Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Podsumowanie

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

Streszczenie

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Wprowadzenie

SNF2 family chromatin remodeling complexes include a central SNF2-like ATPase subunit ^1,2. Some SNF2-like ATPases function as single subunit enzymes, while others function as the catalytic subunit of larger multi-subunit complexes. Elucidating the molecular mechanisms by which each of the subunits of chromatin remodeling complexes contribute to their activities requires the ability to perform biochemical assays that dissect the remodeling process.

ATP-dependent nucleosome remodeling by the human INO80 complex and other chromatin remodeling enzymes can be envisioned as a multi-step process that starts with binding of the remodeling enzyme to nucleosomes, followed by activation of its DNA- and/or nucleosome-dependent ATPase, translocation of the remodeling enzyme on nucleosomal DNA, and eventual repositioning of nucleosomes ^1,2. Understanding the molecular details of the ATP-dependent chromatin remodeling process requires dissection of the remodeling reaction into its individual steps and definition of the contributions of individual subunits of the chromatin remodeling complex to each step of the reaction. Such analyses require the ability to analyze nucleosome remodeling and other activities using defined molecular substrates in vitro.

In a previous JOVE protocol, we described procedures used to generate INO80 chromatin remodeling complexes and subcomplexes with defined subunit compositions ³. Here, we present three biochemical assays that enable quantitative analysis of the nucleosome binding, DNA- and nucleosome-activated ATPase, and nucleosome remodeling activities associated with such complexes.

Protokół

1. ATP-dependent Nucleosome Remodeling Assays

To measure ATP-dependent nucleosome remodeling activities, immunopurified INO80 or INO80 subcomplexes are incubated with ATP and a mononucleosomal substrate, which contains a single nucleosome positioned at one end of a 216-bp, ³²P-labeled DNA fragment. The reaction products are then subjected to electrophoresis in native poly-acrylamide gels.

