Chapter 7 Histone Deacetylase Activity Assay Lirong Peng, Zhigang Yuan, and Edward Seto Abstract Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl groups from the ε-amino groups of conserved lysine residues in the amino terminal tail of histones. Accumulating evidence suggests that many, if not all, HDACs can also deacetylate nonhistone proteins. Through deacetylating histones and nonhistone proteins, HDACs regulate a variety of cellular processes including gene transcription, cell differentiation, DNA damage responses, and apoptosis. Aberrant HDACs are implicated in many human diseases and, therefore, it is important to have a consistent and reliable assay for analyzing HDAC activities. The focus of this chapter is to provide up-to-date, easy-to-follow, approaches and techniques, for the assay of HDAC enzymatic activities. Key words HDAC assay, HDAC2, Sirtuin, SIRT1, Protein deacetylation, Core histone, BocLys(Ac)-AMC, Radioactive assay, Fluorometric assay
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Introduction Reversible lysine acetylation is an important posttranslational regulatory mechanism in cells. The lysine acetylation status of proteins is regulated dynamically through the opposing action of histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDAC enzymes catalyze the removal of acetyl groups from lysine residues in histone and nonhistone proteins. Deacetylation may lead to a change in the conformation and/or activity of the substrates. Through deacetylation, dynamic modification of lysine residues near the N-termini of histones plays an important role in the regulation of chromatin structure and gene expression. In general, hypoacetylation of histones is linked to transcriptional repression. Besides histones, HDACs also regulate biological processes, including DNA repair, cell cycle control, cell differentiation, and apoptosis, through deacetylating nonhistone proteins [1–3]. Eighteen mammalian HDACs have been identified so far. Based on protein size, sequence similarity, and organization of the protein domains, they are divided into three classes [1, 2].
Srikumar P. Chellappan (ed.), Chromatin Protocols, Methods in Molecular Biology, vol. 1288, DOI 10.1007/978-1-4939-2474-5_7, © Springer Science+Business Media New York 2015
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Class I HDACs, which include HDAC1, HDAC2, HDAC3, and HDAC8, are highly related to the yeast RPD3 protein. Class II HDACs contain HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10, and are homologues of the yeast HDA1 protein. The Class II enzymes have been subdivided further into two subclasses, IIa (HDAC4, 5, 7, and 9) and IIb (HDAC6 and 10). HDAC11 uniquely shares sequence homology within the catalytic domains of both Class I and II HDACs. Class III HDACs, also called sirtuins (SIR2-like proteins), include SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7. Sirtuins are structurally and evolutionarily unrelated to and mechanistically distinct from the Class I and II HDACs. They couple deacetylation of substrates to cleavage of NAD+ to form nicotinamide and O-acetyl-ADP-ribose. Therefore, the Class III HDAC/sirtuin activity assay requires the addition of NAD+ as a cofactor. With increased interest in HDAC enzymes over the last decade, it is very important to have readily available, reliable, and efficient approaches to analyze their activities. Currently, several different kinds of HDAC activity assays have been established, including radioactive and nonradioactive methods [4–7]. The classic radiometric HDAC activity assay directly detects the release of free radioactive isotope-labeled acetate from the deacetylation reaction using a scintillation counter. In contrast, the colorimetric, fluorogenic, and bioluminogenic methods are based on a two-step enzymatic reaction. The HDAC substrate (an acetylated lysine side chain or peptide) is conjugated with either a colored compound (i.e., paranitroanilide), a fluorescent group (i.e., 7-amino-4-methylcoumarin [AMC]), or an aminoluciferin. After deacetylation by an HDAC, the substrate becomes sensitive to trypsin. Once the deacetylated substrate is cleaved by trypsin, a free chromophore, fluorophore, or aminoluciferin is released. In the colorimetic assay, the colored product absorbs a certain wavelength that can be recorded by a spectrometer. In the fluorescent assay, the production of fluorophore can be measured using a fluorescence reader. In the bioluminescent assay, the free aminoluciferin is processed by luciferase to generate a photophore that can be recorded by a luminometer. The signal of photophore and fluorophore is directly proportional to the deacetylation activity of the sample. For analysis of sirtuin activity, another way is to employ nicotinamidase to measure the product of nicotinamide from the sirtuin-mediated deacetylation reaction (see the SIRTainty™ Class III HDAC Assay kit of Millipore). In this chapter, we present practical step-by-step protocols for scientists outside of the HDAC field who are interested in incorporating HDAC studies into their research. We describe both the classic radioactive method and the widely used fluorometric method, using representative members of classical and sirtuin families: HDAC2 and SIRT1.
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Materials For the radioactive HDAC activity assay, researchers need to prepare isotope-labeled substrates by themselves. For the nonradioactive assay, dozens of colorimetric and fluorometric HDAC/Sirtuin activity assay kits are available from multiple companies (e.g., Millipore, Active Motif, Enzo Life Sciences, and Sigma). Substrates, reagents, and even enzymes can be purchased with a kit or separately. Researchers can choose the products and the commercial source according to the choice of substrate and HDACs. However, all of the reagents and materials, except substrates, can be easily prepared in a laboratory following the protocols described here.
2.1 Reagents for Purification of Enzymatically Active HDACs
2.1.1 To Purify HDAC2 from HEK293T Cells
The biological activity of HDACs is tightly modulated by the binding of cofactors and posttranslational modifications, particularly phosphorylation [8]. Although all Class I and II HDACs can be expressed easily as recombinant proteins in bacteria, we have found that, with the exception of HDAC8, HDAC proteins produced in E. coli display very little enzymatic activities. Unlike bacterially expressed HDACs, HDAC proteins isolated from mammalian or insect cells retain their native conformation, modification, and interaction with cofactors. Therefore, enzymatically active HDACs that are useful for most activity assays can be obtained from immunoaffinity-purified proteins expressed in mammalian cells or insect cells [9]. Researchers can decide the source of HDACs according to their own need and limitations. We present protocols for the expression and purification of enzymatically active HDAC2 from mammalian and insect cells, and enzymatically active SIRT1 from bacteria. Identical approaches can be applied to other HDAC members. Whole HDAC activity from cells could also be examined using nuclear extracts according to the protocol described here. 1. Mammalian expression plasmid pME18S-Flag-HDAC2 that expresses a Flag-tagged HDAC2. 2. HEK293T cells for the transfection recipient. 3. Lipofectamine 2000 (Invitrogen, cat # 11668019). 4. Phosphate-buffered saline (PBS): 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 (pH 7.4). 5. Cell lysis buffer: 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.5 % NP-40, 10 % glycerol. 6. EDTA-free protease inhibitor cocktail (Roche, cat # 11617900). 7. Anti-Flag M2 agarose (Sigma, cat # A2220). 8. 1× HDAC assay buffer: 10 mM Tris–HCl (pH 8.0), 150 mM NaCl, 10 % glycerol. 9. Flag peptide to elute Flag-HDAC2 from agarose (Sigma, cat # F3290).
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2.1.2 To Purify HDAC2 from Sf9 Insect Cells
1. Baculovirus expression plasmid pFastBac-HTa-HDAC2 that expresses a 6× His-tagged HDAC2. Recombinant baculovirus encoding HDAC2 generated according to the manufacturer’s protocol. 2. Sf 9 cells. 3. Cell lysis buffer: 50 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1 mM MgCl2, 5 mM β-mercaptoethanol, 10 % glycerol, and 0.5 % Triton X-100. 4. Ni-NTA (nitrilo-triacetic acid) resin (Qiagen). 5. Buffer A: 20 mM Tris–HCl (pH 8.5), 1 M KCl, 20 mM imidazole, 10 % glycerol, 5 mM β-mercaptoethanol. 6. Buffer B: 20 mM Tris–HCl (pH 8.5), 1 M KCl, 10 % glycerol, 5 mM β-mercaptoethanol. 7. Buffer C: 20 mM Tris–HCl (pH 8.5), 100 mM KCl, 200 mM imidazole, 10 % glycerol, 5 mM β-mercaptoethanol.
