- Email: [email protected]
Purification and enzymatic assay of class I histone deacetylase enzymes
CHAPTER TWO
Purification and enzymatic assay of class I histone deacetylase enzymes Mark K. Adamsa, Charles A.S. Banksa, Sayem Miaha, Maxime Killera,†, Michael P. Washburna,b,* a
Stowers Institute for Medical Research, Kansas City, MO, United States Department of Pathology & Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, United States *Corresponding author: e-mail address: [email protected] b
Contents 1. Introduction 2. Histone deacetylases and HDAC inhibitors 3. Purifying protein for HDAC assay 3.1 Choosing an expression system 3.2 Considerations to minimize assay artifacts 4. HDAC activity assay 4.1 Progression toward a high-throughput fluorescence-based HDAC activity assay 4.2 Assay considerations 5. Protocol 5.1 Equipment 5.2 Reagents 5.3 Buffer preparation 5.4 Cell culture and transfection for recombinant protein production 5.5 Recombinant protein isolation 5.6 HDAC activity assay 6. Analysis 7. Summary Acknowledgments References
†
24 25 26 26 28 29 29 30 32 32 32 33 34 35 36 36 37 37 37
Current address: Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Hamburg, Germany.
Methods in Enzymology, Volume 626 ISSN 0076-6879 https://doi.org/10.1016/bs.mie.2019.07.014
#
2019 Elsevier Inc. All rights reserved.
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Mark K. Adams et al.
Abstract The reversible acetylation of histones has a profound influence on transcriptional status. Histone acetyltransferases catalyze the addition of these chemical modifications to histone lysine residues. Conversely, histone deacetylases (HDACs) catalyze the removal of these acetyl groups from histone lysine residues. As modulators of transcription, HDACs have found themselves as targets of several FDA-approved chemotherapeutic compounds which aim to inhibit enzyme activity. The ongoing efforts to develop targeted and isoform-specific HDAC inhibitors necessitates tools to study these modifications and the enzymes that maintain an equilibrium of these modifications. In this chapter, we present an optimized workflow for the isolation of recombinant protein and subsequent assay of class I HDAC activity. We demonstrate the application of this assay by assessing the activities of recombinant HDAC1, HDAC2, and SIN3B. This assay system utilizes readily available reagents and can be used to assess the activity and responsiveness of class I HDAC complexes to HDAC inhibitors.
1. Introduction The high frequency at which cancer-associated mutations within chromatin modifying enzymes are observed (Morgan & Shilatifard, 2015) suggests that chromatin modifications are central to the regulation of gene expression patterns and the development of cancer. Not surprisingly, such enzymes are the focus of many ongoing studies which aim to characterize their roles in pathologies. Further, the enzymes that modify chromatin as well as their regulatory mechanisms have found themselves as targets of chemotherapeutic compounds. Chromatin modifications exist in several forms, with the acetylation of histone lysine residues being among the best characterized (Allis & Jenuwein, 2016). Traditionally associated with transcriptional activation, the addition of acetyl groups to histone lysine residues by histone acetyltransferases (HATs) provides an epigenetic mark which is recognized by several protein domains (Marmorstein & Zhou, 2014; Musselman & Kutateladze, 2011). Histone acetylation is a reversible chemical modification and histone deacetylases (HDACs) are responsible for the removal of this chemical modification. The reversible nature of histone acetylation as well as its profound influence on transcriptional status situates this post-translational modification as an ideal target for chemotherapeutic modulators of transcription. There are currently four FDA-approved HDAC inhibitors (HDACi) that have demonstrated efficacy as parts of multi-component chemotherapeutic treatment strategies (Table 1) (Suraweera, O’Byrne, & Richard, 2018). As HDACs and their potentials as
Purification and enzymatic assay of class I HDACs
25
Table 1 FDA-approved HDAC inhibitors. Inhibitor Alternative names References
Vorinostat
Richon et al. (1998), Khan et al. (2008), and Zolinza Bantscheff et al. (2011) Suberoylanilide Hydroxamic Acid (SAHA)
Romidepsin Istodax Chromadax FK228
Furumai et al. (2002) and Bantscheff et al. (2011)
Belinostat
Beleodaq PXD101
Khan et al. (2008) and Bantscheff et al. (2011)
Panobinostat Farydak LBH589
Khan et al. (2008) and Bantscheff et al. (2011)
Inhibitor influence on Class I HDAC activity is described in the provided references.
targets of chemotherapeutics are the subjects of ongoing study, it is essential that convenient assays are available with which the functional attributes of these enzymes and their responses to chemotherapeutic agents can be examined. Here, we describe an optimized protocol for the expression and in vitro analysis of class I HDAC enzymes. This assay allows for the screening of enzyme activity as well as enzyme responsiveness to HDAC inhibitors. To demonstrate the workflow, we have focused on the expression, purification, and assay of the Sin3 HDAC complex component SIN3B as well as the catalytic subunits of the Sin3 complex, HDAC1 and HDAC2. SIN3B serves as a scaffolding component of Sin3 complexes, protein complexes that are conserved from yeast to mammals. As Sin3 complexes have been previously shown to be responsive to some but not all HDAC inhibitors (Becher et al., 2014), HDAC complexes containing SIN3B are ideal models for the demonstration of complex responsiveness to HDAC inhibitors.
2. Histone deacetylases and HDAC inhibitors Histone deacetylases are represented by 18 separate enzymes organized into 4 distinct classes (Classes I, II, III, and IV). Classes I, II, and IV are metal-dependent enzymes that have a Zn2+ ion within the catalytic pocket (Lombardi, Cole, Dowling, & Christianson, 2011; Seto & Yoshida, 2014). While not all details regarding a catalytic mechanism have been described for these Zn2+-dependent enzymes, it is accepted that the removal of lysine acetyl groups is coordinated by histidine and/or tyrosine residues and the
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Mark K. Adams et al.
metal ion present within the active site pocket (Lombardi et al., 2011; Seto & Yoshida, 2014). Class III HDACs are represented by sirtuins and utilize a Zn2+-independent catalytic mechanism (Sauve, 2010). In regard to histone acetylation status, class I HDACs (HDAC1, HDAC2, HDAC3, HDAC8) are of interest as they have been shown to localize within the nucleus (Emiliani, Fischle, Van Lint, Al-Abed, & Verdin, 1998; Hu et al., 2000; Taplick et al., 2001; Van den Wyngaert et al., 2000; Wilting et al., 2010). Class I HDACs also share homology with the yeast enzyme Rpd3 (Yang & Seto, 2008), an enzyme that has a demonstrated role in the removal of histone lysine acetyl groups (Kadosh & Struhl, 1998; Rundlett et al., 1996). Thus, these enzymes likely play important and conserved roles in the modulation of histone acetylation status. While HDAC1 and HDAC2 possess intrinsic enzymatic activities (Hassig et al., 1998), they typically exist as the catalytic components of several large protein complexes, including Sin3, NuRD, and CoREST complexes. Additionally, it is likely that HDAC1/2 are components of other protein complexes that are poorly characterized (Bantscheff et al., 2011). HDAC3 is found within NCoR/SMRT complexes (Guenther et al., 2000) while HDAC8 is not a known component of any defined protein complexes. The dependence of class I HDACs, as well as classes II and IV, on Zn2+ ions is exploited by chemotherapeutic HDAC inhibitors (Seto & Yoshida, 2014; Wu, Lu, Cao, & Zhang, 2011). Early HDAC inhibitors produced broad spectrum effects, like those associated with trichostatin A (TCA). To minimize off-target effects associated with HDACi application, recently developed inhibitors only influence the activities of specific enzymes or enzymes if they exist in specific HDAC complexes (Bradner et al., 2010; Lauffer et al., 2013). As we progress toward targeted HDAC inhibitors, readily available and flexible HDAC activity assay systems will be needed to assess the efficacy of these compounds.
3. Purifying protein for HDAC assay 3.1 Choosing an expression system Prior to the analysis of HDAC activity, one must first decide whether endogenous or recombinant protein will be examined. Endogenous HDACs and HDAC complexes can be easily isolated from human cells (Becher et al., 2014). Additionally, recombinant protein production systems, such as baculovirus-mediated expression in insect cells (Hassig et al., 1998) and mammalian expression vector systems (Banks et al., 2018), have been used
27
Purification and enzymatic assay of class I HDACs
to produce enzymatically active HDACs. The analysis of endogenous and recombinant protein both have benefits and shortcomings which must be considered. To analyze the activity of endogenous protein, HDAC complexes are first enriched from cell extracts and immobilized using immunoprecipitation assays and antibodies which recognize HDAC complex components (Becher et al., 2014) (Fig. 1). A benefit of this approach is that transfection reagents and epitope tags are not required. This approach can also be used to isolate proteins of interest from cells which are difficult to transfect. However, this approach is often hampered by a lack of properly validated antibodies. Therefore, antibodies must be tested for suitability in immunoprecipitation assays and the specificity of epitope recognition by the antibody must be examined. The elution of protein complexes from affinity purification matrices in active conformations can also be challenging due to harsh elution conditions often required during the immunoprecipitation of proteins. However, this issue can be avoided as proteins do not 1
Recombinant protein CMV HDAC Halo transient transfection
fluorescent substrate
Halo capture and elution PROTEIN PRODUCTION
multi-well plate
excitation: 355 nm emission: 460 nm
PROTEIN ISOLATION
IP
2
develop with trypsin
DEACETYLATE SUBSTRATE
plate reader
Endogenous protein Protein preparation
HDAC activity assay
Fig. 1 Enrichment and assay of HDAC enzymes. HDAC enzymes can be acquired via (1) expression of recombinant protein or (2) isolation of endogenous protein. (1) For the production and isolation of recombinant protein, an open reading frame encoding the protein of interest along with an affinity tag is transiently expressed in mammalian cells using commercially available transfection reagents. Recombinant protein is enriched using affinity tag capture and eluted from the affinity purification matrix. (2) To isolate endogenous HDACs, antibodies that recognize the protein of interest are immobilized on Dynabeads™ Protein A or Dynabeads™ Protein G (Life Technologies). Cell lysates are incubated with the immobilized antibody and washed to remove contaminating proteins. For the assay of endogenous protein, elution of purified protein is unnecessary and HDAC activity can be performed while protein is immobilized on the affinity purification matrix. Using a multi-well plate platform, isolated recombinant or endogenous proteins are incubated at 37 °C with a fluorescent substrate, Boc-Lys(Ac)-AMC, in the presence or absence of HDAC inhibitors. Reactions are developed with trypsin and fluorescence is examined using a fluorescent plate reader with an excitation wavelength of 355 nm and an emission wavelength of 460 nm.