1. To generate the ³²P-labeled, '601' DNA fragment, amplify from pGEM-3Z-601⁴ a 216 bp DNA fragment containing an end-positioned 601 nucleosome positioning sequence, using the oligonucleotides 5′-ACAGGATGTATATATCTGACACGTGCCTGG and 5′-AATACTCAAGCTTGGATGCCTGCAG as forward and reverse primers.
   1. Set up a 100 μl PCR reaction as described in Table 1.
   2. Perform the PCR reactions in a thermal cycler using the following program: 1 min at 96 °C, followed by 45 sec at 94 °C, 30 sec at 57 °C, 60 sec at 72 °C for 30 cycles; ending with 7 min at 72 °C.
   3. After the PCR reaction is finished, remove the unincorporated nucleotides by passing the reaction products twice through nuclease-free spin columns.
   4. Run 5 μl of the purified PCR product in a 1.2% agarose gel to confirm that the PCR reaction generated the desired ~216 bp DNA fragment.
   5. Dilute 5 μl of the purified PCR product 20-fold, and measure the DNA concentration using a UV spectrophotometer. The average yield is ~40 ng/μl.
   6. Measure the radioactivity of 1 μl of the purified PCR product in a scintillation counter. Estimate the labeling efficiency by calculating the cpm/ng. A successful labeling reaction is expected to yield ~15,000 cpm/ng of 601 DNA fragment.
2. Transfer Hela cell nucleosomes onto the labeled 601 DNA using a serial dilution method ⁵.
   1. Mix 2 pmol of ³²P-labeled 601 DNA fragment with 6 µg of Hela nucleosomes (prepared as described ⁵) in 50 µl of a buffer containing 1.0 M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.1 mM PMSF, and 1 mM DTT and incubate at 30 °C for 30 min.
   2. Sequentially dilute the mixture to 0.8 M, 0.6 M, and 0.4 M NaCl by dilution with 12.5 µl, 20.8 µl, and 41.6 µl, respectively, of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1 mM PMSF, and 1 mM DTT, with a 30 min incubation at 30 °C after each dilution.
   3. Further dilute the mixture to 0.2 M and then to 0.1 M NaCl by addition of 125 µl and then 250 µl of the same buffer containing 0.1% Nonidet P-40, 20% glycerol, and 200 µg/ml BSA, with a 30 min incubation at 30 °C between the final two dilutions. Store the mononucleosome substrate at 4 °C for up to 3 months.
3. Perform ATP-dependent nucleosome sliding reactions in a total volume of 10 µl. The reaction components are listed in Table 2. Since the optimal NaCl concentration for INO80 nucleosome remodeling activity is ~50 mM NaCl (unpublished results), adjust the total concentration of NaCl in each reaction to 50 mM, taking into account the amount of NaCl contained in preparations of nucleosomes and enzyme.
   1. Before beginning to set up the assays, cast native poly-acrylamide gels (18 x 16 cm). To prepare a single gel containing 5% acrylamide (acrylamide:bisacrylamide 37.5:1), 0.5x TBE (45 mM Tris borate, 1 mM EDTA), 0.01% ammonium persulfate (APS), and 0.001% N,N,N´,N´-tetramethylethylenediamine (TEMED), mix the ingredients listed in Table 3. Allow the gel to polymerize for at least 2 hr at RT.
   2. Meanwhile, for each reaction to be performed, combine in a pre-chilled siliconized 1.5 ml microcentrifuge tube ~20 nM INO80 or INO80 subcomplex (prepared as described ³) with an amount of EB100 buffer (Table 4) sufficient to give a volume of 4.75 μl. Immediately re-freeze any remaining INO80-containing fractions in powdered dry ice or liquid nitrogen.
   3. Set up a master cocktail with the rest of the ingredients, scaling up by a factor of 'X' (where X = total number of reactions +3). The amount of each ingredient needed for a single 10 μl reaction is listed in Table 5; the recipe should be scaled up according to the number of reactions to be performed. Mix well by tapping the tube or by pipetting up and down with a Pipetman and spin the tube for a few seconds in a benchtop microcentrifuge.
   4. Dispense 5.25 µl of the master cocktail to each of the reaction tubes set up in step 1.3.2. Mix well by pipetting up and down. Start the reactions by transferring reaction tubes to a 30 °C heat block or water bath and incubate for 2 hr.
   5. Meanwhile, prepare 'removing mix' cocktail containing competitor DNA and nucleosomes, scaling up by a factor of X (X = total number of reactions + 4). The amount of each ingredient needed to prepare 1.5 µl removing mix for a single reaction is listed in Table 6; the recipe should be scaled up depending on the number of assays to be performed.
   6. Terminate reactions by adding 1.5 μl of the removing mix. Mix well, spin down, and incubate at 30 °C for a further 30 min.
   7. Meanwhile, pre-run the native polyacrylamide gel in a vertical electrophoresis unit at 100 V for 30 min at 4 °C, using 0.5x TBE as running buffer with a magnetic stir bar inside the lower chamber to maintain constant buffer circulation.
   8. To load the sample, add 2.5 µl of loading dye containing 3x TBE, 30% glycerol, 0.25% Bromophenol Blue, and 0.25% Xylene Cyanol FF. Mix well, briefly spin the samples, and load onto the gel using loading tips.
   9. Run the gel at 200 V for 4.5 hr at 4 °C with buffer circulation.
   10. To detect the signal, transfer the gel to a stack of two sheets of filter paper. Wrap the filter paper with the gel on top using clear plastic wrap, and then expose it to a storage phosphor screen at 4 °C for the desired time.
   11. Scan the screen with an isotope imaging scanner system and analyze the data using suitable software.

2. Mononucleosome Binding Assays

To assay the binding affinity of a given INO80 complex for mononucleosomes, perform an Electrophoretic Mobility Shift Assay (EMSA) using the mononucleosomal substrate generated in Step 1.2.

1. Set up the reaction mixes for binding assays as described for nucleosome remodeling assays but omit the ATP and removing mix from the reactions; incubate at 30 °C for 30 min.
2. Add 2.5 µl of loading dye to each reaction mixture, and apply to a native polyacrylamide gel containing 3.5% acrylamide (acrylamide:bis 37.5:1), 1% Glycerol, 0.5x TBE, 0.01% APS, and 0.001% TEMED.
3. Using 0.5x TBE as running buffer, run the gel at 200 V for 2.5 hr at 4 °C with buffer circulation and expose to a storage phosphor screen.

3. DNA- and Nucleosome-dependent ATPase Assays

Perform ATPase assays in 5 μl reaction mixtures containing 20 mM Tris-HCl (pH 7.5), 60 mM NaCl, 6.6 mM MgCl₂, 0.8 mM EDTA, 0.015% Nonidet P-40, 2.5% glycerol, 0.1 mg/ml BSA, 1 mM DTT, 0.1 mM PMSF, 2 mM ATP, 2 μCi of [α-³²P] ATP (3,000 Ci/mmol). For each INO80 complex or amount of INO80 complex to be assayed set up three parallel reactions, one containing EB100 buffer to measure DNA- or nucleosome-independent ATPase, one containing closed circular plasmid DNA (5,000 bp, ~30 nM) to measure DNA-dependent ATPase, and one containing Hela oligonucleosomes (~185 nM) to measure nucleosome-dependent ATPase. Set up all reactions on ice.