2.1.3 To Purify SIRT1 from E. coli
1. Bacterial expression plasmid pGEX-5X1-SIRT1 that generates a GST-tagged SIRT1 recombinant protein. 2. Competent BL21/DE3 bacteria. 3. 0.1 M IPTG. 4. STE buffer: 10 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA. 5. 10 % N-lauryl sarcosine. 6. 1 M DTT. 7. Sonicator (Sonic Dismembrator 60, Fisher Scientific). 8. 10 % Triton X-100. 9. Glutathione-agarose beads (Sigma, cat # G4510). 10. Elution buffer: 10 mM glutathione in 50 mM Tris–HCl (pH 8.0), 1 mM DTT (freshly made).
2.1.4 HeLa Nuclear Extract
HeLa nuclear extracts (2.5 mg/ml, 20 mM HEPES [pH7.5], 350 mM NaCl, 20 % glycerol, 1 mM MgCl2, 0.1 mM EGTA, 5 mM DTT, 1 % NP-40, proteasome inhibitor) will be used as a positive control of enzymatic active HDACs. The extract can be purchased from Millipore (cat # 12-309) or other companies or it can be prepared as previously described [10].
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Acetylated core histones or synthetic acetylated histone H4 peptides were originally used as substrates for HDACs [4, 11]. However, besides histone tails, an increasing number of nonhistone proteins are found to be the natural substrates for HDACs. Different substrates, p53 peptides for example, have been developed to test the deacetylase activity of HDACs [7, 12, 13]. Non-peptide-acetylated
Substrates
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lysine moieties, i.e., Boc-Lys(Ac) (Nα-[t-butoxycarbonyl]-Nωacetyl lysine amide), are widely used in commercial HDAC activity assay kits because they work for most HDACs. HDACs differ in substrate affinity towards different substrates. Based on Enzo Life Sciences literatures, Class I, IIb, and III HDACs are generally most sensitive to acetylated p53 (379-382) peptides, next to acetylated p53 (317-320) peptides, followed by acetylated lysine 16 of histone H4, and the least to Lys-Ac. Class IIa HDACs are the exception. Class IIa HDACs (HDAC4, 5, 7, and 9) have undetectable activity with peptide substrates in vitro, but are active with acetylated lysine moieties, i.e., trifluoroacetyl-lysine [14]. Therefore, when analyzing the activities of Class IIa HDACs, Boc-Lys(Tfa)-AMC ([S]-tertbutyl1-[4-methyl-2-oxo-2H-chromen-7-ylamino]-1-oxo-6-[2,2,2trifluoroacetamido]hexan-2–ylcarbamate) can be chosen as the substrate. Sirtuins are more sensitive to acetylated p53 peptides than to acetyl-lysine side chains (Lys-Ac) and H4-AcK16 peptides. Individually, HDAC8 has a higher activity with the dually acetylated p53 peptides (379-382: RHK[Ac]K[Ac]) and the H4-AcK16 peptides than with singly acetylated p53 peptides (379-382: RHKK[Ac] and 317-320: QPKK[Ac]). HDAC10 and HDAC11 have higher preference for p53 (379-382) peptides than p53 (317-320) peptides. These substrates can be radioactively, colorimetrically, or fluorescently labeled dependent on the choice of assays. 2.2.1 Radioactive Substrates
1. HeLa cells.
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3. 2 M sodium butyrate (NaB).
2. 10 mg/ml cyclohexamide (CHX). 4. 1.0 mCi/ml [3H] acetic acid (Perkin Elmer, cat # 3499232). 5. IB buffer: 10 mM Tris–HCl (pH 7.4), 2 mM MgCl2, 3 mM CaCl2, 10 mM sodium butyrate, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Prepare immediately prior to use. 6. NIB buffer: 1 % Nonidet P-40 in IB buffer. Prepare immediately prior to use. 7. Storage buffer: 10 mM HEPES (pH 7.5), 1 mM EDTA, 10 mM KCl, 0.2 mM PMSF, and 10 % glycerol. 8. 5 M NaCl. 9. SnakeSkin® Pleated Dialysis Tubing (Pierce, cat #68053).
2.2.2 Nonradioactive Substrates
Although radioactive HDAC assays are very sensitive, they have many shortcomings including labor consumption, batch-to-batch variation of labeling, exposure to radioactivity, and the need for radioactive waste management [15, 16]. As alternatives to radiolabeled histones or peptides, nonradioactive labeled substrates have been developed. HDAC activity can be readily detected by measuring the optical density or fluorescence using a spectrometer.
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In this protocol, the fluorophore-conjugated Boc-Lys[Ac]-AMC (N-[4-Methyl-7-coumarinyl]-Nα-[t-butoxycarbonyl]-Nω-acetyl lysine amide) is used as the substrate. We have used the product from Enzo Life Biosciences (cat # ALX-260-137-M001) in this protocol. However, it is also available from other companies including Biovision (cat # K330-100) and BACHEM (cat # I-1875.0250). 2.2.3 Materials Needed for Enzymatic Reaction and Detection
1. 5× HDAC buffer: 50 mM Tris–HCl (pH 8.0), 750 mM NaCl, 50 % glycerol.
Radioactive Assay
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2. Stop solution: 1 M HCl and 0.4 M acetic acid. 4. Plastic scintillation vials (Pony vial, 6 ml, Perkin Elmer, cat # 6000592). 5. Gas scintillation counter (Matrix 96 beta counter, Canberra Packard).
Nonradioactive Assay
1. 10× HDAC assay buffer: 250 mM Tris–HCl (pH 8.0), 1.37 M NaCl, 27 mM KCl, 10 mM MgCl2, 10 mg/ml BSA. 2. Developer buffer: 10 mg/ml trypsin (Sigma, cat # T4799) in 50 mM Tris–HCl (pH8.0), 10 mM NaCl, 2 μM Trichostatin A, or 5 mM nicotinamide (see Note 1). 3. 50 mM β-nicotinamide adenine dinucleotide hydrate (NAD+) (Sigma, cat # N1636, only added for Class III sirtuin assays). 4. Nunc™ 96 well polystyrene plates, black (Sigma, cat # P8741). 5. A plate reader capable of the excitation of 350–380 nm and the detection of the emission of 450–480 nm (Envision 2103 multilabel reader, Perkin Elmer).
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Methods
3.1 Express and Purify Enzymatic Active HDACs 3.1.1 Purification of HDAC2 from HEK293T Cells
1. Transfect one 10-cm plate of HEK293T cells with 10 μg of pME18S-Flag-HDAC2 plasmid using Lipofectamine 2000 according to the manufacturer’s manual. In a separate transfection, use pME18S vector as a negative control. 2. At 36 h post-transfection, wash cells twice with 1× PBS. Harvest cells in 1 ml fresh 1× PBS in a 1.5-ml tube. After centrifugation at 1,500 × g for 5 min, lyse cell pellet in five packed-cell-pellet volumes of cell lysis buffer in the presence of an EDTA-free protease inhibitor cocktail for 30 min at 4 °C. (It is preferable not to include EDTA in the preparation of cell lysate.) Remove cell debris by centrifugation at 10,000 × g for 15 min at 4 °C, and transfer the supernatant to a prechilled 1.5-ml tube (see Note 2).
