Chapter Two - Purification and enzymatic assay of class I histone deacetylase enzymes

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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.

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 CellEquipmentSourceCatalog number
1.150 mm platesTPP®93150
2.Falcon™ Cell ScrapersCorning®353089
3.Microcentrifuge TubesVWR™87003-294
4.PrecisionGlide™ NeedleBecton, Dickinson and Company305110
5.DynaMag™-2 MagnetThermo Scientific12321D
6.Micro Bio-Spin® ColumnsBio-Rad Laboratories, Inc.7326204
7.384-well microplate, BlackGreiner Bio-One781097
8.SpectraMax® Gemini™ XSMolecular Devices0112-0059
9.Odyssey® CLx ImagerLI-COR®9140

Reagents

Empty CellReagentSourceCatalog 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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  • Cited by (2)

    • Differential Complex Formation via Paralogs in the Human Sin3 Protein Interaction Network

      2020, Molecular and Cellular Proteomics
      Citation Excerpt :

      To identify enriched proteins over negative AP controls, Z-statistic values of ≥ 3 and log2 fold change values ≥ 2 were selected as filter values. HDAC activity assays of transiently produced proteins were performed as described (29). Briefly, ∼1 × 107 293T cells were plated in 150 mm dishes and cultured in 25 ml DMEM + 10% fetal bovine serum + 1 × GlutaMAX Supplement.

    Current address: Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg, Hamburg, Germany.

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