1. Biochemistry and Chemical Biology
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Diverse nucleosome Site-Selectivity among histone deacetylase complexes

  1. Zhipeng A Wang
  2. Christopher J Millard
  3. Chia-Liang Lin
  4. Jennifer E Gurnett
  5. Mingxuan Wu
  6. Kwangwoon Lee
  7. Louise Fairall
  8. John WR Schwabe  Is a corresponding author
  9. Philip A Cole  Is a corresponding author
  1. Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, United States
  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, United States
  3. Leicester Institute of Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, United Kingdom
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Cite this article as: eLife 2020;9:e57663 doi: 10.7554/eLife.57663

Abstract

Histone acetylation regulates chromatin structure and gene expression and is removed by histone deacetylases (HDACs). HDACs are commonly found in various protein complexes to confer distinct cellular functions, but how the multi-subunit complexes influence deacetylase activities and site-selectivities in chromatin is poorly understood. Previously we reported the results of studies on the HDAC1 containing CoREST complex and acetylated nucleosome substrates which revealed a notable preference for deacetylation of histone H3 acetyl-Lys9 vs. acetyl-Lys14 (Wu et al, 2018). Here we analyze the enzymatic properties of five class I HDAC complexes: CoREST, NuRD, Sin3B, MiDAC and SMRT with site-specific acetylated nucleosome substrates. Our results demonstrate that these HDAC complexes show a wide variety of deacetylase rates in a site-selective manner. A Gly13 in the histone H3 tail is responsible for a sharp reduction in deacetylase activity of the CoREST complex for H3K14ac. These studies provide a framework for connecting enzymatic and biological functions of specific HDAC complexes.

Introduction

Reversible histone acetylation (Cole, 2008) regulates a range of major fundamental biological processes including gene expression, DNA replication, cell growth, and differentiation (Jamaladdin et al., 2014). Histone acetyltransferases (HATs) catalyze Lys sidechain acetylation on the N-terminal tails of each of the four core histones (Weinert et al., 2018), H2A, H2B, H3, and H4 that comprise nucleosomes, the building blocks of chromatin (Helin and Dhanak, 2013Figure 1A). Enzymological and cellular analysis has shown that particular HATs such as p300/CBP, PCAF/GCN5, and Myst family members display varying degrees of site selectivity in the context of histone tail peptides and/or chromatin (Shortt et al., 2017). For example, p300/CBP is best known to acetylate Lys18 and Lys27 in histone H3 (Dancy and Cole, 2015), whereas PCAF/GCN5 prefers to acetylate Lys14 and Lys9 of histone H3 (Ali et al., 2018). Histone deacetylases (HDACs) remove acetyl-Lys modifications in chromatin and other proteins and belong to two major families in humans (Taunton et al., 1996), classical HDACs which include 11 Zn metallohydrolase enzymes and seven sirtuins (Wang et al., 2017), that catalyze NAD-dependent Lys deacetylation (Falkenberg and Johnstone, 2014). The class I HDACs, HDAC1, HDAC2, and HDAC3 are relatively similar at the amino acid sequence level and show overlapping pharmacological sensitivities to the benzamide class of HDAC inhibitors represented by entinostat (Yang et al., 2019).

Histone H3 tail acetylation and HDAC complexes.

(A) Different acetylation sites on H3 N-terminal tail studied in this manuscript; (B) Components of four well- established HDAC1 complexes CoREST (LSD1, HDAC1, CoREST1), NuRD (MTA1, HDAC1, RBBP4), Sin3B (Sin3, HDAC1, RBBP4), MiDAC (MIDEAS, HDAC1, DNTTP1), and one HDAC3 complex SMRT (GPS2-NCoR2 chimera, HDAC3, and TBL1 ).

HDAC1 and HDAC2 (Kelly and Cowley, 2013) are the most similar HDACs and are interchangeably found in a variety of multisubunit protein complexes including CoREST, Sin3(A/B), NuRD, and MiDAC whereas HDAC3 is commonly associated with the SMRT/NCoR complex (Bantscheff et al., 2011Figure 1B). The CoREST (Humphrey et al., 2001), Sin3 (Laherty et al., 1997), NuRD (Xue et al., 1998), (Zhang et al., 1999), and SMRT (Guenther et al., 2000), (Wen et al., 2000), (Li et al., 2000) complexes are generally believed to be involved in silencing of gene expression and are recruited by various transcription factors to chromatin in a cell type and cell state dependent manner (Ooi and Wood, 2007). Less is known about the MiDAC complex although it has been associated with mitotic processes (Bantscheff et al., 2011). Recent structural studies on each of these five HDAC complexes have provided insights into their distinct molecular architectures (Millard et al., 2017) (CoREST (Song et al., 2020), NuRD (Millard et al., 2016), Sin3A (Clark et al., 2015), MiDAC (Itoh et al., 2015), and SMRT (Watson et al., 2012a) and inositol phosphates have been shown to have varying roles in regulating their stabilities and catalytic activities (Millard et al., 2013).

Histone substrate site-selectivity experiments with HDAC enzymes have generally involved analyzing the isolated HDAC polypeptides or catalytic domains and acetyl-Lys containing histone peptide tails rather than the more physiological HDAC complexes and nucleosome substrates (Zhang et al., 2016), (Riester et al., 2007), (Gurard-Levin et al., 2009). Such studies have typically shown little to no amino acid sequence site-selectivities for HDACs. As the physiological enzyme forms of HDAC1-3 exist in multisubunit complexes (Millard et al., 2017) and the natural substrates are chromatin comprised of nucleosomes, histone octamers wrapped by 146 bp of double stranded DNA, the biological relevance of the free HDAC and histone tail substrate peptide experiments is unclear. Recent studies on the CoREST complex and acetylated nucleosome substrates revealed a notable preference for deacetylation of histone H3 acetyl-Lys9 vs. acetyl-Lys14 (Wu et al., 2018). This was especially interesting because the CoREST complex also includes the enzyme LSD1 which specifically demethylates methyl-Lys4 in histone H3 and this activity is strongly inhibited when the H3 tail also contains acetyl-Lys14 but not acetyl-Lys9 (Kalin et al., 2018). These properties suggest a biological role in histone H3 marked by Lys14 acetylation and Lys4 methylation to protect against CoREST complex silencing. The CoREST complex enzymatic studies also raise the possibility that different HDAC complexes may show distinct deacetylase kinetics and nucleosome site selectivities.

In this study, we evaluate the catalytic actions of five purified class I HDAC complexes CoREST, MiDAC, NuRD, Sin3B, and SMRT with five different histone H3 acetylated mononucleosomes as well as the corresponding free histone H3 substrates. We find that the kinetics of the HDAC complexes across the range of free acetylated histone H3 substrates are fairly similar, whereas there are large differences between the complexes with respect to deacetylase rates and site-selectivity with nucleosome substrates. Among these complexes, the CoREST complex shows a special resistance to H3K14ac which we find is largely driven by the preceding Gly13 residue.