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3. Subject the supernatant to immunoprecipitation reactions with anti-Flag M2 agarose. Add 30 μl anti-Flag M2 agarose (prewashed with cell lysis buffer) per ml of cell lysate, and agitate for 2 h at 4 °C. 4. Two hours later, pellet immune complexes by centrifugation at 10,000 × g for 2 min at 4 °C. Carefully remove supernatant, and wash immune complexes four times using cell lysis buffer. Subsequently, wash the immune complexes three times in 1× HDAC assay buffer. Beads containing the immunoprecipitated HDAC2 are now ready to be used in the enzymatic assay. 5. (Optional) Flag-HDAC2 can be eluted with 50 μl of 1 mg/ml of Flag peptide in 25 mM Tris–HCl (pH 7.1), 120 mM NaCl, and 10 % glycerol. Dialyze protein against 25 mM Tris–HCl (pH 7.1), 120 mM NaCl, and 10 % glycerol, and distribute into 1.5-ml centrifuge tubes. Store materials at −80 °C. The HDAC sample should be refrozen immediately at −20 or −70 °C after each use to reduce loss of activity. 3.1.2 Purification of HDAC2 from Sf9 Insect Cells
1. To produce a high-titer stock baculovirus encoding HDAC2, infect Sf9 cells with recombinant virus at a multiplicity of infection (MOI) of 0.01–0.1. Once 15–20 % cell survival is achieved, harvest the virus-containing cell supernatant by centrifugation at 2,000 × g for 3 min at room temperature. 2. Infect Sf9 cells with viral particles (virus/cell ratio of 10:1), and harvest cells at 48 h post-infection by centrifugation at 500 × g for 2 min at room temperature. 3. Lyse cells in ice-cold cell lysis buffer containing 1× EDTA-free protease inhibitor. Centrifuge at 10,000 × g for 10 min. 4. Incubate the supernatant with Ni-NTA resin on a rotator for 1 h at 4 °C. 5. Wash resin successively with ten volumes of buffer A and two volumes of buffer B. 6. Elute protein with buffer C. 7. Determine protein concentration by the Bradford method using the Bio-Rad dye reagent with BSA as a standard. Analyze purified HDAC2 protein by SDS-PAGE and Coomassie blue staining.
3.1.3 Purification of SIRT1 from E. coli
1. Transform BL21/DE3 bacteria with pGEX-5X1-SIRT1. Grow a 6-ml culture in the presence of antibiotics overnight. 2. Dilute 100-fold of the overnight bacteria culture into 250 ml LB broth with antibiotics, and incubate at 37 °C until the OD600 reaches 0.6 (about 2 h 15 min). 3. Add IPTG to the culture to a final concentration of 0.5 mM, and incubate bacteria for 4–8 h.
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4. Pellet cells by centrifugation at 6,500 × g for 10 min at 4 °C. 5. Resuspend pellet in 5 ml of ice-cold STE buffer. Addition of 100 μg/ml lysozyme and 1× protease inhibitor to the STE buffer is optional. 6. Add DTT to a final concentration of 5 mM. 7. Add N-lauryl sarcosine to a final concentration of 1.5 % from a 10 % stock. 8. Mix cells thoroughly and sonicate at 4 °C until the lysate becomes clear. Typically, sonicating the bacteria five times for 15 s each at the output setting of 4 is sufficient. 9. Add Triton X-100 to a final concentration of 3 % from a 10 % stock. 10. Centrifuge insoluble fractions at 10,000 × g at 4 °C for 10 min. 11. Aliquot supernatants into microfuge tubes (100–200 μl each), snap freeze in liquid nitrogen (or dry ice with ethanol), and store at −80 °C (optional). 12. Pre-swell 30 μl glutathione-agarose bead slurry, and mix with the supernatant. Rotate to mix at 4 °C for 2 h or overnight. 13. Wash beads with PBS five times to remove unbound and nonspecifically bound proteins. 14. Elute fusion protein by adding 100 μl elution buffer. Repeat at least twice. 15. Analyze individual fractions by SDS–PAGE and Coomassie blue staining; pool fractions containing the highest amounts of purified recombinant protein, aliquot, and store at −80 °C (see Fig. 1).
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Fig. 1 A representative experiment to show purification of an enzymatically active GST-SIRT1. Coomassie blue staining showing the purified GST-SIRT1
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1. Prepare one confluent 15-cm plate of HeLa cells. 2. Wash cells once with 1× PBS. 3. Incubate cells with 20 ml PBS containing 200 μl of 10 mg/ml CHX for 10 min at 37 °C. 4. Aspirate PBS and incubate cells with 20 ml PBS containing 200 μl of 10 mg/ml CHX, 100 μl NaB, and 400 μl of 1.0 mCi/ ml [3H] acetic acid for 1 h at 37 °C. 5. Collect cells and chill on ice for 10 min. 6. Centrifuge at 1,500 × g for 5 min at 4 °C to pellet cells. The following steps are done at 4 °C or on ice. 7. Wash cells with 5 ml PBS containing 25 μl of 2 M NaB three times. 8. Wash cells twice with 1 ml NIB buffer (see Note 3). 9. Wash cells once with 1 ml NIB buffer containing 100 mM NaCl. 10. Wash cells once with 1 ml IB buffer containing 100 mM NaCl. 11. Wash cells twice with 500 μl of IB buffer containing 400 mM NaCl. 12. From steps 7–11, pellet cells by centrifugation at 500 × g for 5 min at 4 °C. 13. Extract the nuclei pellet with 0.25 ml of 0.2 N H2SO4 for 90 min on ice. 14. Centrifuge at 30,000 × g for 90 min at 4 °C. 15. Dialyze supernatant against 500 ml of storage buffer overnight at 4 °C. Aliquot purified core histones and store at −80 °C (see Notes 3–5).
3.3 HDAC Activity Assay 3.3.1 Radioactive HDAC Activity Assay
Here we describe a detailed protocol for small-scale labeling and isolation of core histones from HeLa cells with [3H] acetic acid, which is performed routinely in our laboratory and is adapted from Carmen et al. [4]. In this protocol, we have used radiolabeled acetylated core histones as the substrates. However, histones can be replaced with radiolabeled acetylated H4 or p53 peptide substrates in this assay. 1. For each reaction, the following reagents are mixed in a 1.5-ml tube in a final volume of 200 μl, containing 40 μl of 5× HDAC buffer, [3H]-labeled core histones (about 10,000 cpm), 0.5 μg of HDAC2 or 1 μg of GST-SIRT1 (add 1 mM NAD+ cofactor if the analyzed enzyme is sirtuin), and ddH2O (see Note 6). For the negative control, HDAC reaction can be heated to 95 °C for 5 min to destroy any enzymatic activity or skip the addition of the enzyme into the reaction. 2. Incubate reactions for 2 h at 30 °C. 3. Stop reaction by the addition of 50 μl of stop solution.