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need to be eluted from the matrix for activity assays. Instead, activity assays can be performed while proteins are immobilized on the affinity purification matrix (Fig. 1). As antibodies suitable for immunoprecipitation assays are not available for all components of HDAC complexes, the assay of recombinant protein is often the only feasible approach to isolate and examine the properties of HDACs. This offers several advantages over the study of endogenous protein, including the capacity to produce high levels of protein, the ability to introduce affinity tags, and high-level purity of isolated proteins. During protein purification steps, glycine- and urea-based elution methods should be avoided to prevent disruption of protein-protein interactions and protein folding. As enzymes must be isolated in active conformations, recombinant protein isolation systems must be chosen that have gentle elution procedures. While many suitable mammalian expression systems exist, we find that the Promega Flexi® Cloning System offers the highest level of convenience for the cloning of open reading frames (Blommel, Martin, Wrobel, Steffen, & Fox, 2006). This system is based on the ability of SgfI and PmeI restriction enzymes to recognize rare eight base pair sequences and allows for easy transfer of open reading frames between expression vectors. As the HaloTag® purification system is based on a covalent capture technique, stringent wash steps can be included without a decrease in protein yield. Additionally, a gentle protein elution with TEV protease is incorporated and can be performed at 4 °C. Thus, this system is ideal for the isolation of pure recombinant proteins in active states.
3.2 Considerations to minimize assay artifacts Epitope tag interference should always be considered when producing recombinant protein. This is true for recombinant HDACs as there are well documented influences on enzyme functional properties by epitope tags. The N-terminus of HDAC1 is essential for proper protein-protein interactions (Taplick et al., 2001) and results obtained by us and others show that placement of epitope tags at the N-termini of HDAC1, HDAC2, and HDAC3 negatively influences catalytic properties (Banks et al., 2018; Li et al., 2004; Taplick et al., 2001). However, epitope tag placement on the C-termini of HDAC1 and HDAC2 does not disrupt activity or interaction networks (Banks et al., 2018; Taplick et al., 2001). Therefore, recombinant forms of these proteins should have epitope tags place on C-termini.
Purification and enzymatic assay of class I HDACs
29
As an alternative to the direct analysis of HDAC enzymes, the expression and isolation of peripheral HDAC complex components can be performed. This avoids potential issues associated with the placement of epitope tags on the enzymatic subunits of HDAC complexes. Secondly, there is evidence that some HDAC inhibitors target enzymes only if they are present in specific protein complexes. For example, the HDAC inhibitor MS-275 is a weak inhibitor of HDAC1/2 that exists in Sin3 complexes but a strong inhibitor HDAC1/2 present in other complexes (Bantscheff et al., 2011; Becher et al., 2014). The direct isolation of HDAC1/2 from mammalian cells enriches for all protein complexes within which these proteins exist. Purifying proteins that are unique components of distinct HDAC complexes allows for the analysis of specific HDAC complexes and is a more suitable approach when the properties of specific complexes are to be examined. Enzyme purification conditions should be optimized for each assay. Factors, such as temperature, should be considered during all steps of the procedure. It has been shown that the activities of HDAC1 and HDAC3 decrease if purification procedures are performed at room temperature (Li et al., 2004). Therefore, it is recommended to perform all protein preparation steps on ice and at 4°C.
4. HDAC activity assay 4.1 Progression toward a high-throughput fluorescence-based HDAC activity assay As HDACs have long been recognized as important modulators of transcriptional activity, systems have been developed to assess the enzymatic properties of these enzymes. Early assays measured the release of [3H]-acetic acid from [3H]-acetyl histones (Kwon, Owa, Hassig, Shimada, & Schreiber, 1998; Sambucetti et al., 1999; Smith, Martin-Brown, Florens, Washburn, & Workman, 2010). However, this assay system required the use of radioactive isotopes and laborious techniques to isolate and label histones (Yoshida, Kijima, Akita, & Beppu, 1990). These early assays were also limited to the analysis of HDACs which recognize histones as substrates while not being suitable for HDACs that recognize non-histone proteins as substrates. In 1976, an assay system was developed to measure the activity of chymotrypsin (Zimmerman, Yurewicz, & Patel, 1976) and, later, other proteases (Morita et al., 1977; Zimmerman, Ashe, Yurewicz, & Patel, 1977). Hydrolysis of the substrate used in these assays released 7-amino-4-methylcoumarin (AMC), a fluorescent molecule whose
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Mark K. Adams et al.
abundance could be used as a measure of enzyme activity. Recognizing the barriers associated with the tritium-based HDAC assays, groups began developing fluorescence-based assays to measure HDAC activity that utilized principles of these previously developed protease assays. Wegener et al. developed an HDAC activity assay with the intention of creating a system suitable for high-throughput applications (Wegener, Wirsching, Riester, & Schwienhorst, 2003). To make this assay suitable for HDAC activity analysis, novel substrates were developed that exploit the inability of trypsin to cleave at the C-terminus of acetylated lysine residues. The fluorescent AMC molecule can only be released from the substrate if the acetyl group present on the substrate’s lysine residue is first removed by an HDAC enzyme (Fig. 2A). Thus, AMC abundance and fluorescent intensity provides a read-out of HDAC activity. The process of measuring activity during this assay follows a two-step reaction. In the first step, substrate is incubated with the protein sample whose activity is to be assayed. After stopping the reaction with a pan-HDAC inhibitor, such as trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA, Vorinostat), reactions are incubated with trypsin. Cleavage at the C-terminus of the substrate lysine residue from which acetyl groups have been removed liberates AMC, after which fluorescence can be measured (Fig. 2A).
4.2 Assay considerations Some considerations should be made when optimizing assay conditions. First, the enzyme substrate Boc-Lys(Ac)-AMC, as well as SAHA and many other HDAC inhibitors, have low solubility in aqueous and ethanol solutions. Therefore, these compounds must be reconstituted in dimethyl sulfoxide (DMSO). As DMSO itself may function as an HDAC inhibitor (Marks & Breslow, 2007), final concentrations of this compound must be maintained at low levels. DMSO concentrations presented in the provided protocol do not inhibit HDAC enzymes. Further, Boc-Lys(Ac)-AMC has been commonly utilized as a substrate in assays as it is recognized by HDAC1, HDAC2, and HDAC3; however, it is poorly recognized by other HDACs like HDAC8 (Nott, Watson, Robinson, Crepaldi, & Riccio, 2008; Vaidya et al., 2012; Wegener et al., 2003). It should be noted that other commercially available assay systems and substrates exist that may be better suited for assays of specific HDAC enzymes, such as the FLUOR DE LYS® fluorescent assay system (Enzo Life Sciences, Inc.).
A
O
Boc-Lys(Ac)-AMC
O
O
NH
O
RFU x 100 (HDAC activity)
O
NH
O
B
460 nm
O NH
O
7-Amino-4-methylcoumarin
NH
3. Detect
HDAC1
O NH
O
1. Deacetylate
NH
O
O
5.0
DMSO SAHA (HDAC inhibitor) *
4.0 3.0 2.0 1.0
Trypsin
SIN3B-Halo
NH2
Halo
0
O
2. Develop
C
D
DMSO
WB: anti-HDAC1
HDAC2-Halo
controls
2
WB: anti-HDAC2
4.0 3.0
1 2.0 1.0 HDAC2-Halo adjusted
HDAC2-Halo
Halo-HDAC2
HDAC1-Halo adjusted
HDAC1-Halo
Halo-HDAC1
HeLa nuclear extract
0
Halo
Band intensities (a.u.)