Results

Production of H3 acetylated nucleosomes and HDAC complexes

Semisynthetic X. laevis histone H3 proteins (Wang et al., 2015) mono-acetylated at positions Lys9, Lys14, Lys18, Lys23, and Lys27 were prepared using F40 sortase (Piotukh et al., 2011). In this approach, the N-terminal tails aa1-34 were prepared as synthetic peptides containing the acetyl-Lys and terminating in a depsipeptide linkage between Thr and Gly and the H3 globular domain aa34-135 was produced recombinantly. F40 sortase treatment of the H3 peptide and H3 globular domain catalyzes transpeptidation leading to ligation of the fragments to produce pure, scarless full-length modified histone H3s (Figure 2A–D and Figure 2—figure supplements 12). Western blot analysis with the site-specific relevant acetyl-Lys antibodies demonstrated that each of the semisynthetic histone H3s contained the designated marks (Figure 3—figure supplement 1 and Wu et al., 2018). The semisynthetic acetylated H3s were incorporated into mononucleosomes containing 146 bp DNA 601 Widom sequence (Luger et al., 1997Figure 2—figure supplement 3A–B). The HDAC complexes CoREST (LSD1, HDAC1, CoREST1), NuRD (MTA1, HDAC1, RBBP4), Sin3B (Sin3, HDAC1, RBBP4), MiDAC (MIDEAS, HDAC1, DNTTIP1), and SMRT (GPS2-NCOR2 chimera, HDAC3, and TBL1) were expressed in HEK293F cells by transient transfection of three plasmids encoding the relevant proteins (Figure 3—figure supplement 2A). The details of how these complexes have been arrived at and are produced have been described previously (Song et al., 2020), (Millard et al., 2016), (Clark et al., 2015), (Itoh et al., 2015), (Watson et al., 2012a), (Zhang et al., 2018), (Watson et al., 2012b), (Portolano et al., 2014). In general, the two non-HDAC proteins in each case were selected based on the following criteria: 1) A set of proteins that included both well-established HDAC and nucleosome binding partners for a given complex, 2) Efficient transient co-expression of soluble proteins in HEK293F that stay associated by immunoaffinity chromatography and lead to relatively pure and concentrated complexes in peak fractions (>50% purity), 3) The ability of the core complexes to be reproducibly isolated as monodisperse peaks in appropriate stoichiometries after size exclusion chromatography. The HDAC complexes employed here were shown to be relatively pure and in the expected stoichiometries by SDS-PAGE (Figure 3—figure supplement 2B).

Figure 2 with 3 supplements see all
The semi-synthesis of full-length histone H3 with site-specific acetylations.

(A) Synthesis of H3 proteins with site-specific acetylations; gH3: globular region of histone H3; (B) MALDI-MS for a semi-synthetic histone H3 product, using H3K27ac as an example, *: an unknown minor impurity; (C) SDS-PAGE of all the H3 proteins with acetylations, as H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K9acR8G, H3K14acG13R; (D) Native 6% TBE gel of the nucleosome folding results with acetylated H3s, each showing ≥95% purity. #: minor free DNA band.

Table 1
Rate constants of different HDAC1/HDAC3 complexes on various histone proteins with site-specific acetylation.

Kinetic values shown are ± S.E.M. For each enzymatic reaction on histone proteins: the semi-synthetic H3K9/14/18/23/27ac histone H3 proteins (1.0 μM) were incubated with different HDAC1 and HDAC3 complexes at the following concentrations (2 nM CoREST, 10 nM MiDAC, 5 nM NuRD, 4 nM Sin3B, 2 nM SMRT, and 5 nM HDAC1, n = 2–5) under a reaction buffer of 50 mM HEPES 7.5 containing 100 mM KCl, 100 μM IP6, and 0.2 mg/mL BSA at 37 °C. The reaction time was counted, and multiple samples were taken at different time points between 0–30 min. The complexes include CoREST, NuRD, Sin3B, MiDAC, and SMRT, together with free HDAC1 enzyme. The kinetic data were divided by a factor of 5 in order to be comparable with the kinetic data on nucleosomes.

Normalized
V/[E] (min−1)
HDAC1CoREST(RCOR1)MiDAC(MiDEAS)NuRD(MTA1)Sin3B(Sin3)SMRT(NCoR2)
H3K9ac0.30 ± 0.0462.1 ± 0.380.42 ± 0.0860.84 ± 0.380.56 ± 0.150.44 ± 0.10
H3K14ac0.65 ± 0.402.8 ± 0.180.79 ± 0.291.5 ± 0.581.1 ± 0.0930.78 ± 0.34
H3K18ac1.1 ± 0.271.8 ± 0.370.45 ± 0.290.86 ± 0.321.2 ± 0.241.5 ± 0.57
H3K23ac0.64 ± 0.102.6 ± 0.550.22 ± 0.0370.36 ± 0.120.93 ± 0.200.65 ± 0.32
H3K27ac1.1 ± 0.252.0 ± 0.350.43 ± 0.0381.5 ± 0.481.1 ± 0.311.8 ± 0.80

Deacetylase assays with HDAC complexes

Each of the HDAC complexes was assayed with isolated acetylated histone H3 proteins or acetylated nucleosomes as substrates, using Western blot to monitor the disappearance of the requisite acetyl-Lys modification. The five HDAC complexes displayed robust catalytic activities with isolated acetylated H3 substrates with velocities (V/E) averaging in the range of 0.5–2 min−1 and modest differences (largely <3 fold) in acetyl-Lys site-selectivity among K9ac, K14ac, K18ac, K23ac, and K27ac. These HDAC complexes show similar deacetylase activities as the isolated commercial HDAC1 (averaging ~0.8 min−1) with free histone H3 substrates. These results are generally consistent with the prior studies that have indicated that local amino acid context within peptide substrates does not have a large impact on HDAC-catalyzed deacetylation kinetics (Table 1, Figure 4—figure supplements 16A–E,G).

In contrast to the results with isolated H3 substrates, the results with acetylated nucleosome substrates showed dramatic differences among the HDAC complexes. The HDAC complex velocities (V/E) with acetylated nucleosomes varied over 200-fold, ranging from <0.005 min−1 to 1.2 min−1. The MiDAC complex showed the fastest velocities with V/E up to 1.2 min−1 whereas the NuRD and Sin3B complexes showed V/E ~ 0.007 min−1. The CoREST and SMRT complexes were intermediate with V/E values averaging in the range of 0.03–0.1 min−1 (Figure 3).

Figure 3 with 2 supplements see all
Typical results for Western-blot based deacetylation activity assay using different complexes on nucleosome with H3K9ac (n = 2 for each assay).

Among the five HDAC complexes, there were distinct substrate selectivity features that are notable. CoREST showed similar velocities (V/E 0.06–0.1 min−1) for K9ac, K18ac, K23ac, and K27ac that were markedly greater than the V/E for K14ac (<0.005 min−1). In contrast, the MiDAC complex velocity for deacetylating K9ac (V/E 1.2 min−1) was only about 2-fold greater than for K14ac (V/E 0.52 min−1). However, the MiDAC complex deacetylated K23ac at a much lower rate (V/E 0.048 min−1) relative to K9ac removal and in general, MiDAC deacetylase activity decreased when comparing more C-terminal acetyl-Lys sites in H3 with more N-terminal locations (Figure 4A–F). The one HDAC3 complex investigated here, SMRT, showed the highest rates of deacetylase activities on K9ac and K27ac and in general had more similar rates across the five sites compared with CoREST and MiDAC. The activities of NuRD and Sin3B displayed very low similar deacetylase activity at every H3 acetyl-Lys site (Table 2, Figure 4—figure supplements 16A–F).