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Fig. 2 A representative experiment to determine HDAC activity using radiolabeled core histones. Cells were transfected with either Flag-HDAC2 or empty vector for 48 h, lysed, and immunoprecipitated with anti-Flag M2 agarose beads. Immunoprecipitated samples then were subjected to HDAC assays as described using approximately 8,000 cpm core histone as substrate. The expression of Flag-HDAC2 was monitored by Western blotting using anti-Flag antibodies
4. Extract released [3H]-acetate by adding 0.4 ml ethyl acetate with mixing. Centrifuge at 10,000 × g for 3 min at room temperature to separate phases. 5. Transfer 200 μl of the upper (organic) phase to a scintillation vial with 3 ml scintillation liquid and measure CPM in a scintillation counter (see Note 7). 6. HDAC activity = (counts from test sample [cpm] − counts from control [cpm]) / volume of sample (μl) (see Fig. 2). 3.3.2 Fluorometric HDAC Activity Assay
This fluorometric protocol is described using the GST-SIRT1 enzyme and the Boc-Lys(Ac)-AMC substrate. However, BocLys(Ac)-AMC can be replaced with p53-AMC (Enzo Life Sciences, BML-KI177-0005) in this assay. This protocol can also be modified to evaluate the activity of Class I/II HDACs with the omission of NAD+ (see Notes 8 and 9). 1. Dissolve the substrate of Boc-Lys(Ac)-AMC in DMSO to give a 50 mM solution. 2. Add 0.4 μl of substrates and 6 μl of 50 mM NAD+ (NAD+ could be omitted if the HDAC of interest belongs to Class I/II) in 1× HDAC buffer within a 96-well black plate. 3. Add 1 μg of GST-SIRT1 and 1× HDAC buffer to give a total volume of 100 μl per well with final concentration of 200 μM Boc-Lys(Ac)-AMC and 3 mM NAD+ (see Notes 10 and 11), and mix well. For the background control, add 1× HDAC
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Fig. 3 A representative fluorometric experiment using non-radiolabeled substrates to measure GST-SIRT1 activity
buffer into the substrates. For the negative control, add GST instead of GST-SIRT1 into the reaction. In addition, GSTSIRT1 is added into a well together with 5 mM nicotinamide to set another negative control (see Fig. 3). For the positive control, add 5 μg of HeLa extract per well. 4. Incubate the plate for 1 h at 32 °C. 5. Add 100 μl of developer buffer and mix well (see Note 1). Incubate the plate for 20 min at 37 °C. 6. Read plate within 60 min. Measure the release of fluorescence in a fluorimeter plate reader with the excitation wavelength of 350–380 nm and the emission wavelength of 440–480 nm. 7. The fluorescent signals are recorded against background wells that contain substrates but without the enzyme. All fluorescence readings are expressed in relative fluorescence units (RFU) (see Note 12, 13, and Fig. 3). Overall, there are different methods to examine the activity of HDACs, especially for sirtuins. Researchers can choose the assay according to their needs. Radioactive methods are sensitive but labor consuming. Nonradioactive methods are particularly more suitable for large-scale drug screenings. Alternatively, if a researcher wants to detect the deacetylation activity of an HDAC against a specific target, i.e., the deacetylation effect of SIRT2 on tubulin, Western blotting would be a viable choice. The acetylated targets can be detected by Western blotting with target-specific acetylation antibody or pan-acetylated lysine antibodies. Pan-acetylated lysine
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antibodies are available from multiple companies, including Millipore and Cell Signaling, and make up one of the most convenient reagents to study acetylation/deacetylation. It is wise to test several different pan-acetylated lysine antibodies against the protein of interest since each antibody targets different acetylated lysines with different efficiencies. Since the basal acetylation level of many proteins may be too low to be detectable by standard procedures, a common strategy to induce acetylation is by treating cells with 10 mM nicotinamide, 100 ng/ml Trichostatin A, or both, before cell harvest.
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Notes 1. For the sirtuin assay, 5 mM nicotinamide will be added into the developer buffer to inhibit the enzymatic reaction. For the Class I/II HDAC assay, 2 μM Trichostatin A should be added instead. 2. To maximize the release of HDACs from nuclei, cells can be sonicated three times for 15 s each at the output setting of 4 (Sonic Dismembrator 60, Fisher Scientific). 3. Since PMSF is readily degraded in water-based solutions, it is important that IB buffer, NIB buffer, and storage buffer are freshly made. Alternatively, complete EDTA-free protease inhibitor cocktail tablets (Roche, cat # 11836170001) can be used in place of PMSF. 5. To ensure that the isolated core histones are labeled correctly, an aliquot of core histones can be measured for specific activity using a scintillation counter. Using this protocol, the counts for 10 μl of core histones are approximately 10,000 cpm. 6. For many HDACs, core histones or histone H4 peptides work well as a substrate. However, some HDACs may require mononucleosome instead of free histones as a substrate. The preparation of mononucleosome substrates has been described elsewhere [3, 10, 14]. 7. The upper phase should be very carefully transferred to the scintillation vial using a pipet tip to avoid background count carryover from interphase contamination. 8. GST-SIRT1 used in the assay can be replaced with an immunoprecipitated SIRT1 protein from transfected mammalian cells as described for HDAC2. 9. To maintain the enzymatic activity, aliquot the purified HDACs or HeLa nuclear extract, snap freeze in liquid nitrogen, and store at −80 °C. Thaw reagents just before use. Aliquot unused material into prechilled microcentrifuge tubes and snap freeze in liquid nitrogen prior to storage at −80 °C.
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10. It is recommended to run every sample and control at least in duplicate. 11. Although the described fluorometric assay is performed within a regular 96-well plate, it could also be modified to be performed on a ½-volume 96-well plate by decreasing half of the reaction volume and half of the developer buffer. 12. For the fluorometric assay, black plate is often used. However, white plates may be chosen to a get higher signal. If the signal is too strong, clear plates could be used instead, and the reading would decrease about fivefold. 13. Developer buffer and NAD+ may exhibit some autofluorescence. Background reading of the substrates without enzymes is usually high. Therefore, it is necessary to subtract the reading of the HDAC samples to that of no-enzyme control. Different readers and 96-well plates have different sensitivities in the detection of fluorophores. Researchers can optimize the instrument setting and the choice of plates by using non-acetylated Boc-Lys-AMC standards (Enzo Life Sciences). In brief, incubate different dilutions of a non-acetylated substrate standard (i.e., 0–50 μM) with an equal volume of developer buffer and read the fluorescence as described in the protocol. Adjust the parameters of the reader according to the range of the standards. References 1. Yang XJ, Seto E (2008) The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men. Nat Rev Mol Cell Biol 9(3):206–218 2. Glozak MA, Sengupta N, Zhang X, Seto E (2005) Acetylation and deacetylation of nonhistone proteins. Gene 363:15–23 3. Peng L, Seto E (2011) Deacetylation of nonhistone proteins by HDACs and the implications in cancer. Handb Exp Pharmacol 206:39–56 4. Carmen AA, Rundlett SE, Grunstein M (1996) HDA1 and HDA3 are components of a yeast histone deacetylase (HDA) complex. J Biol Chem 271(26):15837–15844 5. Heltweg B, Jung M (2003) A homogeneous nonisotopic histone deacetylase activity assay. J Biomol Screen 8:89–95 6. Hoffmann K et al (2000) First non-radioactive assay for in vitro screening of histone deacetylase inhibitors. Pharmazie 55:601–606 7. Wegener D et al (2003) A fluorogenic histone deacetylase assay well suited for high-throughput activity screening. Chem Biol 10:61–68 8. Heltweg B, Trapp J, Jung M (2005) In vitro assays for the determination of histone deacetylase activity. Methods 36(4):332–337
9. Rezai-Zadeh N, Tsai SC, Wen YD, Yao YL, Yang WM, Seto E (2004) Histone deacetylases: purification of the enzymes, substrates, and assay conditions. Methods Enzymol 377: 167–179 10. Dignam JD, Lebovitz RM, Roeder RG (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11: 1475–1489 11. Sun JM, Spencer VA, Chen HY, Li L, Davie JR (2003) Measurement of histone acetyltransferase and histone deacetylase activities and kinetics of histone acetylation. Methods 31(1):12–23 12. Borra MT, Smith BC, Denu JM (2005) Mechanism of human SIRT1 activation by resveratrol. J Biol Chem 280(17):17187–17195 13. Halley F, Reinshagen J, Ellinger B, Wolf M, Niles AL, Evans NJ, Kirkland TA, Wagner JM, Jung M, Gribbon P, Gul S (2011) A bioluminogenic HDAC activity assay: validation and screening. J Biomol Screen 16(10):1227–1235 14. Lahm A, Paolini C, Pallaoro M, Nardi MC, Jones P, Neddermann P, Sambucini S, Bottomley MJ, Lo Surdo P, Carfí A, Koch U, De Francesco R, Steinkühler C, Gallinari P (2007) Unraveling
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the hidden catalytic activity of vertebrate class IIa histone deacetylases. Proc Natl Acad Sci U S A 104(44):17335–17340 15. Utley RT, Owen-Hughes TA, Juan LJ, Cote J, Adams CC, Workman JL (1996) In vitro analysis of transcription factor binding to nucleosomes
and nucleosome disruption/displacement. Methods Enzymol 274:276–291 16. Kolle D, Brosch G, Lechner T, Lusser A, Loidl P (1998) Biochemical methods for analysis of histone deacetylases. Methods 15(4): 323–331
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Introduction Reversible lysine acetylation is an important posttranslational regulatory mechanism in cells. The lysine acetylation status of proteins is regulated dynamically through the opposing action of histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDAC enzymes catalyze the removal of acetyl groups from lysine residues in histone and nonhistone proteins. Deacetylation may lead to a change in the conformation and/or activity of the substrates. Through deacetylation, dynamic modification of lysine residues near the N-termini of histones plays an important role in the regulation of chromatin structure and gene expression. In general, hypoacetylation of histones is linked to transcriptional repression. Besides histones, HDACs also regulate biological processes, including DNA repair, cell cycle control, cell differentiation, and apoptosis, through deacetylating nonhistone proteins [1–3]. Eighteen mammalian HDACs have been identified so far. Based on protein size, sequence similarity, and organization of the protein domains, they are divided into three classes [1, 2].