HDAC2
2
TBS
Halo-HDAC1 1.27E+06 Halo-HDAC2 1.28E+06 HDAC1-Halo 1.73E+06 HDAC2-Halo 8.00E+05
SAHA (HDAC inhibitor)
HDAC1
5.0
RFU x 10000 (HDAC activity)
1
Halo-HDAC2
HDAC1-Halo
Halo-HDAC1
Purification
Fig. 2 Assay of catalytic and peripheral HDAC complex components. (A) Boc-Lys(Ac)AMC is a synthetic enzyme substrate that, upon incubation with an HDAC enzyme, is deacetylated (1). After removal of the acetyl group from the substrate’s lysine residue, trypsin hydrolyzes the substrate (2) and releases the fluorescent molecule AMC (3). (B) Activity of SIN3B-HaloTag®-purified samples (SIN3B-Halo) or HaloTag® controlpurified samples (Halo) isolated from 293T cells transiently expressing SIN3B-HaloTag® or HaloTag® as a control. HaloTag® control samples were transfected with an expression vector previously described (Banks, Boanca, Lee, Florens, & Washburn, 2015) that expresses HaloTag® alone. To analyze activity, 2.5 μL (of 100 μL) of affinity-purified protein was distributed to each reaction well. SIN3B-containing complexes possess measurable HDAC activity and this activity is inhibited by the HDAC inhibitor SAHA. *, P < 0.01. Mean SD, n ¼ 4. (C–D) Expression and activity of recombinant HDAC1 or HDAC2 transiently expressed in 293T cells. Cells were transfected with expression constructs to express HDAC1/2 with N- or C-terminal HaloTag® and protein was isolated from cell lysate with Magne® HaloTag® Beads. (C) 10 μL of protein sample (of 100 μL total volume) were loaded per lane and probed with rabbit-anti-HDAC1 or rabbit-anti-HDAC2 antibody. Blots were probed with IRDye® 800CW Goat anti-Rabbit IgG secondary antibody and fluorescence was examined using an Odyssey® CLx Imager. Absorbance units were calculated using LiCor Image Studio™. (D) Activity and normalized activity of HDAC1 and HDAC2. 5 μL of eluted protein sample (of 100 μL total volume) were used for each reaction. Normalized activity was calculated by dividing RFU values by protein absorbance units in (C) (HDAC1-Halo adjusted and HDAC2-Halo adjusted). TBS and protein isolates from cells transiently expressing HaloTag® alone were used as negative controls for HDAC activity. HeLa nuclear extract was used as a positive control for HDAC activity. Mean SD, n ¼ 3. Placement of HaloTag® on the N-termini of proteins results in the inhibition of HDAC activity. HDAC1 and HDAC2 are partially inhibited by 10 μM SAHA. Panels (C–D): Figures are reproduced, with permission from Banks, C. A. S., Miah, S., Adams, M. K., Eubanks, C. G., Thornton, J. L., Florens, L., et al. (2018). Differential HDAC1/2 network analysis reveals a role for prefoldin/CCT in HDAC1/2 complex assembly. Scientific Reports, 8, 13712. https://doi.org/10.1038/s41598-018-32009-w.
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5. Protocol 5.1 Equipment Equipment
1.
Source
150 mm plates
TPP
Catalog number
®
93150 ®
353089
2.
Falcon™ Cell Scrapers
Corning
3.
Microcentrifuge Tubes
VWR™
87003-294
4.
PrecisionGlide™ Needle
Becton, Dickinson and Company
305110
5.
DynaMag™-2 Magnet
Thermo Scientific
12321D
®
6.
Micro Bio-Spin Columns
Bio-Rad Laboratories, Inc.
7326204
7.
384-well microplate, Black
Greiner Bio-One
781097
®
8.
SpectraMax Gemini™ XS
Molecular Devices
0112-0059
9.
Odyssey® CLx Imager
LI-COR®
9140
5.2 Reagents
Reagent
Source ®
Catalog number ®
1.
IGEPAL CA-630
Sigma-Aldrich
2.
AcTEV™ protease
Thermo Scientific 12575015
3.
Boc-Lys(Ac)-AMC
ApexBio
A8713
4.
DMSO
Sigma-Aldrich®
D2650-100ML
5.
SAHA
Cayman Chemical 10009929
6.
Trypsin from porcine pancreas
Sigma-Aldrich®
T4799-5G
7.
293T cells
ATCC®
CRL-11268™
8. 9.
DMEM without L-glutamine Fetal Bovine Serum (FBS)
10. Gibco™ GlutaMAX™ Supplement
Corning
®
Peak Serum
I8896
15-013-CV ®
PS-FB1
Thermo Scientific 35050061
33
Purification and enzymatic assay of class I HDACs
Reagent
Source
Catalog number
11. Gibco™ Opti-MEM™
Thermo Scientific 31985-062
12. Lipofectamine™ LTX Reagent with PLUS™ Reagent
Thermo Scientific 15338100
13. pFN21A HaloTag® CMV Flexi® Vector
Promega
G2821
14. pFC14A HaloTag® CMV Flexi® Vector
Promega
G9651
15. Mammalian Lysis Buffer
Promega
G938A
16. Protease Inhibitor Cocktail
Promega
G652A
17. Magne HaloTag Beads
Promega
G7281
18. Pierce™ BCA Protein Assay Kit
Thermo Scientific 23225
19. HDAC1 Antibody (rabbit polyclonal)
Proteintech®
®
®
20. HDAC2 Antibody (rabbit polyclonal)
®
Proteintech
21. IRDye® 800CW Goat anti-Rabbit IgG LI-COR® Secondary Antibody
10197-1-AP 12922-3-AP 926-32211
5.3 Buffer preparation 5.3.1 Recombinant protein isolation buffers 3.1.1. Prepare Affinity Purification Wash Buffer. To prepare, add 50 μL of IGEPAL® CA-630 to 100 mL TBS (pH 7.4). The final IGEPAL® CA-630 concentration is 0.05%. Chill buffer on ice prior to use. 3.1.2. Prepare AcTEV™ Elution Buffer. To prepare 1 mL, add 935 μL sterile H2O to a microcentrifuge tube. Add 50 μL of 20 AcTEV™ Protease Buffer, 10 μL of 0.1 M DTT, and 5 μL AcTEV™ Protease. Mix gently and store on ice. 100μL will be needed for each protein purification. 5.3.2 HDAC activity assay buffers 3.2.1. Prepare a 30 mM Substrate Stock Solution. Resuspend 50 mg of Boc-Lys(Ac)-AMC in 3.7 mL DMSO. Distribute into 100 μL aliquots. 3.2.2. Prepare 1 mM Substrate Working Solution. Add 100 μL of 30 mM Substrate Stock Solution from step 3.2.1 to 2.9 mL TBS (pH 7.4). Store on ice.
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Mark K. Adams et al.
3.2.3. Prepare 100 mM SAHA Stock Solution. Add 25 mg SAHA to 946 μL DMSO. 3.2.4. Prepare 200 μM SAHA Working Solution. Add 5 μL SAHA Stock Solution from step 3.2.3 to 2.5 mL TBS (pH 7.4). 3.2.5. Prepare Negative Control Solution. Add 5 μL DMSO to 2.5 mL TBS (pH 7.4). 3.2.6. Prepare 50 mg/mL Trypsin Solution. Add 100 mg trypsin to 2 mL TBS (pH 7.4). 5.3.3 Notes Affinity Purification Wash Buffer can be prepared and stored at 4 °C. AcTEV™ Elution Buffer must be made immediately prior to protein isolation. Substrate Stock Solution aliquots can be stored at 20°C. Substrate Working Solution, SAHA Stock Solution, SAHA Working Solution, Negative Control Solution, and Trypsin Solution must be prepared fresh immediately prior to activity assay.
5.4 Cell culture and transfection for recombinant protein production 4.1. Culture 293T cells in DMEM supplemented with GlutaMAX™ and FBS to a final concentration of 10%. 4.2. Seed cells at a density of 1 107 cells per 150 mm culture plate. The total volume of media should be 30 mL. Culture cells for 24 h at 37 °C and 5% CO2. 4.3. Dilute expression vector with Opti-MEM™. For each plate of cells, prepare 7.5 μg of expression vector in 6.6 mL Opti-MEM™ within a 50 mL conical tube. Invert tube to mix. For our examples, we have expressed N- and C-terminally tagged HDAC1 or HDAC2 using pFN21A HaloTag® CMV Flexi® Vector and pFC14A HaloTag® CMV Flexi® Vector derivatives described in Banks et al. (2018). SIN3B isoform 2 (CDS of NM_001297595) was cloned into AsiSI and PmeI sites of pFC14A HaloTag® CMV Flexi® Vector and contains a C-terminal HaloTag®. Note that the amount of expression vector used can be optimized. In our examples, we use less vector DNA than recommended by the manufacturer’s guidelines. 4.4. To the diluted expression vector, add 7.5 μL PLUS™ Reagent. Invert tube to mix. Incubate at room temperature for 5 min. 4.5. Add 50 μL of Lipofectamine® LTX Reagent to the tube. Invert tube to mix. Incubate at room temperature for 30 min. While other transfection reagents can be used, Lipofectamine® LTX Reagent is preferred as it is not necessary to change media after addition of transfection reagent.
Purification and enzymatic assay of class I HDACs
35
4.6. Gently add the DNA:lipid complex to cells and swirl plates. Culture for 36–48 h at 37 °C and 5% CO2. 4.7. Remove media and gently wash cells with 10 mL PBS (pH 7.4). Aspirate PBS and replace with 10 mL fresh PBS. Resuspend cells using a Falcon™ cell scraper and transfer to a 15 mL conical tube. 4.8. Centrifuge cells for 10 min at 500g and 4 °C. Remove supernatant and store cells on ice until protein isolation is performed. Alternately, cell pellets can be frozen and stored at 80 °C. Continue to Section 5.5.