Figure 4 with 6 supplements see all
Typical results for Western-blot based deacetylation activity assay with 5 nM MiDAC on different nucleosomes with site-specific acetylation.

(A) nucleosome with H3K9ac, n = 2, V/E = 1.2 ± 0.19 min−1; (B) nucleosome with H3K14ac, n = 3, V/E = 0.52 ± 0.034 min−1; (C) nucleosome with H3K18ac, n = 2, V/E = 0.17 ± 0.0051 min−1; (D) nucleosome with H3K23ac, n = 4, V/E = 0.048 ± 0.0071 min−1; (E) nucleosome with H3K27ac, n = 5, V/E = 0.18 ± 0.036 min−1, and (F) the curve fitting for kinetics analysis.

Table 2
Rate constants of different HDAC1 and HDAC3 complexes on various nucleosomes with site-specific acetylation on H3.

Kinetic values shown are ± S.E.M. For each enzymatic reaction on nucleosomes: the semi-synthetic H3K9/14/18/23/27ac nucleosomes (100 nM) were incubated with different HDAC1 and HDAC3 complexes at the following concentrations (30 nM CoREST, 5 nM MiDAC, 80 nM NuRD, 45 nM Sin3B, 60 nM SMRT, and 60 nM HDAC1, n = 2–5), under a reaction buffer of 50 mM HEPES 7.5 containing 100 mM KCl, 100 μM IP6, and 0.2 mg/mL BSA at 37 °C. The reaction time was counted, and multiple samples were taken at different time points generally between 0–120 min (The MiDAC complex on H3K9ac was between 0–90 min). The complexes include CoREST, NuRD, Sin3B, MiDAC, and SMRT, together with free HDAC1 enzyme.

V/[E] (min−1)HDAC1CoREST(RCOR1)MiDAC(MiDEAS)NuRD(MTA1)Sin3B(Sin3)SMRT(NCoR2)
H3K9acN.D.0.10 ± 0.0121.2 ± 0.190.012 ± 0.00320.0050 ± 0.00430.046 ± 0.0045
H3K14acN.D.<0.0050.52 ± 0.0340.0091 ± 0.00080<0.0050.019 ± 0.0022
H3K18acN.D.0.062 ± 0.00190.17 ± 0.00510.0046 ± 0.000880.0082 ± 0.00300.021 ± 0.0039
H3K23acN.D.0.067 ± 0.0100.048 ± 0.00710.0071 ± 0.000830.0084 ± 0.00330.0081 ± 0.0028
H3K27acN.D.0.093 ± 0.00880.18 ± 0.0360.0077 ± 0.00280.0089 ± 0.000530.039 ± 0.0027

CoREST and MiDAC analysis on mutant acetylated nucleosomes

Three out of the five acetyl-Lys positions in the H3 tail examined here contain an Arg directly preceding the Lys (K9, K18, and K27) whereas Lys14 is preceded by Gly, the smallest amino acid. Given that among the five HDAC complexes studied here, K14ac cleavage from nucleosomes is in all cases slower than K9ac (most dramatically with the CoREST complex) we swapped amino acid residues at the 8 and 13 positions in H3. That is, we prepared K9ac nucleosomes containing R8G and K14ac nucleosomes bearing G13R (Figure 5, Figure 5—figure supplements 1 and 23A–D). Fortunately, the corresponding K9ac and K14ac selective antibodies were still able to recognize these mutant nucleosomes and could be used in HDAC assays. We performed deacetylase assays on these nucleosomes using the two most robust nucleosome HDAC complexes in our hands, the CoREST complex and MiDAC complex. The kinetic measurements with the CoREST complex showed striking differences relative to WT acetylated nucleosomes. The K9acR8G nucleosomes displayed a ~ 3 fold deacetylase rate reduction compared with WT K9ac (K9acR8G V/E 0.029 min−1 vs WT K9ac V/E 0.1 min−1). Remarkably, K14acG13R nucleosomes showed a > 17 fold increase in deacetylase rate vs. WT K14ac (K14acG13R V/E 0.084 min−1 vs. WT K14ac V/E < 0.005 min−1). The near matching of V/E deacetylase values of the CoREST complex on K14acG13R and WT K9ac nucleosomes suggests a profound rejection of a Gly at the H3 13th position in nucleosomes by the CoREST complex although it is more tolerated at the 8 position of H3. The kinetic studies with the MiDAC complex showed much less sensitivity to changes in Arg8 and Gly13. In fact, K9acR8G nucleosome deacetylation by the MiDAC complex matched that of WT K9ac nucleosomes (V/E 1.2 min−1 for both). K14acG13R nucleosome deacetylation by MiDAC was accelerated by 2.5 fold relative to WT K14ac nuclesomes, indicating a minor preference for Arg at the H3 13th position.

Figure 5 with 3 supplements see all
Scheme and kinetics for CoREST and MiDAC complex activity on nucleosome with mutations H3K9acR8G (n = 3 for CoREST assay, n = 5 for MiDAC assay) and 14acG13R (n = 6 for CoREST assay, n = 3 for MiDAC assay).

(A) The sequence of H3K9acR8G comparing with WT-H3K9ac; (B) The sequence of H3K14acG13R comparing with WT-H3K14ac; (C) Table for the rate constants of different complexes on nucleosomes with H3K9ac/9acR8G/14ac/14acG13R. The reaction conditions were similar to the enzymatic reactions on H3K9/14/18/23/27ac nucleosomes described in Table 1.

NuRD (HDAC1-MTA1-RBBP4) deacetylation of H4K16ac nucleosomes

The very slow rates of nucleosome H3 deacetylation by the RBBP4 containing NuRD and Sin3B complexes prompted us to consider that the scaffold protein RBBP4 might be impeding the catalytic activity by obstructing the H3 tails (Millard et al., 2016). We thus examined whether a nucleosome containing acetylation on histone H4 might be an improved substrate for an RBBP4 complex. However, the deacetylation rate (V/E) for NuRD was low and similar to those of the acetylated H3 nucleosomes (Figure 6, Figure 6—figure supplement 1A,B). These results suggest that the low nucleosome deacetylase activity of RBBP4-containing HDAC complexes may be unrelated to RBBP4/H3 interaction.

Figure 6 with 1 supplement see all
Western-blot and kinetics of NuRD complex activity on nucleosome with H4K16ac.

(A) Western-blot based deacetylation activity assay with 40 nM NuRD complex (HDAC1-MTA1-RBBP4) on H4K16ac nucleosomes blot with site-specific acetylation antibody and anti-H3 antibody to show the overall nucleosome amount remaining unchanged (n = 3); (B) rate constant of NuRD complex deacetylation on H4K16ac nucleosome. The reaction conditions were similar to the enzymatic reactions on H3K9/14/18/23/27ac nucleosomes described in Table 1.