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Class I HDACs, which include HDAC1, HDAC2, HDAC3, and HDAC8, are highly related to the yeast RPD3 protein. Class II HDACs contain HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10, and are homologues of the yeast HDA1 protein. The Class II enzymes have been subdivided further into two subclasses, IIa (HDAC4, 5, 7, and 9) and IIb (HDAC6 and 10). HDAC11 uniquely shares sequence homology within the catalytic domains of both Class I and II HDACs. Class III HDACs, also called sirtuins (SIR2-like proteins), include SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7. Sirtuins are structurally and evolutionarily unrelated to and mechanistically distinct from the Class I and II HDACs. They couple deacetylation of substrates to cleavage of NAD+ to form nicotinamide and O-acetyl-ADP-ribose. Therefore, the Class III HDAC/sirtuin activity assay requires the addition of NAD+ as a cofactor. With increased interest in HDAC enzymes over the last decade, it is very important to have readily available, reliable, and efficient approaches to analyze their activities. Currently, several different kinds of HDAC activity assays have been established, including radioactive and nonradioactive methods [4–7]. The classic radiometric HDAC activity assay directly detects the release of free radioactive isotope-labeled acetate from the deacetylation reaction using a scintillation counter. In contrast, the colorimetric, fluorogenic, and bioluminogenic methods are based on a two-step enzymatic reaction. The HDAC substrate (an acetylated lysine side chain or peptide) is conjugated with either a colored compound (i.e., paranitroanilide), a fluorescent group (i.e., 7-amino-4-methylcoumarin [AMC]), or an aminoluciferin. After deacetylation by an HDAC, the substrate becomes sensitive to trypsin. Once the deacetylated substrate is cleaved by trypsin, a free chromophore, fluorophore, or aminoluciferin is released. In the colorimetic assay, the colored product absorbs a certain wavelength that can be recorded by a spectrometer. In the fluorescent assay, the production of fluorophore can be measured using a fluorescence reader. In the bioluminescent assay, the free aminoluciferin is processed by luciferase to generate a photophore that can be recorded by a luminometer. The signal of photophore and fluorophore is directly proportional to the deacetylation activity of the sample. For analysis of sirtuin activity, another way is to employ nicotinamidase to measure the product of nicotinamide from the sirtuin-mediated deacetylation reaction (see the SIRTainty™ Class III HDAC Assay kit of Millipore). In this chapter, we present practical step-by-step protocols for scientists outside of the HDAC field who are interested in incorporating HDAC studies into their research. We describe both the classic radioactive method and the widely used fluorometric method, using representative members of classical and sirtuin families: HDAC2 and SIRT1.
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Materials For the radioactive HDAC activity assay, researchers need to prepare isotope-labeled substrates by themselves. For the nonradioactive assay, dozens of colorimetric and fluorometric HDAC/Sirtuin activity assay kits are available from multiple companies (e.g., Millipore, Active Motif, Enzo Life Sciences, and Sigma). Substrates, reagents, and even enzymes can be purchased with a kit or separately. Researchers can choose the products and the commercial source according to the choice of substrate and HDACs. However, all of the reagents and materials, except substrates, can be easily prepared in a laboratory following the protocols described here.
2.1 Reagents for Purification of Enzymatically Active HDACs
2.1.1 To Purify HDAC2 from HEK293T Cells
The biological activity of HDACs is tightly modulated by the binding of cofactors and posttranslational modifications, particularly phosphorylation [8]. Although all Class I and II HDACs can be expressed easily as recombinant proteins in bacteria, we have found that, with the exception of HDAC8, HDAC proteins produced in E. coli display very little enzymatic activities. Unlike bacterially expressed HDACs, HDAC proteins isolated from mammalian or insect cells retain their native conformation, modification, and interaction with cofactors. Therefore, enzymatically active HDACs that are useful for most activity assays can be obtained from immunoaffinity-purified proteins expressed in mammalian cells or insect cells [9]. Researchers can decide the source of HDACs according to their own need and limitations. We present protocols for the expression and purification of enzymatically active HDAC2 from mammalian and insect cells, and enzymatically active SIRT1 from bacteria. Identical approaches can be applied to other HDAC members. Whole HDAC activity from cells could also be examined using nuclear extracts according to the protocol described here. 1. Mammalian expression plasmid pME18S-Flag-HDAC2 that expresses a Flag-tagged HDAC2. 2. HEK293T cells for the transfection recipient. 3. Lipofectamine 2000 (Invitrogen, cat # 11668019). 4. Phosphate-buffered saline (PBS): 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 (pH 7.4). 5. Cell lysis buffer: 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.5 % NP-40, 10 % glycerol. 6. EDTA-free protease inhibitor cocktail (Roche, cat # 11617900). 7. Anti-Flag M2 agarose (Sigma, cat # A2220). 8. 1× HDAC assay buffer: 10 mM Tris–HCl (pH 8.0), 150 mM NaCl, 10 % glycerol. 9. Flag peptide to elute Flag-HDAC2 from agarose (Sigma, cat # F3290).
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2.1.2 To Purify HDAC2 from Sf9 Insect Cells
1. Baculovirus expression plasmid pFastBac-HTa-HDAC2 that expresses a 6× His-tagged HDAC2. Recombinant baculovirus encoding HDAC2 generated according to the manufacturer’s protocol. 2. Sf 9 cells. 3. Cell lysis buffer: 50 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1 mM MgCl2, 5 mM β-mercaptoethanol, 10 % glycerol, and 0.5 % Triton X-100. 4. Ni-NTA (nitrilo-triacetic acid) resin (Qiagen). 5. Buffer A: 20 mM Tris–HCl (pH 8.5), 1 M KCl, 20 mM imidazole, 10 % glycerol, 5 mM β-mercaptoethanol. 6. Buffer B: 20 mM Tris–HCl (pH 8.5), 1 M KCl, 10 % glycerol, 5 mM β-mercaptoethanol. 7. Buffer C: 20 mM Tris–HCl (pH 8.5), 100 mM KCl, 200 mM imidazole, 10 % glycerol, 5 mM β-mercaptoethanol.