5.5 Recombinant protein isolation 5.1. Thaw cells on ice and resuspend in 300 μL chilled Mammalian Lysis Buffer containing a protease inhibitor cocktail. Pass through a PrecisionGlide™ needle 5–10 times. Centrifuge at 18,000 g for 10 min at 4 °C in a tabletop centrifuge. Protease inhibitor cocktails that contain AEBSF should be avoided when HaloTag® is used for protein purification as this molecule interferes with capture of the HaloTag®. 5.2. Transfer supernatant to a clean microcentrifuge tube containing 700 μL chilled PBS. Invert tube to mix. Centrifuge at 18,000g for 10 min at 4 °C in a tabletop centrifuge. 5.3. Transfer supernatant to a clean microcentrifuge tube on ice. Leave the final 10–20 μL of supernatant to avoid disturbing pellet. 5.4. Quantify protein using a Pierce™ BCA Protein Assay Kit and BSA protein standards. 5.5. Transfer 2 mg supernatant to a clean microcentrifuge tube containing pre-washed 100 μL Magne® HaloTag® Beads. Incubate with gentle rotation for 2 h at 4 °C. 5.6. Place tubes on DynaMag™-2 Magnet and remove supernatant. Add 1 mL chilled Affinity Purification Wash Buffer and invert to mix. 5.7. Place tubes on DynaMag™-2 Magnet and remove supernatant. 5.8. Perform steps 5.6–5.7 two additional times for a total of three washes. 5.9. Add 1 mL chilled TBS and invert to mix. Place tubes on DynaMag™-2 Magnet and remove supernatant. 5.10. Resuspend in 100μL AcTEV™ Elution Buffer and rotate overnight at 4°C. 5.11. Place tubes on DynaMag™-2 Magnet and transfer supernatant to a microcentrifuge tube containing a Micro Bio-Spin® column on ice. 5.12. Centrifuge at 500 g for 1 min at 4 °C. The flow through contains the purified protein. 5.13. Snap freeze purified protein in liquid N2 and store at 80°C. Alternatively, proceed immediately to HDAC activity assay.
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Mark K. Adams et al.
5.6 HDAC activity assay 6.1. Refer to Section 5.3.2 for buffer preparation. 6.2. Dilute 2.5 to 10 μL purified protein with TBS (pH 7.4). The final volume should be equal to 42.5 μL for each reaction assay. 6.3. To each well of a 384-well black microplate that will be used during the activity assay, add the 42.5 μL of diluted protein sample. 6.4. Add 2.5 μL Negative Control Solution or 200μM SAHA Working Stock Solution to each well. For this and all remaining steps, it is suggested that a multi-channel pipette be used to avoid pipetting error. The addition of SAHA (or another HDACi) at this step allows for the determination of enzyme response to HDAC inhibitors. Different HDACis, such as those found in Table 1, can be analyzed and assays can be performed using a range of HDACi concentrations. In the provided example, SAHA is used at a concentration of 10μM. The Negative Control Solution accounts for the DMSO found within the HDACi solution and should be added to wells in which no HDACi is present. 6.5. Add 5 μL Substrate Working Solution to each well. The total volume in each well after addition of substrate should be 50μL. Cover plate and invert to mix. Centrifuge plate at 200 g for 1 min. Incubate 1 h at 37 °C with gentle rotation. 6.6. Stop the reaction with the addition of a pan-HDAC inhibitor. Add 2.5 μL 200 μM SAHA working stock solution to each well. Incubate 5 min at 37 °C with gentle rotation. In the provided example, SAHA is used to stop the enzymatic reaction as it is known that Sin3 complexes are responsive to SAHA. Other pan-HDAC inhibitors, such as trichostatin A, can also be used to stop the enzymatic reaction. 6.7. Hydrolyze the deacetylated substrate and release AMC. Add 6μL Trypsin Solution to each well. Incubate 1 h at 37°C with gentle rotation. During this step, trypsin digests the substrate which releases AMC. Digestion only occurs if acetyl groups have been removed from the substrate by the analyzed enzyme. 6.8. Measure fluorescence on a SpectraMax® Gemini™ XS or comparable instrument. During data acquisition, use an excitation wavelength of 355 nm and an emission wavelength of 460 nm.
6. Analysis Examination of enzyme activities and inhibition is as simple as reviewing relative fluorescent units (RFUs) for each reaction (Fig. 2B, D).
Purification and enzymatic assay of class I HDACs
37
Biological replicates should be examined for each sample and a statistical analysis, such as an unpaired t-test (Fig. 2B), can be performed to determine the significance of differences in HDAC activity between treatment groups. If comparing RFU values between protein samples, it is important that values be normalized to account for variations in protein abundance between samples. If the molecular weight and amount of protein in each sample is known, then activities can be normalized to protein concentration. However, contaminating proteins, such as TEV protease introduced during protein elution, may prevent the accurate measure of recombinant protein concentration. Additionally, proteins concentrations are often below the detection limits of commercially available protein quantification kits. In such instances, concentrations of proteins possessing affinity tags can be estimated using intensity values of proteins on Western blots (Fig. 2C and D).
7. Summary As HDACs continue to be studied as potential targets of chemotherapeutic agents, tools and systems must be established for the adequate characterization of these enzymes. We describe a straight-forward HDAC activity assay that utilizes commercially available materials. While we demonstrate an approach based on the recombinant expression of HaloTagged proteins, the described HDAC activity assay system is flexible and can be used with other affinity purification systems as well as with endogenous protein. This system can be used for the direct analysis of purified HDAC enzymes (Fig. 2C and D) as well as non-enzymatic subunits of HDAC complexes (Fig. 2B). Thus, the catalytic properties of specific HDAC complexes can be examined and their responsiveness to HDACis can be assessed.
Acknowledgments Research reported in this publication was supported by the Stowers Institute for Medical Research and the National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers F32GM122215 (M.K.A.) and R01GM112639 (M.P.W.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Original data underlying this manuscript can be accessed from the Stowers Original Data Repository at https:// www.stowers.org/research/publications/libpb-1444.
References Allis, C. D., & Jenuwein, T. (2016). The molecular hallmarks of epigenetic control. Nature Reviews Genetics, 17(8), 487–500. https://doi.org/10.1038/nrg.2016.59.
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Banks, C. A. S., Boanca, G., Lee, Z. T., Florens, L., & Washburn, M. P. (2015). Proteins interacting with cloning scars: A source of false positive protein-protein interactions. Scientific Reports, 5, 8530. https://doi.org/10.1038/srep08530. Banks, C. A. S., Miah, S., Adams, M. K., Eubanks, C. G., Thornton, J. L., Florens, L., et al. (2018). Differential HDAC1/2 network analysis reveals a role for prefoldin/CCT in HDAC1/2 complex assembly. Scientific Reports, 8, 13712. https://doi.org/10.1038/ s41598-018-32009-w. Bantscheff, M., Hopf, C., Savitski, M. M., Dittmann, A., Grandi, P., Michon, A. M., et al. (2011). Chemoproteomics profiling of HDAC inhibitors reveals selective targeting of HDAC complexes. Nature Biotechnology, 29(3), 255–265. https://doi.org/10.1038/ nbt.1759. Becher, I., Dittmann, A., Savitski, M. M., Hopf, C., Drewes, G., & Bantscheff, M. (2014). Chemoproteomics reveals time-dependent binding of histone deacetylase inhibitors to endogenous repressor complexes. ACS Chemical Biology, 9(8), 1736–1746. https://doi. org/10.1021/cb500235n. Blommel, P. G., Martin, P. A., Wrobel, R. L., Steffen, E., & Fox, B. G. (2006). High efficiency single step production of expression plasmids from cDNA clones using the flexi vector cloning system. Protein Expression and Purification, 47(2), 562–570. https://doi.org/ 10.1016/j.pep.2005.11.007. Bradner, J. E., West, N., Grachan, M. L., Greenberg, E. F., Haggarty, S. J., Warnow, T., et al. (2010). Chemical phylogenetics of histone deacetylases. Nature Chemical Biology, 6(3), 238–243. https://doi.org/10.1038/nchembio.313. Emiliani, S., Fischle, W., Van Lint, C., Al-Abed, Y., & Verdin, E. (1998). Characterization of a human RPD3 ortholog, HDAC3. Proceedings of the National Academy of Sciences of the United States of America, 95(6), 2795–2800. https://doi.org/10.1073/ pnas.95.6.2795. Furumai, R., Matsuyama, A., Kobashi, N., Lee, K. H., Nishiyama, M., Nakajima, H., et al. (2002). FK228 (depsipeptide) as a natural prodrug that inhibits class i histone deacetylases. Cancer Research, 62(17), 4916–4921. Retrieved from http://cancerres aacrjournals.org. Guenther, M. G., Lane, W. S., Fischle, W., Verdin, E., Lazar, M. A., & Shiekhattar, R. (2000). A core SMRT corepressor complex containing HDAC3 and TBL1, a WD40-repeat protein linked to deafness. Genes & Development, 14(9), 1048–1057. Retrieved from http://genesdev.cshlp.org/. Hassig, C. A., Tong, J. K., Fleischer, T. C., Owa, T., Grable, P. G., Ayer, D. E., et al. (1998). A role for histone deacetylase activity in HDAC1-mediated transcriptional repression. Proceedings of the National Academy of Sciences of the United States of America, 95(7), 3519–3524. https://doi.org/10.1073/pnas.95.7.3519. Hu, E., Chen, Z., Fredrickson, T., Zhu, Y., Kirkpatrick, R., Zhang, G. F., et al. (2000). Cloning and characterization of a novel human class I histone deacetylase that functions as a transcription repressor. Journal of Biological Chemistry, 275(20), 15254–15264. https:// doi.org/10.1074/jbc.M908988199. Kadosh, D., & Struhl, K. (1998). Histone deacetylase activity of Rpd3 is important for transcriptional repression in vivo. Genes & Development, 12(6), 797–805. https://doi.org/ 10.1101/gad.12.6.797. Khan, N., Jeffers, M., Kumar, S., Hackett, C., Boldog, F., Khramtsov, N., et al. (2008). Determination of the class and isoform selectivity of small-molecule histone deacetylase inhibitors. Biochemical Journal, 409(2), 581–589. https://doi.org/10.1042/BJ20070779. Kwon, H. J., Owa, T., Hassig, C. A., Shimada, J., & Schreiber, S. L. (1998). Depudecin induces morphological reversion of transformed fibroblasts via the inhibition of histone deacetylase. Proceedings of the National Academy of Sciences of the United States of America, 95(7), 3356–3361. https://doi.org/10.1073/pnas.95.7.3356.
