Chapter Two - Purification and enzymatic assay of class I histone deacetylase enzymes
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 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.
Section snippets
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
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 to produce enzymatically active HDACs. The analysis of endogenous and
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
Equipment
Empty Cell Equipment Source Catalog number 1. 150 mm plates TPP® 93150 2. Falcon™ Cell Scrapers Corning® 353089 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
Reagents
Empty Cell Reagent Source Catalog number 1. IGEPAL® CA-630
Analysis
Examination of enzyme activities and inhibition is as simple as reviewing relative fluorescent units (RFUs) for each reaction (Fig. 2B, D). 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
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
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
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Mechanism of assembly, activation and lysine selection by the SIN3B histone deacetylase complex
2023, Nature Communications
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Current address: Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Hamburg, Germany.