2.1.3 To Purify SIRT1 from E. coli
1. Bacterial expression plasmid pGEX-5X1-SIRT1 that generates a GST-tagged SIRT1 recombinant protein. 2. Competent BL21/DE3 bacteria. 3. 0.1 M IPTG. 4. STE buffer: 10 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA. 5. 10 % N-lauryl sarcosine. 6. 1 M DTT. 7. Sonicator (Sonic Dismembrator 60, Fisher Scientific). 8. 10 % Triton X-100. 9. Glutathione-agarose beads (Sigma, cat # G4510). 10. Elution buffer: 10 mM glutathione in 50 mM Tris–HCl (pH 8.0), 1 mM DTT (freshly made).
2.1.4 HeLa Nuclear Extract
HeLa nuclear extracts (2.5 mg/ml, 20 mM HEPES [pH7.5], 350 mM NaCl, 20 % glycerol, 1 mM MgCl2, 0.1 mM EGTA, 5 mM DTT, 1 % NP-40, proteasome inhibitor) will be used as a positive control of enzymatic active HDACs. The extract can be purchased from Millipore (cat # 12-309) or other companies or it can be prepared as previously described [10].
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Acetylated core histones or synthetic acetylated histone H4 peptides were originally used as substrates for HDACs [4, 11]. However, besides histone tails, an increasing number of nonhistone proteins are found to be the natural substrates for HDACs. Different substrates, p53 peptides for example, have been developed to test the deacetylase activity of HDACs [7, 12, 13]. Non-peptide-acetylated
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lysine moieties, i.e., Boc-Lys(Ac) (Nα-[t-butoxycarbonyl]-Nωacetyl lysine amide), are widely used in commercial HDAC activity assay kits because they work for most HDACs. HDACs differ in substrate affinity towards different substrates. Based on Enzo Life Sciences literatures, Class I, IIb, and III HDACs are generally most sensitive to acetylated p53 (379-382) peptides, next to acetylated p53 (317-320) peptides, followed by acetylated lysine 16 of histone H4, and the least to Lys-Ac. Class IIa HDACs are the exception. Class IIa HDACs (HDAC4, 5, 7, and 9) have undetectable activity with peptide substrates in vitro, but are active with acetylated lysine moieties, i.e., trifluoroacetyl-lysine [14]. Therefore, when analyzing the activities of Class IIa HDACs, Boc-Lys(Tfa)-AMC ([S]-tertbutyl1-[4-methyl-2-oxo-2H-chromen-7-ylamino]-1-oxo-6-[2,2,2trifluoroacetamido]hexan-2–ylcarbamate) can be chosen as the substrate. Sirtuins are more sensitive to acetylated p53 peptides than to acetyl-lysine side chains (Lys-Ac) and H4-AcK16 peptides. Individually, HDAC8 has a higher activity with the dually acetylated p53 peptides (379-382: RHK[Ac]K[Ac]) and the H4-AcK16 peptides than with singly acetylated p53 peptides (379-382: RHKK[Ac] and 317-320: QPKK[Ac]). HDAC10 and HDAC11 have higher preference for p53 (379-382) peptides than p53 (317-320) peptides. These substrates can be radioactively, colorimetrically, or fluorescently labeled dependent on the choice of assays. 2.2.1 Radioactive Substrates
1. HeLa cells.
Radioactive Acetylated Core Histones
3. 2 M sodium butyrate (NaB).
2. 10 mg/ml cyclohexamide (CHX). 4. 1.0 mCi/ml [3H] acetic acid (Perkin Elmer, cat # 3499232). 5. IB buffer: 10 mM Tris–HCl (pH 7.4), 2 mM MgCl2, 3 mM CaCl2, 10 mM sodium butyrate, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Prepare immediately prior to use. 6. NIB buffer: 1 % Nonidet P-40 in IB buffer. Prepare immediately prior to use. 7. Storage buffer: 10 mM HEPES (pH 7.5), 1 mM EDTA, 10 mM KCl, 0.2 mM PMSF, and 10 % glycerol. 8. 5 M NaCl. 9. SnakeSkin® Pleated Dialysis Tubing (Pierce, cat #68053).
2.2.2 Nonradioactive Substrates
Although radioactive HDAC assays are very sensitive, they have many shortcomings including labor consumption, batch-to-batch variation of labeling, exposure to radioactivity, and the need for radioactive waste management [15, 16]. As alternatives to radiolabeled histones or peptides, nonradioactive labeled substrates have been developed. HDAC activity can be readily detected by measuring the optical density or fluorescence using a spectrometer.
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In this protocol, the fluorophore-conjugated Boc-Lys[Ac]-AMC (N-[4-Methyl-7-coumarinyl]-Nα-[t-butoxycarbonyl]-Nω-acetyl lysine amide) is used as the substrate. We have used the product from Enzo Life Biosciences (cat # ALX-260-137-M001) in this protocol. However, it is also available from other companies including Biovision (cat # K330-100) and BACHEM (cat # I-1875.0250). 2.2.3 Materials Needed for Enzymatic Reaction and Detection
1. 5× HDAC buffer: 50 mM Tris–HCl (pH 8.0), 750 mM NaCl, 50 % glycerol.
Radioactive Assay
3. Ethyl acetate.
2. Stop solution: 1 M HCl and 0.4 M acetic acid. 4. Plastic scintillation vials (Pony vial, 6 ml, Perkin Elmer, cat # 6000592). 5. Gas scintillation counter (Matrix 96 beta counter, Canberra Packard).
Nonradioactive Assay
1. 10× HDAC assay buffer: 250 mM Tris–HCl (pH 8.0), 1.37 M NaCl, 27 mM KCl, 10 mM MgCl2, 10 mg/ml BSA. 2. Developer buffer: 10 mg/ml trypsin (Sigma, cat # T4799) in 50 mM Tris–HCl (pH8.0), 10 mM NaCl, 2 μM Trichostatin A, or 5 mM nicotinamide (see Note 1). 3. 50 mM β-nicotinamide adenine dinucleotide hydrate (NAD+) (Sigma, cat # N1636, only added for Class III sirtuin assays). 4. Nunc™ 96 well polystyrene plates, black (Sigma, cat # P8741). 5. A plate reader capable of the excitation of 350–380 nm and the detection of the emission of 450–480 nm (Envision 2103 multilabel reader, Perkin Elmer).
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Methods
3.1 Express and Purify Enzymatic Active HDACs 3.1.1 Purification of HDAC2 from HEK293T Cells
1. Transfect one 10-cm plate of HEK293T cells with 10 μg of pME18S-Flag-HDAC2 plasmid using Lipofectamine 2000 according to the manufacturer’s manual. In a separate transfection, use pME18S vector as a negative control. 2. At 36 h post-transfection, wash cells twice with 1× PBS. Harvest cells in 1 ml fresh 1× PBS in a 1.5-ml tube. After centrifugation at 1,500 × g for 5 min, lyse cell pellet in five packed-cell-pellet volumes of cell lysis buffer in the presence of an EDTA-free protease inhibitor cocktail for 30 min at 4 °C. (It is preferable not to include EDTA in the preparation of cell lysate.) Remove cell debris by centrifugation at 10,000 × g for 15 min at 4 °C, and transfer the supernatant to a prechilled 1.5-ml tube (see Note 2).