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Lauffer, B. E. L., Mintzer, R., Fong, R., Mukund, S., Tam, C., Zilberleyb, I., et al. (2013). Histone deacetylase (HDAC) inhibitor kinetic rate constants correlate with cellular histone acetylation but not transcription and cell viability. Journal of Biological Chemistry, 288(37), 26926–26943. https://doi.org/10.1074/jbc.M113.490706. Li, J., Staver, M. J., Curtin, M. L., Holms, J. H., Frey, R. R., Edalji, R., et al. (2004). Expression and functional characterization of recombinant human HDAC1 and HDAC3. Life Sciences, 74(22), 2693–2705. https://doi.org/10.1016/j.lfs.2003.09.070. Lombardi, P. M., Cole, K. E., Dowling, D. P., & Christianson, D. W. (2011). Structure, mechanism, and inhibition of histone deacetylases and related metalloenzymes. Current Opinion in Structural Biology, 21(6), 735–743. https://doi.org/10.1016/j.sbi.2011.08.004. Marks, P. A., & Breslow, R. (2007). Dimethyl sulfoxide to vorinostat: Development of this histone deacetylase inhibitor as an anticancer drug. Nature Biotechnology, 25(1), 84–90. https://doi.org/10.1038/nbt1272. Marmorstein, R., & Zhou, M. M. (2014). Writers and readers of histone acetylation: Structure, mechanism, and inhibition. Cold Spring Harbor Perspectives in Biology, 6(7), a018762. https://doi.org/10.1101/cshperspect.a018762. Morgan, M. A., & Shilatifard, A. (2015). Chromatin signatures of cancer. Genes & Development, 29(3), 238–249. https://doi.org/10.1101/gad.255182.114. Morita, T., Kato, H., Iwanaga, S., Takada, K., Kimura, T., & Sakakibara, S. (1977). New fluorogenic substrates for α-thrombin, factor Xa, kallikreins, and urokinase. The Journal of Biochemistry, 82(5), 1495–1498. https://doi.org/10.1093/oxfordjournals.jbchem.a131840. Musselman, C. A., & Kutateladze, T. G. (2011). Handpicking epigenetic marks with PHD fingers. Nucleic Acids Research, 39(21), 9061–9071. https://doi.org/10.1093/nar/gkr613. Nott, A., Watson, P. M., Robinson, J. D., Crepaldi, L., & Riccio, A. (2008). S-nitrosylation of histone deacetylase 2 induces chromatin remodelling in neurons. Nature, 455(7211), 411–415. https://doi.org/10.1038/nature07238. Richon, V. M., Emiliani, S., Verdin, E., Webb, Y., Breslow, R., Rifkind, R. A., et al. (1998). A class of hybrid polar inducers of transformed cell differentiation inhibits histone deacetylases. Proceedings of the National Academy of Sciences of the United States of America, 95(6), 3003–3007. https://doi.org/10.1073/pnas.95.6.3003. Rundlett, S. E., Carmen, A. A., Kobayashi, R., Bavykin, S., Turner, B. M., & Grunstein, M. (1996). HDA1 and RPD3 are members of distinct yeast histone deacetylase complexes that regulate silencing and transcription. Proceedings of the National Academy of Sciences of the United States of America, 93(25), 14503–14508. https://doi.org/10.1073/pnas.93.25.14503. Sambucetti, L. C., Fischer, D. D., Zabludoff, S., Kwon, P. O., Chamberlin, H., Trogani, N., et al. (1999). Histone deacetylase inhibition selectively alters the activity and expression of cell cycle proteins leading to specific chromatin acetylation and antiproliferative effects. Journal of Biological Chemistry, 274(49), 34940–34947. https://doi.org/10.1074/ jbc.274.49.34940. Sauve, A. A. (2010). Sirtuin chemical mechanisms. Biochimica et Biophysica Acta (BBA)—Proteins and Proteomics, 1804(8), 1591–1603. https://doi.org/10.1016/j.bbapap.2010.01.021. Seto, E., & Yoshida, M. (2014). Erasers of histone acetylation: The histone deacetylase enzymes. Cold Spring Harbor Perspectives in Biology, 6(4), a018713. https://doi.org/ 10.1101/cshperspect.a018713. Smith, K. T., Martin-Brown, S. A., Florens, L., Washburn, M. P., & Workman, J. L. (2010). Deacetylase inhibitors dissociate the histone-targeting ING2 subunit from the Sin3 complex. Chemistry & Biology, 17(1), 65–74. https://doi.org/10.1016/j.chembiol.2009.12.010. Suraweera, A., O’Byrne, K. J., & Richard, D. J. (2018). Combination therapy with histone deacetylase inhibitors (HDACi) for the treatment of cancer: Achieving the full therapeutic potential of HDACi. Frontiers in Oncology, 8, 92. https://doi.org/10.3389/ fonc.2018.00092.
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Purification and enzymatic assay of class I histone deacetylase enzymes Mark K. Adamsa, Charles A.S. Banksa, Sayem Miaha, Maxime Killera,†, Michael P. Washburna,b,* a
Stowers Institute for Medical Research, Kansas City, MO, United States Department of Pathology & Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, United States *Corresponding author: e-mail address: [email protected] b
Contents 1. Introduction 2. Histone deacetylases and HDAC inhibitors 3. Purifying protein for HDAC assay 3.1 Choosing an expression system 3.2 Considerations to minimize assay artifacts 4. HDAC activity assay 4.1 Progression toward a high-throughput fluorescence-based HDAC activity assay 4.2 Assay considerations 5. Protocol 5.1 Equipment 5.2 Reagents 5.3 Buffer preparation 5.4 Cell culture and transfection for recombinant protein production 5.5 Recombinant protein isolation 5.6 HDAC activity assay 6. Analysis 7. Summary Acknowledgments References
†
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Current address: Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Hamburg, Germany.
Methods in Enzymology, Volume 626 ISSN 0076-6879 https://doi.org/10.1016/bs.mie.2019.07.014
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2019 Elsevier Inc. All rights reserved.
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Abstract The reversible acetylation of histones has a profound influence on transcriptional status. Histone acetyltransferases catalyze the addition of these chemical modifications to histone lysine residues. Conversely, histone deacetylases (HDACs) catalyze the removal of these acetyl groups from histone lysine residues. As modulators of transcription, HDACs have found themselves as targets of several FDA-approved chemotherapeutic compounds which aim to inhibit enzyme activity. The ongoing efforts to develop targeted and isoform-specific HDAC inhibitors necessitates tools to study these modifications and the enzymes that maintain an equilibrium of these modifications. In this chapter, we present an optimized workflow for the isolation of recombinant protein and subsequent assay of class I HDAC activity. We demonstrate the application of this assay by assessing the activities of recombinant HDAC1, HDAC2, and SIN3B. This assay system utilizes readily available reagents and can be used to assess the activity and responsiveness of class I HDAC complexes to HDAC inhibitors.
1. Introduction The high frequency at which cancer-associated mutations within chromatin modifying enzymes are observed (Morgan & Shilatifard, 2015) suggests that chromatin modifications are central to the regulation of gene expression patterns and the development of cancer. Not surprisingly, such enzymes are the focus of many ongoing studies which aim to characterize their roles in pathologies. Further, the enzymes that modify chromatin as well as their regulatory mechanisms have found themselves as targets of chemotherapeutic compounds. Chromatin modifications exist in several forms, with the acetylation of histone lysine residues being among the best characterized (Allis & Jenuwein, 2016). Traditionally associated with transcriptional activation, the addition of acetyl groups to histone lysine residues by histone acetyltransferases (HATs) provides an epigenetic mark which is recognized by several protein domains (Marmorstein & Zhou, 2014; Musselman & Kutateladze, 2011). Histone acetylation is a reversible chemical modification and histone deacetylases (HDACs) are responsible for the removal of this chemical modification. The reversible nature of histone acetylation as well as its profound influence on transcriptional status situates this post-translational modification as an ideal target for chemotherapeutic modulators of transcription. There are currently four FDA-approved HDAC inhibitors (HDACi) that have demonstrated efficacy as parts of multi-component chemotherapeutic treatment strategies (Table 1) (Suraweera, O’Byrne, & Richard, 2018). As HDACs and their potentials as
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Table 1 FDA-approved HDAC inhibitors. Inhibitor Alternative names References
Vorinostat
Richon et al. (1998), Khan et al. (2008), and Zolinza Bantscheff et al. (2011) Suberoylanilide Hydroxamic Acid (SAHA)
Romidepsin Istodax Chromadax FK228
Furumai et al. (2002) and Bantscheff et al. (2011)
Belinostat
Beleodaq PXD101
Khan et al. (2008) and Bantscheff et al. (2011)
Panobinostat Farydak LBH589
Khan et al. (2008) and Bantscheff et al. (2011)
Inhibitor influence on Class I HDAC activity is described in the provided references.
targets of chemotherapeutics are the subjects of ongoing study, it is essential that convenient assays are available with which the functional attributes of these enzymes and their responses to chemotherapeutic agents can be examined. Here, we describe an optimized protocol for the expression and in vitro analysis of class I HDAC enzymes. This assay allows for the screening of enzyme activity as well as enzyme responsiveness to HDAC inhibitors. To demonstrate the workflow, we have focused on the expression, purification, and assay of the Sin3 HDAC complex component SIN3B as well as the catalytic subunits of the Sin3 complex, HDAC1 and HDAC2. SIN3B serves as a scaffolding component of Sin3 complexes, protein complexes that are conserved from yeast to mammals. As Sin3 complexes have been previously shown to be responsive to some but not all HDAC inhibitors (Becher et al., 2014), HDAC complexes containing SIN3B are ideal models for the demonstration of complex responsiveness to HDAC inhibitors.