Discussion

A principal finding of this study is that distinct HDAC1 complexes can show sharply different deacetylase activities toward different nucleosome substrates. This would appear to suggest that the unique biological consequences of the various HDAC1 complexes are not simply dependent on where they are recruited to in chromatin and by which particular transcription factor (Watson et al., 2012b). Rather, the different catalytic properties and site-selectivities of the HDAC1 complexes likely drive different patterns of histone deacetylation and chromatin states. Although it is possible that the closely related paralog HDAC2 replaces HDAC1 in some population of the HDAC complexes analyzed here, because of the massive overexpression of HDAC1 we believe this to be a very minor pool. Prior published structural studies of these complexes and mass spectroscopic analyses are also consistent with this idea (Song et al., 2020), (Millard et al., 2016; Itoh et al., 2015; Millard et al., 2013; Zhang et al., 2018), (Portolano et al., 2014). Furthermore, because HDAC1 and HDAC2 have very high sequence homology, we believe it is likely that HDAC1 and HDAC2 will behave in similar ways in terms of the nucleosome deacetylation assays carried out here.

The data with the CoREST complex selectivity stand out for the strong resistance to one of the five H3 tail acetylation sites, H3K14ac, investigated (Wu et al., 2018). It is likely not a coincidence that K14ac blocks the removal of H3K4 methylation as this could allow doubly modified H3K4me1/2 and H3K14ac tails to be immune from the silencing action of the CoREST complex. H3K4 methylation is known to be commonly found in the context of enhancer elements in DNA (Hsu et al., 2018), which is also associated with H3K14ac (Karmodiya et al., 2012), possibly insulating such K4 methylation by CoREST. Although this has not been rigorously evaluated with JMJ family demethylases, a yeast homolog JHD2 has been shown to be more weakly able to demethylate H3K4me3 in the context of H3K14ac (Maltby et al., 2012).

The Gly/Arg swaps at positions 8 and 13 in histone H3 clearly demonstrate that the presence of Gly13 rather than an Arg residue is a principal determinant of the meager rate of CoREST-mediated deacetylation of K14ac in nucleosome substrates. However, each of the five class I HDAC complexes examined here were relatively ineffective at deacetylating K14ac in the context of nucleosomes presumably at least partly due to Gly13. That this K14ac deselection is not true in isolated H3 indicates that the molecular recognition of Lys14ac by these HDAC complexes is altered when the H3 tail is presented to the HDAC1 active site in a chromatin context. We are unsure if this represents kcat or KM differences conferred by histone H3 Gly13 vs. Arg13 because of the challenges of making such measurements with nucleosome substrates. We speculate that these effects may not be reflected in overall affinities between the various HDAC complexes and particular acetylated nucleosomes, but rather are driven by subtle local differences between the HDAC complex active sites and the nucleosomal substrates. The cellular relevance of these findings is supported by the fact that H3K14ac is a relatively abundant mark at baseline (Sidoli and Garcia, 2017) and is rather unaffected by class I HDAC inhibitor treatment compared with H3K9ac and H3K27ac which show substantial increases with such inhibitors (Schölz et al., 2015). It is also possible that robust histone acetyltransferase targeting H3K14 contributes to the elevated H3K14 acetylation levels in cells.

The MiDAC complex showed far and away the speediest average deacetylation rate across the five acetyl-Lys marks looked at in nucleosomes. Impressively, MiDAC deacetylated K9ac nucleosomes (V/E 1.2 min−1) more rapidly than K9ac in free H3 histones. Unlike CoREST, NuRD, Sin3B, and SMRT which are thought to be transcriptional silencers and recruited to chromatin by transcription factors, MiDAC is not known to be employed in this way. Furthermore, its ground state affinity to chromatin is thought to be quite high (Itoh et al., 2015). It is interesting that there was a modest trend for the rate of deacetylation to be highest closest to the N-terminus of H3 in the context of nucleosome substrates. We speculate that this may represent a greater accessibility of the extreme N-terminus of the H3 tail in chromatin to the HDAC1 active site in this complex.

In contrast to MiDAC, the NuRD and Sin3B complexes showed the lowest deacetylation rates with H3 acetylated nucleosomes. There are several possible reasons for this. It is plausible the NuRD and Sin3B may have a more extreme requirement for recruitment to nucleosomes by a transcription factor compared with the other HDAC complexes looked at here (Zhang et al., 2018), (Walsh, 2006). This could offer the advantage of preventing gene silencing except when triggered for by a cellular stimulus (Millard et al., 2017). In addition, there could be an additional macromolecular component or post-translational modification (Wang, 2019) lacking in our system that would unleash the deacetylase activity or alter substrate selectivities of any of the HDAC complexes on nucleosomes (Hudson et al., 2015). Indeed, NuRD and the other HDAC complexes investigated here have been reported to have a variety of additional protein subunits depending on cell type and cell state (Millard et al., 2016), (Lai and Wade, 2011), (Yang and Seto, 2008). For the NuRD complex, however, it is established that the chromatin remodelling module is rather loosely associated, and we do not expect that its absence would greatly influence HDAC activity. In addition to roles for transcription factors and additional subunits, the NuRD or Sin3B complexes could show very high site-selectivity on one or more sites in histones beyond the acetylated-Lys that we have examined thus far in H3 or H4. Future studies will be needed to determine if one or more of these possibilities is operative.

We have focused on classical HDAC acetyl-Lys selectivities in chromatin. However, there is reason to believe that the NAD-dependent sirtuins may also show site-selectivities. Recent studies on Sirt7 have shown that this enzyme shows a strong preference for H3K36ac in the context of chromatin (Wang et al., 2019). It will be important in future experiments to extend the analysis of deacetylase actions across the full complement of the HDAC enzymes to create a deeper understanding of the connections between reversible acetylation in health and disease states.

Materials and methods

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Gene (X. laevis)Histone H2A, H2B, H4 (X. laevis)DOI: 10.7554/eLife.37231Construct shared by Cynthia Wolberger (Johns Hopkins)
Gene (X. laevis)Histone gH3 (X. laevis)DOI: 10.7554/eLife.37231
DOI: 10.1021/ja205630g
Construct shared by Dirk Schwarzer (Tübingen)
Gene (S. aureus)F40-sortase (Originally mutated fromS. aureus sortase A)DOI: 10.7554/eLife.37231
DOI: 10.1021/ja205630g
Construct shared by Dirk Schwarzer (Tübingen)
Gene (E. coli)146 bp 601 DNA (Widom sequence forE. coliexpression)DOI: 10.7554/eLife.37231Construct shared by Song Tan (Penn State)
Strain, strain background (E. coli)BL21 Rosetta (DE3) pLysS (E. coli)Sigma-AldrichCat# 70956–4Competent cells
Cell line (Homo-sapiens)HEK293F (Homo-sapiens)Thermo Fisher ScientificRRID:CVCL_6642Transfected with plasmids to express different HDAC1 complexes. Mycoplasma tested negative. Authenticated by the venfor. Not on ICLAC misidentified cell line registry.
AntibodyAnti-H3K9ac (Rabbit)Cell SignalingCat# 9671, RRID:AB_331532WB (1:2000)
AntibodyAnti-H3K14ac (Rabbit)EMP MilliporeCat# 07–353, RRID:AB_310545WB (1:2000)
AntibodyAnti-H3K18ac (Rabbit)EMP MilliporeCat# 07–354, RRID:AB_ 441945WB (1:2000)
AntibodyAnti-H3K23ac (Rabbit)EMP MilliporeCat# 07–355, RRID:AB_310546WB (1:2000)
AntibodyAnti-H3K27ac (Rabbit)Cell SignalingCat# 8173, RRID:AB_10949503WB (1:2000)
AntibodyAnti-H3 (Rabbit)AbcamCat# ab1791, RRID:AB_302613WB (1:5000)
AntibodyAnti-H4K16ac (Rabbit)Active MotifCat# 39167, RRID:AB_2636968WB (1:5000)
AntibodyHRP conjugated anti-Rabbit secondary (Goat)Cell SignalingTechnology Cat# 7074, RRID:AB_2099233WB (1:2000)
Peptide, recombinant proteinHDAC1/HSP70BPS BioscienceCat# 50051
Peptide, recombinant proteinNucleosome with H4K16acEpicypherCat# 16–0354
Chemical compound20 Fmoc-amino acidsEMP Millipore
DOI: 10.7554/eLife.37231
Fmoc-Gly-OH as example: Cat# 852001
Software, algorithmImageJDOI: 10.7554/eLife.37231
Download fromimagej.nih.gov/ij/
RRID:SCR_003070
Software, algorithmGraphPad Prism EightDOI: 10.7554/eLife.37231
Download fromwww.graphpad.com
RRID:SCR_002798