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3. Subject the supernatant to immunoprecipitation reactions with anti-Flag M2 agarose. Add 30 μl anti-Flag M2 agarose (prewashed with cell lysis buffer) per ml of cell lysate, and agitate for 2 h at 4 °C. 4. Two hours later, pellet immune complexes by centrifugation at 10,000 × g for 2 min at 4 °C. Carefully remove supernatant, and wash immune complexes four times using cell lysis buffer. Subsequently, wash the immune complexes three times in 1× HDAC assay buffer. Beads containing the immunoprecipitated HDAC2 are now ready to be used in the enzymatic assay. 5. (Optional) Flag-HDAC2 can be eluted with 50 μl of 1 mg/ml of Flag peptide in 25 mM Tris–HCl (pH 7.1), 120 mM NaCl, and 10 % glycerol. Dialyze protein against 25 mM Tris–HCl (pH 7.1), 120 mM NaCl, and 10 % glycerol, and distribute into 1.5-ml centrifuge tubes. Store materials at −80 °C. The HDAC sample should be refrozen immediately at −20 or −70 °C after each use to reduce loss of activity. 3.1.2 Purification of HDAC2 from Sf9 Insect Cells
1. To produce a high-titer stock baculovirus encoding HDAC2, infect Sf9 cells with recombinant virus at a multiplicity of infection (MOI) of 0.01–0.1. Once 15–20 % cell survival is achieved, harvest the virus-containing cell supernatant by centrifugation at 2,000 × g for 3 min at room temperature. 2. Infect Sf9 cells with viral particles (virus/cell ratio of 10:1), and harvest cells at 48 h post-infection by centrifugation at 500 × g for 2 min at room temperature. 3. Lyse cells in ice-cold cell lysis buffer containing 1× EDTA-free protease inhibitor. Centrifuge at 10,000 × g for 10 min. 4. Incubate the supernatant with Ni-NTA resin on a rotator for 1 h at 4 °C. 5. Wash resin successively with ten volumes of buffer A and two volumes of buffer B. 6. Elute protein with buffer C. 7. Determine protein concentration by the Bradford method using the Bio-Rad dye reagent with BSA as a standard. Analyze purified HDAC2 protein by SDS-PAGE and Coomassie blue staining.
3.1.3 Purification of SIRT1 from E. coli
1. Transform BL21/DE3 bacteria with pGEX-5X1-SIRT1. Grow a 6-ml culture in the presence of antibiotics overnight. 2. Dilute 100-fold of the overnight bacteria culture into 250 ml LB broth with antibiotics, and incubate at 37 °C until the OD600 reaches 0.6 (about 2 h 15 min). 3. Add IPTG to the culture to a final concentration of 0.5 mM, and incubate bacteria for 4–8 h.
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4. Pellet cells by centrifugation at 6,500 × g for 10 min at 4 °C. 5. Resuspend pellet in 5 ml of ice-cold STE buffer. Addition of 100 μg/ml lysozyme and 1× protease inhibitor to the STE buffer is optional. 6. Add DTT to a final concentration of 5 mM. 7. Add N-lauryl sarcosine to a final concentration of 1.5 % from a 10 % stock. 8. Mix cells thoroughly and sonicate at 4 °C until the lysate becomes clear. Typically, sonicating the bacteria five times for 15 s each at the output setting of 4 is sufficient. 9. Add Triton X-100 to a final concentration of 3 % from a 10 % stock. 10. Centrifuge insoluble fractions at 10,000 × g at 4 °C for 10 min. 11. Aliquot supernatants into microfuge tubes (100–200 μl each), snap freeze in liquid nitrogen (or dry ice with ethanol), and store at −80 °C (optional). 12. Pre-swell 30 μl glutathione-agarose bead slurry, and mix with the supernatant. Rotate to mix at 4 °C for 2 h or overnight. 13. Wash beads with PBS five times to remove unbound and nonspecifically bound proteins. 14. Elute fusion protein by adding 100 μl elution buffer. Repeat at least twice. 15. Analyze individual fractions by SDS–PAGE and Coomassie blue staining; pool fractions containing the highest amounts of purified recombinant protein, aliquot, and store at −80 °C (see Fig. 1).
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Fig. 1 A representative experiment to show purification of an enzymatically active GST-SIRT1. Coomassie blue staining showing the purified GST-SIRT1
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1. Prepare one confluent 15-cm plate of HeLa cells. 2. Wash cells once with 1× PBS. 3. Incubate cells with 20 ml PBS containing 200 μl of 10 mg/ml CHX for 10 min at 37 °C. 4. Aspirate PBS and incubate cells with 20 ml PBS containing 200 μl of 10 mg/ml CHX, 100 μl NaB, and 400 μl of 1.0 mCi/ ml [3H] acetic acid for 1 h at 37 °C. 5. Collect cells and chill on ice for 10 min. 6. Centrifuge at 1,500 × g for 5 min at 4 °C to pellet cells. The following steps are done at 4 °C or on ice. 7. Wash cells with 5 ml PBS containing 25 μl of 2 M NaB three times. 8. Wash cells twice with 1 ml NIB buffer (see Note 3). 9. Wash cells once with 1 ml NIB buffer containing 100 mM NaCl. 10. Wash cells once with 1 ml IB buffer containing 100 mM NaCl. 11. Wash cells twice with 500 μl of IB buffer containing 400 mM NaCl. 12. From steps 7–11, pellet cells by centrifugation at 500 × g for 5 min at 4 °C. 13. Extract the nuclei pellet with 0.25 ml of 0.2 N H2SO4 for 90 min on ice. 14. Centrifuge at 30,000 × g for 90 min at 4 °C. 15. Dialyze supernatant against 500 ml of storage buffer overnight at 4 °C. Aliquot purified core histones and store at −80 °C (see Notes 3–5).
3.3 HDAC Activity Assay 3.3.1 Radioactive HDAC Activity Assay
Here we describe a detailed protocol for small-scale labeling and isolation of core histones from HeLa cells with [3H] acetic acid, which is performed routinely in our laboratory and is adapted from Carmen et al. [4]. In this protocol, we have used radiolabeled acetylated core histones as the substrates. However, histones can be replaced with radiolabeled acetylated H4 or p53 peptide substrates in this assay. 1. For each reaction, the following reagents are mixed in a 1.5-ml tube in a final volume of 200 μl, containing 40 μl of 5× HDAC buffer, [3H]-labeled core histones (about 10,000 cpm), 0.5 μg of HDAC2 or 1 μg of GST-SIRT1 (add 1 mM NAD+ cofactor if the analyzed enzyme is sirtuin), and ddH2O (see Note 6). For the negative control, HDAC reaction can be heated to 95 °C for 5 min to destroy any enzymatic activity or skip the addition of the enzyme into the reaction. 2. Incubate reactions for 2 h at 30 °C. 3. Stop reaction by the addition of 50 μl of stop solution.