2. Histone deacetylases and HDAC inhibitors Histone deacetylases are represented by 18 separate enzymes organized into 4 distinct classes (Classes I, II, III, and IV). Classes I, II, and IV are metal-dependent enzymes that have a Zn2+ ion within the catalytic pocket (Lombardi, Cole, Dowling, & Christianson, 2011; Seto & Yoshida, 2014). While not all details regarding a catalytic mechanism have been described for these Zn2+-dependent enzymes, it is accepted that the removal of lysine acetyl groups is coordinated by histidine and/or tyrosine residues and the
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metal ion present within the active site pocket (Lombardi et al., 2011; Seto & Yoshida, 2014). Class III HDACs are represented by sirtuins and utilize a Zn2+-independent catalytic mechanism (Sauve, 2010). In regard to histone acetylation status, class I HDACs (HDAC1, HDAC2, HDAC3, HDAC8) are of interest as they have been shown to localize within the nucleus (Emiliani, Fischle, Van Lint, Al-Abed, & Verdin, 1998; Hu et al., 2000; Taplick et al., 2001; Van den Wyngaert et al., 2000; Wilting et al., 2010). Class I HDACs also share homology with the yeast enzyme Rpd3 (Yang & Seto, 2008), an enzyme that has a demonstrated role in the removal of histone lysine acetyl groups (Kadosh & Struhl, 1998; Rundlett et al., 1996). Thus, these enzymes likely play important and conserved roles in the modulation of histone acetylation status. While HDAC1 and HDAC2 possess intrinsic enzymatic activities (Hassig et al., 1998), they typically exist as the catalytic components of several large protein complexes, including Sin3, NuRD, and CoREST complexes. Additionally, it is likely that HDAC1/2 are components of other protein complexes that are poorly characterized (Bantscheff et al., 2011). HDAC3 is found within NCoR/SMRT complexes (Guenther et al., 2000) while HDAC8 is not a known component of any defined protein complexes. The dependence of class I HDACs, as well as classes II and IV, on Zn2+ ions is exploited by chemotherapeutic HDAC inhibitors (Seto & Yoshida, 2014; Wu, Lu, Cao, & Zhang, 2011). Early HDAC inhibitors produced broad spectrum effects, like those associated with trichostatin A (TCA). To minimize off-target effects associated with HDACi application, recently developed inhibitors only influence the activities of specific enzymes or enzymes if they exist in specific HDAC complexes (Bradner et al., 2010; Lauffer et al., 2013). As we progress toward targeted HDAC inhibitors, readily available and flexible HDAC activity assay systems will be needed to assess the efficacy of these compounds.
3. Purifying protein for HDAC assay 3.1 Choosing an expression system Prior to the analysis of HDAC activity, one must first decide whether endogenous or recombinant protein will be examined. Endogenous HDACs and HDAC complexes can be easily isolated from human cells (Becher et al., 2014). Additionally, recombinant protein production systems, such as baculovirus-mediated expression in insect cells (Hassig et al., 1998) and mammalian expression vector systems (Banks et al., 2018), have been used
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to produce enzymatically active HDACs. The analysis of endogenous and recombinant protein both have benefits and shortcomings which must be considered. To analyze the activity of endogenous protein, HDAC complexes are first enriched from cell extracts and immobilized using immunoprecipitation assays and antibodies which recognize HDAC complex components (Becher et al., 2014) (Fig. 1). A benefit of this approach is that transfection reagents and epitope tags are not required. This approach can also be used to isolate proteins of interest from cells which are difficult to transfect. However, this approach is often hampered by a lack of properly validated antibodies. Therefore, antibodies must be tested for suitability in immunoprecipitation assays and the specificity of epitope recognition by the antibody must be examined. The elution of protein complexes from affinity purification matrices in active conformations can also be challenging due to harsh elution conditions often required during the immunoprecipitation of proteins. However, this issue can be avoided as proteins do not 1
Recombinant protein CMV HDAC Halo transient transfection
fluorescent substrate
Halo capture and elution PROTEIN PRODUCTION
multi-well plate
excitation: 355 nm emission: 460 nm
PROTEIN ISOLATION
IP
2
develop with trypsin
DEACETYLATE SUBSTRATE
plate reader
Endogenous protein Protein preparation
HDAC activity assay
Fig. 1 Enrichment and assay of HDAC enzymes. HDAC enzymes can be acquired via (1) expression of recombinant protein or (2) isolation of endogenous protein. (1) For the production and isolation of recombinant protein, an open reading frame encoding the protein of interest along with an affinity tag is transiently expressed in mammalian cells using commercially available transfection reagents. Recombinant protein is enriched using affinity tag capture and eluted from the affinity purification matrix. (2) To isolate endogenous HDACs, antibodies that recognize the protein of interest are immobilized on Dynabeads™ Protein A or Dynabeads™ Protein G (Life Technologies). Cell lysates are incubated with the immobilized antibody and washed to remove contaminating proteins. For the assay of endogenous protein, elution of purified protein is unnecessary and HDAC activity can be performed while protein is immobilized on the affinity purification matrix. Using a multi-well plate platform, isolated recombinant or endogenous proteins are incubated at 37 °C with a fluorescent substrate, Boc-Lys(Ac)-AMC, in the presence or absence of HDAC inhibitors. Reactions are developed with trypsin and fluorescence is examined using a fluorescent plate reader with an excitation wavelength of 355 nm and an emission wavelength of 460 nm.
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need to be eluted from the matrix for activity assays. Instead, activity assays can be performed while proteins are immobilized on the affinity purification matrix (Fig. 1). As antibodies suitable for immunoprecipitation assays are not available for all components of HDAC complexes, the assay of recombinant protein is often the only feasible approach to isolate and examine the properties of HDACs. This offers several advantages over the study of endogenous protein, including the capacity to produce high levels of protein, the ability to introduce affinity tags, and high-level purity of isolated proteins. During protein purification steps, glycine- and urea-based elution methods should be avoided to prevent disruption of protein-protein interactions and protein folding. As enzymes must be isolated in active conformations, recombinant protein isolation systems must be chosen that have gentle elution procedures. While many suitable mammalian expression systems exist, we find that the Promega Flexi® Cloning System offers the highest level of convenience for the cloning of open reading frames (Blommel, Martin, Wrobel, Steffen, & Fox, 2006). This system is based on the ability of SgfI and PmeI restriction enzymes to recognize rare eight base pair sequences and allows for easy transfer of open reading frames between expression vectors. As the HaloTag® purification system is based on a covalent capture technique, stringent wash steps can be included without a decrease in protein yield. Additionally, a gentle protein elution with TEV protease is incorporated and can be performed at 4 °C. Thus, this system is ideal for the isolation of pure recombinant proteins in active states.
3.2 Considerations to minimize assay artifacts Epitope tag interference should always be considered when producing recombinant protein. This is true for recombinant HDACs as there are well documented influences on enzyme functional properties by epitope tags. The N-terminus of HDAC1 is essential for proper protein-protein interactions (Taplick et al., 2001) and results obtained by us and others show that placement of epitope tags at the N-termini of HDAC1, HDAC2, and HDAC3 negatively influences catalytic properties (Banks et al., 2018; Li et al., 2004; Taplick et al., 2001). However, epitope tag placement on the C-termini of HDAC1 and HDAC2 does not disrupt activity or interaction networks (Banks et al., 2018; Taplick et al., 2001). Therefore, recombinant forms of these proteins should have epitope tags place on C-termini.
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As an alternative to the direct analysis of HDAC enzymes, the expression and isolation of peripheral HDAC complex components can be performed. This avoids potential issues associated with the placement of epitope tags on the enzymatic subunits of HDAC complexes. Secondly, there is evidence that some HDAC inhibitors target enzymes only if they are present in specific protein complexes. For example, the HDAC inhibitor MS-275 is a weak inhibitor of HDAC1/2 that exists in Sin3 complexes but a strong inhibitor HDAC1/2 present in other complexes (Bantscheff et al., 2011; Becher et al., 2014). The direct isolation of HDAC1/2 from mammalian cells enriches for all protein complexes within which these proteins exist. Purifying proteins that are unique components of distinct HDAC complexes allows for the analysis of specific HDAC complexes and is a more suitable approach when the properties of specific complexes are to be examined. Enzyme purification conditions should be optimized for each assay. Factors, such as temperature, should be considered during all steps of the procedure. It has been shown that the activities of HDAC1 and HDAC3 decrease if purification procedures are performed at room temperature (Li et al., 2004). Therefore, it is recommended to perform all protein preparation steps on ice and at 4°C.