Reagents

20 amino acids were purchased from EMP Millipore. Site specific primary antibodies include anti-H3K9ac (Cell Signaling), anti-H3K14ac (EMP Millipore), anti-H3K18ac (EMP Millipore), anti-H3K23ac (EMP Millipore), anti-H3K27ac (Cell Signaling), anti-H4K16ac (Active Motif) antibodies, as well as anti-histone H3 (Abcam). Secondary antibody as HRP-conjugated anti-rabbit antibody from Cell Signaling. H4K16ac nucleosome was purchased from Epicypher. The free enzyme HDAC1 (in the complex with HSP70) is purchased from BPS Bioscience.

The synthesis of depsipeptide Fmoc-Thr(OtBu)-glycolic acid (TOG)

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The depsipeptide Fmoc-Thr(OtBu)-glycolic acid was synthesized based on a reported two-step protocol (Williamson et al., 2012).

Peptide synthesis

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All the histone H3 peptides 1–34 containing site-specific lysine acetylation (Kac) were synthesized by Fmoc-SPPS by Fmoc-Gly-Wang resin. For 0.2 mmol resin, 0.8 mmol (four eq) Fmoc-amino acid, 3.2 mmol (3.75 eq) HATU and 1.6 mmol (eight eq) N-methylmorpholine (NMM) in 8 mL DMF was added to do the peptide coupling for 90 min, followed by the deprotection of Fmoc with 20% piperidine in dimethylformamide (DMF) for 15 min. The resin was then completely washed with DMF before entering the next coupling cycle. The first depsipeptide TOG was coupled following a similar protocol. After all the coupling steps and deprotection steps, the resin was washed by DMF and dichloromethane (DCM) to dry. Reagent B (5% water, 5% phenol, 2.5% triisopropyl silane (TIPS), 87.5% trifluoroacetic acid (TFA)) for 2.5 hr at room temperature. The crude peptide was concentrated before the addition of cold diethyl ether to precipitate and dried over nitrogen gas flow. After being dissolved in 10% acetonitrile (CH3CN)-water, the crude peptides were further purified by reversed phase HPLC with the C18 column (Varian Dynamax Microsorb 100), with a gradient of 5% CH3CN/0.05% TFA in H2O/0.05% TFA for 2 min and a linear gradient from 5% CH3CN/0.05% TFA to 32% CH3CN/0.05% TFA over 25 min, and the flow rate is 10 mL/min. All the synthetic peptides were lyophilized to powders, followed by the characterization by MALDI-TOF at the Molecular Biology Core Facilities of Dana Farber Cancer Institute (Figure 2—figure supplement 1, Figure 5—figure supplement 1).

  • H3K9ac(1-34) TOG [M + H]+ calculated as m/z 3494.9, observe at m/z 3493.0;

  • Sequence: ARTKQTARKS-TGGKAPRKQL-ATKAARKSAP-A-TOG-G

  • H3K14ac(1-34) TOG [M + H]+ calculated as m/z 3494.9, observed at m/z 3493.5;

  • H3K18ac(1-34) TOG [M + H]+ calculated as m/z 3494.9, observed at m/z 3492.9;

  • H3K23ac(1-34) TOG [M + H]+ calculated as m/z 3494.9, observed at m/z 3493.7;

  • H3K27ac(1-34) TOG [M + H]+ calculated as m/z 3494.9, observed at m/z 3493.1;

  • H3K9acR8G(1-34) TOG [M + H]+ calculated as m/z 3394.8, observed at m/z 3393.8;

  • H3K14acG13R(1-34) TOG [M + H]+ calculated as m/z 3594.1, observed at m/z 3591.7;

Expression and purification of F40 sortase from E. coli

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F40 sortase was purified in a manner reported previously (Wu et al., 2018). The pET21-F40-sortase plasmid was used for the transformation of the E. coli Rosetta (DE3) pLysS strain. A single colony from the LB agar plate was inoculated into 5 mL LB media with 100 mg/L ampicillin at 37 °C overnight, and subsequently inoculated into 1 L large 2YT culture at 37 °C until OD600 reached 0.6. F40 sortase was then induced with 0.25 mM IPTG for 4 hr at 37 °C. The cells were harvested by centrifugation at 4,000 rpm for 15 min and the cell-pellet was resuspended in Lysis buffer containing 20 mM Tris, 0.1% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride (PMSF) at pH 7.5. The cells were then lysed by passing through a french press cell disrupter at about 1,000 psi three times, followed by centrifuging the lysate at 20,000 g for 40 min. The supernatant was loaded to a Ni-NTA Sepharose 6 Fast Flow (GE Healthcare) resin, followed by repeated wash with Wash buffer containing 20 mM Tris, 500 mM NaCl, 20 mM imidazole and 1 mM PMSF at pH 7.5. The F40 sortase was then eluted with Elution buffer containing 20 mM Tris, 500 mM NaCl, 400 mM imidazole and 1 mM PMSF at pH 7.5 in different fractions. All the fractions were analyzed by SDS-PAGE gel and fractions containing F40 sortase were pooled together and dialyzed against Dialysis buffer with 20 mM Tris, 150 mM NaCl, and 5 mM CaCl2 at pH 7.5. After dialysis, F40 sortase (>95% pure based on coomassie stained SDS-PAGE) was concentrated using an Amicon Ultra spin column (10K MWCO, EMD Millipore) to about 1 mM final concentration, and then aliquoted and stored at −80 °C for future usage.