1. For each reaction, combine 10-50 nM of the immunopurified INO80 or INO80 subcomplexes with an amount of EB100 buffer sufficient to give a volume of 2.2 μl in pre-chilled lubricated 1.5 ml microcentrifuge tubes. Immediately re-freeze any INO80-containing fractions in powdered dry ice or liquid nitrogen.
2. Set up a master cocktail. The amount of each ingredient needed for a single reaction is listed in Table 7. Scale up the recipe by scaling up by a factor of 3(X+2) +1, where X = the number of INO80 preparations to be assayed.
3. To prepare 'sub-cocktails' containing buffer only, DNA, or nucleosomes, dispense 2.5(X+2) μl of the master cocktail into three separate tubes. Add 0.3(X+2) μl of either EB100, closed circular plasmid DNA (1.5 µg/µl), or Hela oligonucleosomes (1.5 μg/μl) and mix well.
4. Dispense 2.8 μl of the appropriate sub-cocktail to the enzyme-containing reaction tubes set up in step 3.1. Gently pipette up and down to mix; avoid introducing bubbles.
5. To start reactions, transfer the reaction tubes to a 30 °C heat block.
6. After 5, 15, 30, and 60 min of incubation, spot 0.5 μl of each reaction mixture onto a cellulose polyethyleneimine thin layer chromatography (TLC) plate (20 x 10 cm) in a straight line at least 1.5 cm away from the bottom edge. Immediately return reaction tubes to the 30 °C heat block so multiple time points can be taken from a single tube. After spotting, dry the TLC plates using a blow dryer.
7. Transfer the TLC plates to a glass chamber containing enough 0.375 M potassium phosphate (pH 3.5) to allow the bottom 0.5 cm of the TLC plate to be submerged in the solution.
8. Cover the chamber, and develop until the front of the liquid phase reaches the top of the TLC plates. Immediate dry the plates thoroughly using a blow dryer.
9. Expose the dried TLC plates to a storage Phosphor screen at RT. Scan the screen with an isotope imaging scanner system and determine the amount of radioactive ATP substrate and ADP product.
10. To calculate the amount of ATP hydrolyzed, multiply the % ATP hydrolyzed by the amount of ATP present in the starting reaction mixture using the following formula: pmol ATP hydrolyzed = 10 pmol ATP in starting reaction x [ADP/(ATP+ADP)]

Wyniki

The figures show representative results of biochemical assays used to characterize INO80 activities, including nucleosome sliding (Figure 1) and binding (Figure 2) assays and DNA- or nucleosome-dependent ATPase assays (Figure 3).

The experiment shown in Figure 1 compares the ability of intact INO80 complexes purified through FLAG-Ies2 or FLAG-INO80E and of INO80 subcomplexes purified through either FLAG-Ino80ΔN or Ino80&#...

Dyskusje

To ensure that nucleosome remodeling and ATPase activities we observe in assays depend on the catalytic activity of INO80 complexes, and not on contaminating remodeling and/or ATPase enzymes, we routinely assay nucleosome remodeling and ATPase activity of catalytically inactive versions of INO80 complexes, purified in parallel with wild type INO80 using the same procedure. A negative control reaction lacking ATP should also be performed when assaying nucleosome remodeling activity to test for the presence of contaminatin...

Materiały

Name	Company	Catalog Number	Comments
Protease Inhibitor Cocktail	Sigma	P8340
10x PCR reaction buffer	Roche Applied Science	11435094001
Roche Taq DNA Polymerase	Roche Applied Science	11435094001
NucAway Nuclease-free Spin Columns	Ambion	Cat. # AM10070
ultrapure ATP	USB/Affymetrix	77241 25 UM
bovine serum albumin	Sigma	A9418
40% Acrylamide/Bis 37.5:1	Amresco	0254-500ML
Sonicated salmon sperm DNAs	GE Healthcare	27-4565-01
10% ammonium persulfate (APS)	Thermo Scientific	17874
benzonase	Novagen	Cat. No. 70664
dCTP, [α-32P]- 6,000 Ci/mmol	PerkinElmer	BLU013Z250UC
PCR thermal cycler PTC 200	MJ Research	PTC 200
Hoefer vertical electrophoresis unit	Hoefer	SE600X-15-1.5
lubricated 1.5 ml microcentrifuge tubes	Costar	3207
Storage Phosphor Screen	Molecular Dynamics	63-0034-79
3MM filter paper	Whatman	28458-005	VWR
Typhoon PhosphorImager	GE Healthcare	8600
ImageQuant software	GE Healthcare	ver2003.02
TLC Glass Plates, PEI-Cellulose F	Millipore	5725-7
Immobilon-FL Transfer Membrane 7 x 8.4	Millipore	IPFL07810
General purpose survey meter with end-window or pancake GM (Geiger-Mueller) probe	Biodex	Model 14C