4. HDAC activity assay 4.1 Progression toward a high-throughput fluorescence-based HDAC activity assay As HDACs have long been recognized as important modulators of transcriptional activity, systems have been developed to assess the enzymatic properties of these enzymes. Early assays measured the release of [3H]-acetic acid from [3H]-acetyl histones (Kwon, Owa, Hassig, Shimada, & Schreiber, 1998; Sambucetti et al., 1999; Smith, Martin-Brown, Florens, Washburn, & Workman, 2010). However, this assay system required the use of radioactive isotopes and laborious techniques to isolate and label histones (Yoshida, Kijima, Akita, & Beppu, 1990). These early assays were also limited to the analysis of HDACs which recognize histones as substrates while not being suitable for HDACs that recognize non-histone proteins as substrates. In 1976, an assay system was developed to measure the activity of chymotrypsin (Zimmerman, Yurewicz, & Patel, 1976) and, later, other proteases (Morita et al., 1977; Zimmerman, Ashe, Yurewicz, & Patel, 1977). Hydrolysis of the substrate used in these assays released 7-amino-4-methylcoumarin (AMC), a fluorescent molecule whose
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abundance could be used as a measure of enzyme activity. Recognizing the barriers associated with the tritium-based HDAC assays, groups began developing fluorescence-based assays to measure HDAC activity that utilized principles of these previously developed protease assays. Wegener et al. developed an HDAC activity assay with the intention of creating a system suitable for high-throughput applications (Wegener, Wirsching, Riester, & Schwienhorst, 2003). To make this assay suitable for HDAC activity analysis, novel substrates were developed that exploit the inability of trypsin to cleave at the C-terminus of acetylated lysine residues. The fluorescent AMC molecule can only be released from the substrate if the acetyl group present on the substrate’s lysine residue is first removed by an HDAC enzyme (Fig. 2A). Thus, AMC abundance and fluorescent intensity provides a read-out of HDAC activity. The process of measuring activity during this assay follows a two-step reaction. In the first step, substrate is incubated with the protein sample whose activity is to be assayed. After stopping the reaction with a pan-HDAC inhibitor, such as trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA, Vorinostat), reactions are incubated with trypsin. Cleavage at the C-terminus of the substrate lysine residue from which acetyl groups have been removed liberates AMC, after which fluorescence can be measured (Fig. 2A).
4.2 Assay considerations Some considerations should be made when optimizing assay conditions. First, the enzyme substrate Boc-Lys(Ac)-AMC, as well as SAHA and many other HDAC inhibitors, have low solubility in aqueous and ethanol solutions. Therefore, these compounds must be reconstituted in dimethyl sulfoxide (DMSO). As DMSO itself may function as an HDAC inhibitor (Marks & Breslow, 2007), final concentrations of this compound must be maintained at low levels. DMSO concentrations presented in the provided protocol do not inhibit HDAC enzymes. Further, Boc-Lys(Ac)-AMC has been commonly utilized as a substrate in assays as it is recognized by HDAC1, HDAC2, and HDAC3; however, it is poorly recognized by other HDACs like HDAC8 (Nott, Watson, Robinson, Crepaldi, & Riccio, 2008; Vaidya et al., 2012; Wegener et al., 2003). It should be noted that other commercially available assay systems and substrates exist that may be better suited for assays of specific HDAC enzymes, such as the FLUOR DE LYS® fluorescent assay system (Enzo Life Sciences, Inc.).
A
O
Boc-Lys(Ac)-AMC
O
O
NH
O
RFU x 100 (HDAC activity)
O
NH
O
B
460 nm
O NH
O
7-Amino-4-methylcoumarin
NH
3. Detect
HDAC1
O NH
O
1. Deacetylate
NH
O
O
5.0
DMSO SAHA (HDAC inhibitor) *
4.0 3.0 2.0 1.0
Trypsin
SIN3B-Halo
NH2
Halo
0
O
2. Develop
C
D
DMSO
WB: anti-HDAC1
HDAC2-Halo
controls
2
WB: anti-HDAC2
4.0 3.0
1 2.0 1.0 HDAC2-Halo adjusted
HDAC2-Halo
Halo-HDAC2
HDAC1-Halo adjusted
HDAC1-Halo
Halo-HDAC1
HeLa nuclear extract
0
Halo
Band intensities (a.u.)
HDAC2
2
TBS
Halo-HDAC1 1.27E+06 Halo-HDAC2 1.28E+06 HDAC1-Halo 1.73E+06 HDAC2-Halo 8.00E+05
SAHA (HDAC inhibitor)
HDAC1
5.0
RFU x 10000 (HDAC activity)
1
Halo-HDAC2
HDAC1-Halo
Halo-HDAC1
Purification
Fig. 2 Assay of catalytic and peripheral HDAC complex components. (A) Boc-Lys(Ac)AMC is a synthetic enzyme substrate that, upon incubation with an HDAC enzyme, is deacetylated (1). After removal of the acetyl group from the substrate’s lysine residue, trypsin hydrolyzes the substrate (2) and releases the fluorescent molecule AMC (3). (B) Activity of SIN3B-HaloTag®-purified samples (SIN3B-Halo) or HaloTag® controlpurified samples (Halo) isolated from 293T cells transiently expressing SIN3B-HaloTag® or HaloTag® as a control. HaloTag® control samples were transfected with an expression vector previously described (Banks, Boanca, Lee, Florens, & Washburn, 2015) that expresses HaloTag® alone. To analyze activity, 2.5 μL (of 100 μL) of affinity-purified protein was distributed to each reaction well. SIN3B-containing complexes possess measurable HDAC activity and this activity is inhibited by the HDAC inhibitor SAHA. *, P < 0.01. Mean SD, n ¼ 4. (C–D) Expression and activity of recombinant HDAC1 or HDAC2 transiently expressed in 293T cells. Cells were transfected with expression constructs to express HDAC1/2 with N- or C-terminal HaloTag® and protein was isolated from cell lysate with Magne® HaloTag® Beads. (C) 10 μL of protein sample (of 100 μL total volume) were loaded per lane and probed with rabbit-anti-HDAC1 or rabbit-anti-HDAC2 antibody. Blots were probed with IRDye® 800CW Goat anti-Rabbit IgG secondary antibody and fluorescence was examined using an Odyssey® CLx Imager. Absorbance units were calculated using LiCor Image Studio™. (D) Activity and normalized activity of HDAC1 and HDAC2. 5 μL of eluted protein sample (of 100 μL total volume) were used for each reaction. Normalized activity was calculated by dividing RFU values by protein absorbance units in (C) (HDAC1-Halo adjusted and HDAC2-Halo adjusted). TBS and protein isolates from cells transiently expressing HaloTag® alone were used as negative controls for HDAC activity. HeLa nuclear extract was used as a positive control for HDAC activity. Mean SD, n ¼ 3. Placement of HaloTag® on the N-termini of proteins results in the inhibition of HDAC activity. HDAC1 and HDAC2 are partially inhibited by 10 μM SAHA. Panels (C–D): Figures are reproduced, with permission from Banks, C. A. S., Miah, S., Adams, M. K., Eubanks, C. G., Thornton, J. L., Florens, L., et al. (2018). Differential HDAC1/2 network analysis reveals a role for prefoldin/CCT in HDAC1/2 complex assembly. Scientific Reports, 8, 13712. https://doi.org/10.1038/s41598-018-32009-w.
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5. Protocol 5.1 Equipment Equipment
1.
Source
150 mm plates
TPP
Catalog number
®
93150 ®
353089
2.
Falcon™ Cell Scrapers
Corning
3.
Microcentrifuge Tubes
VWR™
87003-294
4.
PrecisionGlide™ Needle
Becton, Dickinson and Company
305110
5.
DynaMag™-2 Magnet
Thermo Scientific
12321D
®
6.
Micro Bio-Spin Columns
Bio-Rad Laboratories, Inc.
7326204
7.
384-well microplate, Black
Greiner Bio-One
781097
®
8.
SpectraMax Gemini™ XS
Molecular Devices
0112-0059
9.
Odyssey® CLx Imager
LI-COR®
9140
5.2 Reagents
Reagent
Source ®
Catalog number ®
1.
IGEPAL CA-630
Sigma-Aldrich
2.
AcTEV™ protease
Thermo Scientific 12575015
3.
Boc-Lys(Ac)-AMC
ApexBio
A8713
4.
DMSO
Sigma-Aldrich®
D2650-100ML
5.
SAHA
Cayman Chemical 10009929
6.
Trypsin from porcine pancreas
Sigma-Aldrich®
T4799-5G
7.
293T cells
ATCC®
CRL-11268™
8. 9.
DMEM without L-glutamine Fetal Bovine Serum (FBS)
10. Gibco™ GlutaMAX™ Supplement
Corning
®
Peak Serum
I8896
15-013-CV ®
PS-FB1
Thermo Scientific 35050061
33
Purification and enzymatic assay of class I HDACs
Reagent
Source
Catalog number
11. Gibco™ Opti-MEM™
Thermo Scientific 31985-062
12. Lipofectamine™ LTX Reagent with PLUS™ Reagent
Thermo Scientific 15338100
13. pFN21A HaloTag® CMV Flexi® Vector
Promega
G2821
14. pFC14A HaloTag® CMV Flexi® Vector
Promega
G9651
15. Mammalian Lysis Buffer
Promega
G938A
16. Protease Inhibitor Cocktail
Promega
G652A
17. Magne HaloTag Beads
Promega
G7281
18. Pierce™ BCA Protein Assay Kit
Thermo Scientific 23225
19. HDAC1 Antibody (rabbit polyclonal)
Proteintech®
®
®
20. HDAC2 Antibody (rabbit polyclonal)
®
Proteintech
21. IRDye® 800CW Goat anti-Rabbit IgG LI-COR® Secondary Antibody
10197-1-AP 12922-3-AP 926-32211
5.3 Buffer preparation 5.3.1 Recombinant protein isolation buffers 3.1.1. Prepare Affinity Purification Wash Buffer. To prepare, add 50 μL of IGEPAL® CA-630 to 100 mL TBS (pH 7.4). The final IGEPAL® CA-630 concentration is 0.05%. Chill buffer on ice prior to use. 3.1.2. Prepare AcTEV™ Elution Buffer. To prepare 1 mL, add 935 μL sterile H2O to a microcentrifuge tube. Add 50 μL of 20 AcTEV™ Protease Buffer, 10 μL of 0.1 M DTT, and 5 μL AcTEV™ Protease. Mix gently and store on ice. 100μL will be needed for each protein purification. 5.3.2 HDAC activity assay buffers 3.2.1. Prepare a 30 mM Substrate Stock Solution. Resuspend 50 mg of Boc-Lys(Ac)-AMC in 3.7 mL DMSO. Distribute into 100 μL aliquots. 3.2.2. Prepare 1 mM Substrate Working Solution. Add 100 μL of 30 mM Substrate Stock Solution from step 3.2.1 to 2.9 mL TBS (pH 7.4). Store on ice.