Expression of WT-histone H2A, H2B, H4, and histone core region gH3

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Bacterial expression and purification of X. laevis core histones H2A, H2B, globular H3 (gH3; amino acids 34–135 with the first Met cleaved by E. coli), and H4 were performed using a previously reported method (Piotukh et al., 2011), (Luger et al., 1997). Please note that we used gH3 containing Cys110 for K23ac and K27ac semi-synthesis and Ala110 for K9ac, K14ac, K18ac semi-synthesis (Shimko et al., 2011) and the impact of this aa110 difference in the nucleosomes derived from these histone H3 isoforms is assumed to be negligible (Wilkinson and Gozani, 2014). Consequently, H3 C110A has been widely employed in nucleosome chemical biology (Jani et al., 2019) and structural studies (Gatchalian et al., 2017). The pET23-gH3 plasmid was used to transform E. coli Rosetta (DE3) pLysS strain, and a single colony was inoculated into 1 L LB media with 100 mg/L ampicillin at 37 °C. The culture was let to shake and grow at 37 °C until OD600 reached 0.6. Then, gH3 was induced with 0.5 mM IPTGat 37 °C for 3 hr. The cells were then harvested by centrifugation at 4,000 rpm for 15 min and the cell pellet was resuspended in Histone Wash Buffer (50 mM Tris-HCl at pH 7.5, 100 mM NaCl, 1 mM EDTA, 5 mM 2-mercaptoethanol (BME), 0.2 mM phenylmethylsulfonyl fluoride (PMSF) with 1% Triton X-100). The cells were then lysed by passing through a french press cell disrupter at about 1,000 psi three times, followed by centrifugation of the cell lysate at 20,000 g for 40 min. After discarding the supernatants, the pellets were washed with Histone Wash Buffer with 1% Triton X-100 once and Histone Wash Buffer without Triton X-100 twice. The pellets were dissolved in Histone Solubilization Buffer (7 M guanidinium hydrochloride, 20 mM Tris at pH 7.5 and 10 mM dithiothreitol). After centrifuging at 20,000 g for 15 min, the supernatant was dialyzed three times against IEX (ion exchange) buffer (7 M urea, 10 mM Tris pH 7.8, 1 mM EDTA, 0.2 mM PMSF and 5 mM BME). The resulting gH3 solution in IEX buffer was diluted 5-fold in IEX buffer before being loaded onto a tandem Q-SP column (GE healthcare, HiTrap Q HP and HiTrap SP HP, 5 mL each). Both Q and SP column were first equilibrated and washed with IEX buffer containing 100 mM NaCl. Then, gH3 was eluted from the 100 mM - 500 mM NaCl with 100 mM increment. The fractions containing gH3 (>90% pure by coomassie stained SDS-PAGE) were pooled after analyzing each fraction by SDS-PAGE. The combined IEX solution was then dialyzed three times against pure water containing 2 mM BME (3.5K MWCO, Spectra/Por) for 6 hr each. The solution was finally concentrated with an Amicon Ultra spin column (3.5 K MWCO, EMD Millipore) to 10 mL (~100 μM) and stored at 4 °C until usage.

F40 sortase catalyzed histone H3 semi-synthesis

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The histone H3 N-terminal peptide (aa1-34 depsipeptide, 1 mM) and gH3 (aa34-135,~70 μM) was mixed in Sortase Reaction Buffer (50 mM HEPES at pH 7.5, 150 mM NaCl and 5 mM CaCl2). The reaction was initiated by the addition of F40 Sortase (300 μM) and was incubated at 37 °C for overnight. The reaction mixture was centrifuged to precipitate H3 with some sortase, which was then dissolved in 10 mL IEX buffer (7 M urea, 10 mM Tris at pH 7.8, 1 mM EDTA and 5 mM BME). The solution was loaded onto a SP column (GE healthcare, HiTrap SP HP, 1 mL) preequilibrated with IEX buffer containing 100 mM NaCl. After washing with IEX buffer containing 100 mM NaCl, H3 was eluted with a gradient of IEX buffer containing increasing NaCl from 100 to 500 mM. All the fractions were analyzed by SDS-PAGE, and the fractions with pure ligated H3 product (>90% based on coomassie stained SDS-PAGE gels) were pooled and dialyzed three times against pure water with 2 mM BME (3.5K MWCO, Spectra/Por). The solution was then concentrated using an Amicon Ultra spin column (10 K MWCO, EMD Millipore), and was lyophilized to a white powder for storage at −80 °C until usage. Semi-synthesized histone H3 structures were characterized by MALDI-TOF Mass Spectrometry ((matrixed with CHCA) at the Molecular Biology Core Facilities of Dana Farber Cancer Institute (Figure 2—figure supplement 2, Figure 5—figure supplement 1).

  • H3K9ac(C110A): [M + H]+ calculated as m/z 15280.8, observed as m/z 15274.3;

  • H3K14ac(C110A): [M + H]+ calculated as m/z 15280.8, observed as m/z 15278.9;

  • H3K18ac(C110A): [M + H]+ calculated as m/z 15280.8, observed as m/z 15271.6;

  • H3K23ac: [M + H]+ calculated as m/z 15312.8, observed as m/z 15309.6;

  • H3K27ac: [M + H]+ calculated as m/z 15312.8, observed as m/z 15308.6;

  • H3K9acR8G(C110A): [M + H]+ calculated as m/z 15181.6, observed as m/z 15175.1;

  • H3K14acG13R(C110A): [M + H]+ calculated as m/z 15379.9, observed as m/z 15377.1.

Octamer refolding and nucleosome reconstitution

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The octamer refolding and nucleosome assembly were performed following a previous report (Dyer et al., 2004). All four histone proteins H2A, H2B, H3 and H4, each two copies, were dissolved in Unfolding Buffer (7 M guanidine, 20 mM Tris-HCl at pH 7.5 and 10 mM DTT) and quantified by reading A280. The mixed solution with a molar ratio at 1.1: 1.1: 1:1 one was then dialyzed three times against high salt buffer (20 mM Tris at pH 7.5, 2.0 M NaCl, 1 mM EDTA and 5 mM BME). The octamer was purified by size exclusion chromatography using a Superdex 200 10/300 GL column (GE Healthcare). In addition, 146 Widom 601 DNA was prepared by previously reported methods used for nucleosome reassembly (Luger et al., 1997). The 146 bp DNA was obtained from the restriction digests of a specially designed DNA plasmid from E. coli and purified by precipitation with polyethylene glycol. The histone octamer (Figure 2—figure supplement 3B, using H3K9ac and H3K14ac as examples) and Widom DNA were mixed at a 1:1 molar ratio at high salt buffer (10 mM Tris at pH 7.5, 2.0 M KCl, 1 mM EDTA and 1 mM DTT) with a final concentration of 7 μM DNA. Continuous-flow gradient dialysis was used to lower the salt concentration from the mixture to low salt buffer (10 mM Tris at pH 7.5, 250 mM KCl, 1 mM EDTA and 1 mM DTT) by a Minipuls two peristaltic pump (Gilson). The final mixture was subjected to HPLC (Waters, 1525 binary pump, 2489 UV-Vis detector). HPLC using a TEKgel DEAE ion exchange column was employed to purify the final nucleosome product. The liquid phase buffers A and B were as follow: A. TES 250 (10 mM Tris 7.5, 250 mM KCl, 0.5 mM EDTA), B. TES 600 (10 mM Tris 7.5, 600 mM KCl, 0.5 mM EDTA), were used to generate with a 0% B wash for 12 min and a linear gradient from 25% to 75% B over 30 min at 1 mL/min flow rate. The fractions containing the nucleosome were pooled together and dialyzed against a TCS Buffer (20 mM Tris at pH 7.5 and 1 mM DTT, no EDTA) and concentrated to about 20 μM using an Amicon Ultra spin column (10K MWCO, EMD Millipore). The final nucleosome products were analyzed by native TBE gels and all showed over 95% purity based on the fact that free DNA band fluoresces 2.5-fold more brightly than the nucleosome DNA band in this setting (Shahian and Narlikar, 2012).

