v) trichloroacetic acid (TCA) at 4° for several hours. Collect the proteins by centrifugation at 16,000 g for 30 min. 7. Wash the protein pellet extensively in 0.1% HC1 : acetone and twice in acetone at 4 °. Vacuum dry the protein pellet and resuspend in TE (containing 1 mM PMSF, 0.8 ~g/ml pepstatin, 0.6/~g/ml leupeptin). 8. Extract the proteins with perchloric acid (5%) or H2SO4 (0.2 M) at 4 ° with vigorous shaking. Following centrifugation at 16,000 g for 30 min, precipitate the proteins with 30% TCA as described earlier and analyze on SDS-PAGE. Analysis o f DNA. Remove immune complexes from the beads with 1% (w/v) SDS, 100 mM NaHCO3 buffer. Reverse protein-DNA cross-links by heating at 65 ° for 6 hr. Add proteinase K (0.1 mg/ml) and incubate at 37° for 2-3 hr. Precipitate D N A by phenol:chloroform extraction and ethanol precipitation. Notes: Step 1. D N A and protein content followed with [3H]thymidine and [3H]lysine incorporation can be added to the culture medium at 1 and 15 /~Ci/ml, respectively. Triton X-100 is added to the D N A mixture to create micelles of Triton and SDS which prevents SDS disruption of antibody-antigen complexes. 14 Step 4. A high salt wash (0.5 M NaC1) removes nonspecifically bound proteins but care should be taken since this then excludes the analysis of liker histones and other salt-labile proteins. The LiCI wash reduces SDS content and additional TE washes remove any remaining detergents. Step 5. In the original method only the unbound fraction was analyzed, but this was due to technical difficulties regarding the mobility of the protein immunoprecipitated. This method does extend itself to analyze proteins from both the unbound and the bound fractions. UV Cross-Linking The following immunoprecipitation procedure was developed by Kamakaka and Thomas 4 for the analysis of HI-containing chromatin and uses UV cross-linked material. For detailed methodology with respect to UV irradiation, readers are referred to Mirzabekov et al. 25 Procedure 1. Digest 100 p.g cross-linked DNA-protein (300 ~1) with PstI for 12-14 hr and dialyze overnight against 50 mM Tris-HCl, pH 7.5, 5 mM NazEDTA, 150 mM NaC1 (DB buffer). 25 A. D. Mirzabekov, S. G. Bavykin, A. V. Belavsky, V. L. Karpov, O. V. Preobrazhenskaya, V. V. Shick, and K. K. Ebralidse, Methods Enzymol. 170, 386 (1989).

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2. Centrifuge the sample at 30,000 rpm for 30 sec to remove aggregated material before the addition of antisera (10/xl). Incubate overnight at 4° with constant inversion and then add 30 mg (10%, w/v; 50%, v/v) of protein A-Sepharose diluted in DB buffer. Continue the incubation for a further 4 hr at room temperature. 3. Dilute the slurry with 10 ml of the DB buffer containing 0.5% NP40 and centrifuge at 600 g for 10 min. 4. Wash the protein A-Sepharose pellet in 10 ml of 50 mM Tris-HC1, pH 7.5, 5 mM Na2EDTA containing 0.2 M and 0.25 M NaC1. 5. Release bound immunoprecipitated DNA by digestion with proteinase K for 4-12 hr at 42° and purify by phenol extraction followed by chloroform-isoamyl alcohol extraction and ethanol precipitation in the presence of 0.3 M sodium acetate. Resuspend the DNA pellet in distilled H20. 6. Radiolabel immunoprecipitated DNA by nick translation and use to screen various plasmids containing DNA inserts of interest. Notes: Step 3. In the previously described procedure, the unbound material is not analyzed. If this is required, centrifuge the sample first, retain the unbound material, and then resuspend the protein A-Sepharose in the first wash buffer and continue with steps 4-6. Step 6. An alternative approach to analyzing the DNA is that the precipitated material is labeled and then used as the probe to screen various gene sequences. This is advantageous if a large number of gene sequences are to be screened.

Acknowledgment W o r k in t h e a u t h o r s l a b o r a t o r y w a s s u p p o r t e d b y t h e W e l l c o m e T r u s t .

[ 16] M a p p i n g o f N u c l e o s o m e P o s i t i o n s

By

FRITZ

THOMA

The structural and functional subunits of chromatin are nucleosome cores. In a nucleosome core, about 145 bp of DNA are coiled around the outer surface of an octamer of histone proteins, which consists of two H2A. H2B dimers associated with a tetramer of 2(H3-H4). DNA extending from the nucleosome core to the next nucleosome is called linker DNA. It varies from about 20 to 90 bp in length in different organisms or tissues or between individual nucleosomes. Histone HI may be associated METHODS IN ENZYMOLOGY. VOL. 274

Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.