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3.2.3. Prepare 100 mM SAHA Stock Solution. Add 25 mg SAHA to 946 μL DMSO. 3.2.4. Prepare 200 μM SAHA Working Solution. Add 5 μL SAHA Stock Solution from step 3.2.3 to 2.5 mL TBS (pH 7.4). 3.2.5. Prepare Negative Control Solution. Add 5 μL DMSO to 2.5 mL TBS (pH 7.4). 3.2.6. Prepare 50 mg/mL Trypsin Solution. Add 100 mg trypsin to 2 mL TBS (pH 7.4). 5.3.3 Notes Affinity Purification Wash Buffer can be prepared and stored at 4 °C. AcTEV™ Elution Buffer must be made immediately prior to protein isolation. Substrate Stock Solution aliquots can be stored at 20°C. Substrate Working Solution, SAHA Stock Solution, SAHA Working Solution, Negative Control Solution, and Trypsin Solution must be prepared fresh immediately prior to activity assay.
5.4 Cell culture and transfection for recombinant protein production 4.1. Culture 293T cells in DMEM supplemented with GlutaMAX™ and FBS to a final concentration of 10%. 4.2. Seed cells at a density of 1 107 cells per 150 mm culture plate. The total volume of media should be 30 mL. Culture cells for 24 h at 37 °C and 5% CO2. 4.3. Dilute expression vector with Opti-MEM™. For each plate of cells, prepare 7.5 μg of expression vector in 6.6 mL Opti-MEM™ within a 50 mL conical tube. Invert tube to mix. For our examples, we have expressed N- and C-terminally tagged HDAC1 or HDAC2 using pFN21A HaloTag® CMV Flexi® Vector and pFC14A HaloTag® CMV Flexi® Vector derivatives described in Banks et al. (2018). SIN3B isoform 2 (CDS of NM_001297595) was cloned into AsiSI and PmeI sites of pFC14A HaloTag® CMV Flexi® Vector and contains a C-terminal HaloTag®. Note that the amount of expression vector used can be optimized. In our examples, we use less vector DNA than recommended by the manufacturer’s guidelines. 4.4. To the diluted expression vector, add 7.5 μL PLUS™ Reagent. Invert tube to mix. Incubate at room temperature for 5 min. 4.5. Add 50 μL of Lipofectamine® LTX Reagent to the tube. Invert tube to mix. Incubate at room temperature for 30 min. While other transfection reagents can be used, Lipofectamine® LTX Reagent is preferred as it is not necessary to change media after addition of transfection reagent.
Purification and enzymatic assay of class I HDACs
35
4.6. Gently add the DNA:lipid complex to cells and swirl plates. Culture for 36–48 h at 37 °C and 5% CO2. 4.7. Remove media and gently wash cells with 10 mL PBS (pH 7.4). Aspirate PBS and replace with 10 mL fresh PBS. Resuspend cells using a Falcon™ cell scraper and transfer to a 15 mL conical tube. 4.8. Centrifuge cells for 10 min at 500g and 4 °C. Remove supernatant and store cells on ice until protein isolation is performed. Alternately, cell pellets can be frozen and stored at 80 °C. Continue to Section 5.5.
5.5 Recombinant protein isolation 5.1. Thaw cells on ice and resuspend in 300 μL chilled Mammalian Lysis Buffer containing a protease inhibitor cocktail. Pass through a PrecisionGlide™ needle 5–10 times. Centrifuge at 18,000 g for 10 min at 4 °C in a tabletop centrifuge. Protease inhibitor cocktails that contain AEBSF should be avoided when HaloTag® is used for protein purification as this molecule interferes with capture of the HaloTag®. 5.2. Transfer supernatant to a clean microcentrifuge tube containing 700 μL chilled PBS. Invert tube to mix. Centrifuge at 18,000g for 10 min at 4 °C in a tabletop centrifuge. 5.3. Transfer supernatant to a clean microcentrifuge tube on ice. Leave the final 10–20 μL of supernatant to avoid disturbing pellet. 5.4. Quantify protein using a Pierce™ BCA Protein Assay Kit and BSA protein standards. 5.5. Transfer 2 mg supernatant to a clean microcentrifuge tube containing pre-washed 100 μL Magne® HaloTag® Beads. Incubate with gentle rotation for 2 h at 4 °C. 5.6. Place tubes on DynaMag™-2 Magnet and remove supernatant. Add 1 mL chilled Affinity Purification Wash Buffer and invert to mix. 5.7. Place tubes on DynaMag™-2 Magnet and remove supernatant. 5.8. Perform steps 5.6–5.7 two additional times for a total of three washes. 5.9. Add 1 mL chilled TBS and invert to mix. Place tubes on DynaMag™-2 Magnet and remove supernatant. 5.10. Resuspend in 100μL AcTEV™ Elution Buffer and rotate overnight at 4°C. 5.11. Place tubes on DynaMag™-2 Magnet and transfer supernatant to a microcentrifuge tube containing a Micro Bio-Spin® column on ice. 5.12. Centrifuge at 500 g for 1 min at 4 °C. The flow through contains the purified protein. 5.13. Snap freeze purified protein in liquid N2 and store at 80°C. Alternatively, proceed immediately to HDAC activity assay.
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Mark K. Adams et al.
5.6 HDAC activity assay 6.1. Refer to Section 5.3.2 for buffer preparation. 6.2. Dilute 2.5 to 10 μL purified protein with TBS (pH 7.4). The final volume should be equal to 42.5 μL for each reaction assay. 6.3. To each well of a 384-well black microplate that will be used during the activity assay, add the 42.5 μL of diluted protein sample. 6.4. Add 2.5 μL Negative Control Solution or 200μM SAHA Working Stock Solution to each well. For this and all remaining steps, it is suggested that a multi-channel pipette be used to avoid pipetting error. The addition of SAHA (or another HDACi) at this step allows for the determination of enzyme response to HDAC inhibitors. Different HDACis, such as those found in Table 1, can be analyzed and assays can be performed using a range of HDACi concentrations. In the provided example, SAHA is used at a concentration of 10μM. The Negative Control Solution accounts for the DMSO found within the HDACi solution and should be added to wells in which no HDACi is present. 6.5. Add 5 μL Substrate Working Solution to each well. The total volume in each well after addition of substrate should be 50μL. Cover plate and invert to mix. Centrifuge plate at 200 g for 1 min. Incubate 1 h at 37 °C with gentle rotation. 6.6. Stop the reaction with the addition of a pan-HDAC inhibitor. Add 2.5 μL 200 μM SAHA working stock solution to each well. Incubate 5 min at 37 °C with gentle rotation. In the provided example, SAHA is used to stop the enzymatic reaction as it is known that Sin3 complexes are responsive to SAHA. Other pan-HDAC inhibitors, such as trichostatin A, can also be used to stop the enzymatic reaction. 6.7. Hydrolyze the deacetylated substrate and release AMC. Add 6μL Trypsin Solution to each well. Incubate 1 h at 37°C with gentle rotation. During this step, trypsin digests the substrate which releases AMC. Digestion only occurs if acetyl groups have been removed from the substrate by the analyzed enzyme. 6.8. Measure fluorescence on a SpectraMax® Gemini™ XS or comparable instrument. During data acquisition, use an excitation wavelength of 355 nm and an emission wavelength of 460 nm.
6. Analysis Examination of enzyme activities and inhibition is as simple as reviewing relative fluorescent units (RFUs) for each reaction (Fig. 2B, D).
Purification and enzymatic assay of class I HDACs
37
Biological replicates should be examined for each sample and a statistical analysis, such as an unpaired t-test (Fig. 2B), can be performed to determine the significance of differences in HDAC activity between treatment groups. If comparing RFU values between protein samples, it is important that values be normalized to account for variations in protein abundance between samples. If the molecular weight and amount of protein in each sample is known, then activities can be normalized to protein concentration. However, contaminating proteins, such as TEV protease introduced during protein elution, may prevent the accurate measure of recombinant protein concentration. Additionally, proteins concentrations are often below the detection limits of commercially available protein quantification kits. In such instances, concentrations of proteins possessing affinity tags can be estimated using intensity values of proteins on Western blots (Fig. 2C and D).
7. Summary As HDACs continue to be studied as potential targets of chemotherapeutic agents, tools and systems must be established for the adequate characterization of these enzymes. We describe a straight-forward HDAC activity assay that utilizes commercially available materials. While we demonstrate an approach based on the recombinant expression of HaloTagged proteins, the described HDAC activity assay system is flexible and can be used with other affinity purification systems as well as with endogenous protein. This system can be used for the direct analysis of purified HDAC enzymes (Fig. 2C and D) as well as non-enzymatic subunits of HDAC complexes (Fig. 2B). Thus, the catalytic properties of specific HDAC complexes can be examined and their responsiveness to HDACis can be assessed.
Acknowledgments Research reported in this publication was supported by the Stowers Institute for Medical Research and the National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers F32GM122215 (M.K.A.) and R01GM112639 (M.P.W.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Original data underlying this manuscript can be accessed from the Stowers Original Data Repository at https:// www.stowers.org/research/publications/libpb-1444.
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