Expression and purification of HDAC complexes

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The expression and purification of HDAC complexes were performed as described previously (Song et al., 2020), (Millard et al., 2016), (Itoh et al., 2015), (Watson et al., 2012b), (Portolano et al., 2014). For each complex, three plasmids corresponding to the different components of the HDAC complex were co-transfected into HEK293F cells: LSD1 (1–852), HDAC1 (1–482), and Flag tagged CoREST1 (86–485) for the CoREST complex; Flag tagged MTA1 (1–715), HDAC1 (1–482), and RBBP4 (1–425) for the NuRD complex; Flag tagged Sin3B (1–1162), HDAC1 (1–482), RBBP4 (1–425) for the Sin3B complex; Flag tagged MIDEAS (628-887), HDAC1 (1–482), DNTTP1 (1–329) for the MiDAC complex; Flag tagged GPS2-NCoR2 chimera ((1-49) - (220-480)), HDAC3 (1–428), and TBL1 (1–526) for the SMRT complex. After cell lysis by sonication, the complexes were purified by FLAG tag immunoaffinity chromatography and liberated from the column with TEV protease. These mixtures were further purified by gel filtration on either a Superdex 200 Increase 10/300 GL column (MiDAC, SMRT) or a Superose 6 10/300 GL column (CoREST, NuRD, Sin3B). The purified complexes were analyzed by SDS-PAGE stained with coomassie and concentrated to 3–5 μM (Figure 3—figure supplement 2B). After the addition of glycerol to 10%, the complexes are stored at −80°C until further use (Wu et al., 2018). The final HDAC complexes were analyzed by SDS-PAGE gel stained with coomassie and each showed greater than 60% purity.

Analysis of deacetylation of acetylated nucleosomes and acetylated histone H3s

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For all the deacetylation assays, the semi-synthetic H3K9/14/18/23/27ac histone H3 proteins (1.0 μM) or the corresponding nucleosomes (100 nM) assembled in vitro were incubated with different HDAC1 and HDAC3 complexes at different concentrations, under a reaction buffer containing 50 mM HEPES at pH 7.5, 100 mM KCl, 100 μM IP6 (Watson et al., 2016), and 0.2 mg/mL BSA at 37 °C. The complexes include CoREST, NuRD, Sin3B, MiDAC, and SMRT, together with HDAC1 enzyme not involved in specific complex binding from commercial source (BPS Bioscience). The reaction time was counted, and multiple samples were taken at different time points. Typically, 10 μL aliquots of the reaction were quenched with gel loading buffer made by mixing 2 μL 80 mM EDTA and 4 μL 4 x Laemmli sample buffer at each time point. The samples were then boiled for 3 min at 95 °C and loaded onto a 15% SDS-PAGE gel (160 Volts for 35 min). After transferring to nitrocellulose membrane, site-specific antibodies for acetylated H3, such as for anti-H3K9ac, anti-H3K14ac, anti-H3K18ac, anti-H3K23ac and anti-H3K27ac, as well as total H3 antibody, anti-H3, were used to blot the membrane. The specificities of the primary antibodies, anti-H3K23ac and anti-H3K27ac, were validated using histone H3K9ac as a negative control (Figure 3—figure supplement 1), while the specificities of the primary antibodies anti-H3K9ac, anti-H3K14ac and anti-H3K18ac have been confirmed previously (Wu et al., 2018). After treating with the ECL substrate (Bio-Rad), the blotted membrane was and visualized by the G:BOX mini gel imager (Syngene), and then quantified using ImageJ. The intensity values from ImageJ were fit to a single-phase exponential decay curve software (GraphPad Prism Eight). Then, the kinetic data V/E were calculated for nucleosome assays, while normalized V/E were calculated for histone assays (Two H3 molecules to one nucleosome ratio was taken into account for calculation).

Analysis of deacetylation of H4K16ac nucleosomes

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For the H4 deacetylation assays, the commercial H4K16ac nucleosomes (100 nM) was incubated with 40 nM NuRD complexes in a reaction buffer containing 50 mM HEPES at pH 7.5, 100 mM KCl, 100 μM IP6, and 0.2 mg/mL BSA at 37 °C. Multiple samples were taken at different time points and analyzed by Western Blot with anti-H4K16ac antibody, following similar steps of the deacetylation assays of the H3Kac nucleosomes.

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Decision letter

  1. Wilfred A van der Donk
    Reviewing Editor; University of Illinois at Urbana-Champaign, United States
  2. John Kuriyan
    Senior Editor; University of California, Berkeley, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Thank you for submitting your article "Diverse nucleosome site-selectivity among histone deacetylase complexes" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by Wilfred van der Donk as Reviewing Editor and John Kuriyan as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.

Summary:

This work is an extension of previous work from the Cole lab that reported (1) the use of F40 sortase to generate semi-synthetic acetylated H3, (2) its incorporation in nucleosomes, and (3) assays with the LSD1-CoREST-HDAC1 complex. In this work, the authors expand upon this prior work by testing various acetylated nucleosomes against 4 different HDAC1-containing and one HDAC3-containing protein complex (CoREST, NuRD, Sin3B, MiDAC and SMRT). Interestingly, the authors observe that H3K14ac is a poor substrate for the CoREST and Sin3B complexes, and speculate that this may be due to the presence of Gly13 at the P2 position. They demonstrate that mutating Gly13 to Arg13 leads to faster deacetylation by the CoREST complex. Another interesting discovery is that HDAC1 activity at various H3 lysines is modulated by its specific complex. This work is important towards obtaining a molecular level understanding of how various HDAC complexes act on chromatin depending on both enzyme context and substrate context. The reviewers feel this Research Advance is suitable for publication in eLife after minor clarifications.

Revisions:

1) Enzyme activity that is modulated by its context in various complexes is perhaps not too surprising, but gaining information of such modulation is important and valuable. In the current system, the complexes likely bind nucleosomes at different sites and/or allosterically regulate kcat or KM. One aspect of this study that is not well-explained is why complexes were chosen to be limited to three proteins. NuRD, for example, has six known components. The authors should address the choice for a limited set of proteins, and why three components were chosen in each case.

2) The authors propose that HDAC1 activity is inhibited by G13 in the CoREST complex. However, it is unclear why the same residue adjacent to K14ac in the HDAC1 active site would have vastly different effects (CoREST vs MIDAC1). Do the authors have any insights into whether Gly13Arg induces activity due to an effect on kcat or KM (or both)? In future work, determination of binding constants would be valuable.

3) Many of the semisynthetic H3 proteins have additional large unlabeled peaks in the MALDI-TOF mass spectra. These are less visible in the charge states in the ESI spectra due to the large mass range shown but also appear to show up in the western blots (Figure 3—figure supplement 1). The authors should clearly label the additional peaks. It also appears there would be free DNA in the nucleosome preparations. Free DNA is known to inhibit enzymatic activities in some instances, for e.g. for p300. It would be valuable to add a statement to the manuscript as to the percent purity of the nucleosome substrates.