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Fig. 2 A representative experiment to determine HDAC activity using radiolabeled core histones. Cells were transfected with either Flag-HDAC2 or empty vector for 48 h, lysed, and immunoprecipitated with anti-Flag M2 agarose beads. Immunoprecipitated samples then were subjected to HDAC assays as described using approximately 8,000 cpm core histone as substrate. The expression of Flag-HDAC2 was monitored by Western blotting using anti-Flag antibodies
4. Extract released [3H]-acetate by adding 0.4 ml ethyl acetate with mixing. Centrifuge at 10,000 × g for 3 min at room temperature to separate phases. 5. Transfer 200 μl of the upper (organic) phase to a scintillation vial with 3 ml scintillation liquid and measure CPM in a scintillation counter (see Note 7). 6. HDAC activity = (counts from test sample [cpm] − counts from control [cpm]) / volume of sample (μl) (see Fig. 2). 3.3.2 Fluorometric HDAC Activity Assay
This fluorometric protocol is described using the GST-SIRT1 enzyme and the Boc-Lys(Ac)-AMC substrate. However, BocLys(Ac)-AMC can be replaced with p53-AMC (Enzo Life Sciences, BML-KI177-0005) in this assay. This protocol can also be modified to evaluate the activity of Class I/II HDACs with the omission of NAD+ (see Notes 8 and 9). 1. Dissolve the substrate of Boc-Lys(Ac)-AMC in DMSO to give a 50 mM solution. 2. Add 0.4 μl of substrates and 6 μl of 50 mM NAD+ (NAD+ could be omitted if the HDAC of interest belongs to Class I/II) in 1× HDAC buffer within a 96-well black plate. 3. Add 1 μg of GST-SIRT1 and 1× HDAC buffer to give a total volume of 100 μl per well with final concentration of 200 μM Boc-Lys(Ac)-AMC and 3 mM NAD+ (see Notes 10 and 11), and mix well. For the background control, add 1× HDAC
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Fig. 3 A representative fluorometric experiment using non-radiolabeled substrates to measure GST-SIRT1 activity
buffer into the substrates. For the negative control, add GST instead of GST-SIRT1 into the reaction. In addition, GSTSIRT1 is added into a well together with 5 mM nicotinamide to set another negative control (see Fig. 3). For the positive control, add 5 μg of HeLa extract per well. 4. Incubate the plate for 1 h at 32 °C. 5. Add 100 μl of developer buffer and mix well (see Note 1). Incubate the plate for 20 min at 37 °C. 6. Read plate within 60 min. Measure the release of fluorescence in a fluorimeter plate reader with the excitation wavelength of 350–380 nm and the emission wavelength of 440–480 nm. 7. The fluorescent signals are recorded against background wells that contain substrates but without the enzyme. All fluorescence readings are expressed in relative fluorescence units (RFU) (see Note 12, 13, and Fig. 3). Overall, there are different methods to examine the activity of HDACs, especially for sirtuins. Researchers can choose the assay according to their needs. Radioactive methods are sensitive but labor consuming. Nonradioactive methods are particularly more suitable for large-scale drug screenings. Alternatively, if a researcher wants to detect the deacetylation activity of an HDAC against a specific target, i.e., the deacetylation effect of SIRT2 on tubulin, Western blotting would be a viable choice. The acetylated targets can be detected by Western blotting with target-specific acetylation antibody or pan-acetylated lysine antibodies. Pan-acetylated lysine
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antibodies are available from multiple companies, including Millipore and Cell Signaling, and make up one of the most convenient reagents to study acetylation/deacetylation. It is wise to test several different pan-acetylated lysine antibodies against the protein of interest since each antibody targets different acetylated lysines with different efficiencies. Since the basal acetylation level of many proteins may be too low to be detectable by standard procedures, a common strategy to induce acetylation is by treating cells with 10 mM nicotinamide, 100 ng/ml Trichostatin A, or both, before cell harvest.
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Notes 1. For the sirtuin assay, 5 mM nicotinamide will be added into the developer buffer to inhibit the enzymatic reaction. For the Class I/II HDAC assay, 2 μM Trichostatin A should be added instead. 2. To maximize the release of HDACs from nuclei, cells can be sonicated three times for 15 s each at the output setting of 4 (Sonic Dismembrator 60, Fisher Scientific). 3. Since PMSF is readily degraded in water-based solutions, it is important that IB buffer, NIB buffer, and storage buffer are freshly made. Alternatively, complete EDTA-free protease inhibitor cocktail tablets (Roche, cat # 11836170001) can be used in place of PMSF. 5. To ensure that the isolated core histones are labeled correctly, an aliquot of core histones can be measured for specific activity using a scintillation counter. Using this protocol, the counts for 10 μl of core histones are approximately 10,000 cpm. 6. For many HDACs, core histones or histone H4 peptides work well as a substrate. However, some HDACs may require mononucleosome instead of free histones as a substrate. The preparation of mononucleosome substrates has been described elsewhere [3, 10, 14]. 7. The upper phase should be very carefully transferred to the scintillation vial using a pipet tip to avoid background count carryover from interphase contamination. 8. GST-SIRT1 used in the assay can be replaced with an immunoprecipitated SIRT1 protein from transfected mammalian cells as described for HDAC2. 9. To maintain the enzymatic activity, aliquot the purified HDACs or HeLa nuclear extract, snap freeze in liquid nitrogen, and store at −80 °C. Thaw reagents just before use. Aliquot unused material into prechilled microcentrifuge tubes and snap freeze in liquid nitrogen prior to storage at −80 °C.
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10. It is recommended to run every sample and control at least in duplicate. 11. Although the described fluorometric assay is performed within a regular 96-well plate, it could also be modified to be performed on a ½-volume 96-well plate by decreasing half of the reaction volume and half of the developer buffer. 12. For the fluorometric assay, black plate is often used. However, white plates may be chosen to a get higher signal. If the signal is too strong, clear plates could be used instead, and the reading would decrease about fivefold. 13. Developer buffer and NAD+ may exhibit some autofluorescence. Background reading of the substrates without enzymes is usually high. Therefore, it is necessary to subtract the reading of the HDAC samples to that of no-enzyme control. Different readers and 96-well plates have different sensitivities in the detection of fluorophores. Researchers can optimize the instrument setting and the choice of plates by using non-acetylated Boc-Lys-AMC standards (Enzo Life Sciences). In brief, incubate different dilutions of a non-acetylated substrate standard (i.e., 0–50 μM) with an equal volume of developer buffer and read the fluorescence as described in the protocol. Adjust the parameters of the reader according to the range of the standards. References 1. Yang XJ, Seto E (2008) The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men. Nat Rev Mol Cell Biol 9(3):206–218 2. Glozak MA, Sengupta N, Zhang X, Seto E (2005) Acetylation and deacetylation of nonhistone proteins. Gene 363:15–23 3. Peng L, Seto E (2011) Deacetylation of nonhistone proteins by HDACs and the implications in cancer. Handb Exp Pharmacol 206:39–56 4. Carmen AA, Rundlett SE, Grunstein M (1996) HDA1 and HDA3 are components of a yeast histone deacetylase (HDA) complex. J Biol Chem 271(26):15837–15844 5. Heltweg B, Jung M (2003) A homogeneous nonisotopic histone deacetylase activity assay. J Biomol Screen 8:89–95 6. Hoffmann K et al (2000) First non-radioactive assay for in vitro screening of histone deacetylase inhibitors. Pharmazie 55:601–606 7. Wegener D et al (2003) A fluorogenic histone deacetylase assay well suited for high-throughput activity screening. Chem Biol 10:61–68 8. Heltweg B, Trapp J, Jung M (2005) In vitro assays for the determination of histone deacetylase activity. Methods 36(4):332–337
9. Rezai-Zadeh N, Tsai SC, Wen YD, Yao YL, Yang WM, Seto E (2004) Histone deacetylases: purification of the enzymes, substrates, and assay conditions. Methods Enzymol 377: 167–179 10. Dignam JD, Lebovitz RM, Roeder RG (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11: 1475–1489 11. Sun JM, Spencer VA, Chen HY, Li L, Davie JR (2003) Measurement of histone acetyltransferase and histone deacetylase activities and kinetics of histone acetylation. Methods 31(1):12–23 12. Borra MT, Smith BC, Denu JM (2005) Mechanism of human SIRT1 activation by resveratrol. J Biol Chem 280(17):17187–17195 13. Halley F, Reinshagen J, Ellinger B, Wolf M, Niles AL, Evans NJ, Kirkland TA, Wagner JM, Jung M, Gribbon P, Gul S (2011) A bioluminogenic HDAC activity assay: validation and screening. J Biomol Screen 16(10):1227–1235 14. Lahm A, Paolini C, Pallaoro M, Nardi MC, Jones P, Neddermann P, Sambucini S, Bottomley MJ, Lo Surdo P, Carfí A, Koch U, De Francesco R, Steinkühler C, Gallinari P (2007) Unraveling
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the hidden catalytic activity of vertebrate class IIa histone deacetylases. Proc Natl Acad Sci U S A 104(44):17335–17340 15. Utley RT, Owen-Hughes TA, Juan LJ, Cote J, Adams CC, Workman JL (1996) In vitro analysis of transcription factor binding to nucleosomes
and nucleosome disruption/displacement. Methods Enzymol 274:276–291 16. Kolle D, Brosch G, Lechner T, Lusser A, Loidl P (1998) Biochemical methods for analysis of histone deacetylases. Methods 15(4): 323–331