4) Similarly the protein complexes in Figure 3—figure supplement 2B are not homogenous. While purifying complexes using epitope tags from eukaryotic cells is unlikely to yield perfectly homogenous preps, it is important for the authors to mention the percent purity of each complex. Given point 1 above, can they comment whether other components are entirely missing from these complexes and do not have an effect on activity?

5) One concern is that HDAC1 is often exchanged with HDAC2 in many complexes, including NuRD. It appears that the authors did not interrogate HDAC2 directly, but can they confirm that no residual HDAC2 was present in their complexes? Varying degrees of HDAC1/2 may contribute toward some of the differences seen in kinetics data.

6) The authors extend their discovery to the discussion that H3K14ac and H3K4me2 may co-exist at enhancers to prevent CoREST activity. This may be the case, however other enzymes such as KDM4 (Jumonji-domain containing lysine specific demethylase 4) are equally able to demethylate H3K4me2. There is no evidence that these KDMs are sensitive to inhibition by H3K14ac, or that they are excluded from enhancers while CoREST is recruited to enhancers. Therefore, the presented argument may need some additional discussion. Could the presence of H3K14ac at high basal levels equally likely reflect higher degrees of basal HAT activity at this site?

https://doi.org/10.7554/eLife.57663.sa1

Author response

Revisions:

1) Enzyme activity that is modulated by its context in various complexes is perhaps not too surprising, but gaining information of such modulation is important and valuable. In the current system, the complexes likely bind nucleosomes at different sites and/or allosterically regulate kcat or KM. One aspect of this study that is not well-explained is why complexes were chosen to be limited to three proteins. NuRD, for example, has six known components. The authors should address the choice for a limited set of proteins, and why three components were chosen in each case.

There is no doubt that there are multiple versions of each of the endogenous HDAC1/HDAC2 complexes. These differences may be developmentally regulated, cell cycle dependent, tissue specific etc. As the reviewers point out, in general, 3 proteins were included for the complexes studied here, although for the SMRT complex, one of the proteins is a chimeric GPS2-NCOR2 fusion. The selection of what we regard as the core complexes comes from almost a decade of experience of a combination of biochemical and structural studies to develop stable, enzymatically functional complexes that can be purified and can interact efficiently with nucleosomes. The many details of how these complexes have been arrived at and are produced are described in a series of Schwabe lab publications cited in the manuscript that reflect the fruits of these laborious efforts to generate them. In general, the two non-HDAC proteins in each case were selected based on the following criteria: 1) A set of proteins that included both well-established HDAC and nucleosome binding partners for a given complex, 2) Efficient transient co-expression of soluble proteins in HEK293F that could stay associated by immunoaffinity chromatography and lead to relatively pure and concentrated complexes in peak fractions (>50% purity), 3) The ability of the core complexes to be reproducibly isolated as monodisperse peaks in appropriate stoichiometries after size exclusion chromatography. We recognize that it is certainly possible that other subunits, not included in these core complexes, could alter catalytic efficiencies and/or substrate selectivities. We included this point for NuRD in the original manuscript but now make the point more broadly in the revised manuscript. For the NuRD complex, it is established that the chromatin remodelling module is rather loosely associated, and we do not expect that its absence would greatly influence HDAC activity. Moreover, NuRD complexes in which MTA1 is substituted by PWWP2A/B do not associate with the chromatin remodellers. We have revised the manuscript to reflect these additional points.

2) The authors propose that HDAC1 activity is inhibited by G13 in the CoREST complex. However, it is unclear why the same residue adjacent to K14ac in the HDAC1 active site would have vastly different effects (CoREST vs MIDAC1). Do the authors have any insights into whether Gly13Arg induces activity due to an effect on kcat or KM (or both)? In future work, determination of binding constants would be valuable.

We have not measured kcat and KM values due to the challenges involved of attempting such measurements in this complex system including the requirement of working with high concentrations of nucleosomes and our reliance on western blotting and disappearance of acetyl-Lys signals. We do think measuring binding affinities is feasible and plan to do this with fluorescently labeled reagents in future work. Our speculation is that the nucleosome binding affinity will not be strongly affected by H3 Gly13 and that differences in CoREST vs. MiDAC complex deacetylation rates will be related to precise active site complementarity between these catalysts. We have added these comments to the manuscript.

3) Many of the semisynthetic H3 proteins have additional large unlabeled peaks in the MALDI-TOF mass spectra. These are less visible in the charge states in the ESI spectra due to the large mass range shown but also appear to show up in the western blots (Figure 3—figure supplement 1). The authors should clearly label the additional peaks. It also appears there would be free DNA in the nucleosome preparations. Free DNA is known to inhibit enzymatic activities in some instances, for e.g. for p300. It would be valuable to add a statement to the manuscript as to the percent purity of the nucleosome substrates.

We agree that there are some minor impurities in some of the semisynthetic histone H3 MALDI mass spectra (we have not performed ESI in this case), particular in the molecular weight range of the globular H3 fragment. We have now labeled these with asterisks in the revised figures. We think it is unlikely that these impurities at the histone stage will interfere with the nucleosome deacetylation assays as they are likely to be removed at the octamer and/or nucleosome purification stages. We also emphasize that the relative similarity of the deacetylation rates with each of these free acetyl-histone H3 substrates indicate that the impurities probably do not have a large impact on the general activity of HDAC complexes. Regarding the purities of the mononucleosomes with regard to free DNA, we agree that the presence of free DNA could potentially interfere with the deacetylase assays. However, these native DNA gels stained with ethidium bromide indicate <5% free DNA in molar terms. The free DNA band visualized is 10% or less of the relative fluorescence intensity for the nucleosomes among the different preparations. But such analysis overestimates the impurity. The more valid calculation of purity takes into account that free DNA vs. nucleosome bound DNA fluoresces about 2.5 times more brightly due to the packaging with the histone proteins under the conditions of these gels (Shahian and Narlikar, Methods in Mol. Biol. 2012). Thus, we believe that the contaminating free DNA in these preparations is acceptably low for these enzymatic assays. We have summarized each of these points in the revised manuscript.

4) Similarly the protein complexes in Figure 3—figure supplement 2B are not homogenous. While purifying complexes using epitope tags from eukaryotic cells is unlikely to yield perfectly homogenous preps, it is important for the authors to mention the percent purity of each complex. Given point 1 above, can they comment whether other components are entirely missing from these complexes and do not have an effect on activity?

We agree that these HDAC complexes are not completely pure. We have now estimated the purities of each complex based on coomassie staining of the impurity bands relative to the desired specific protein bands within the complex. These estimated purities are as flows: CoREST (84%), NuRD (93%), Sin3B (63%), MIDAC (84%), and SMRT (99%). It is possible that impurities seen or unseen could affect the activities of these complexes but we do not have a simple way to analyze this. However, for various nucleosome deacetylation assays, we have used more than one independent HDAC complex preparation for enzymatic measurements and have seen consistent deacetylase results across different preps.

5) One concern is that HDAC1 is often exchanged with HDAC2 in many complexes, including NuRD. It appears that the authors did not interrogate HDAC2 directly, but can they confirm that no residual HDAC2 was present in their complexes? Varying degrees of HDAC1/2 may contribute toward some of the differences seen in kinetics data.

As the reviewers note, HDAC1 and HDAC2 show overall about 80% amino acid sequence identity and can be interchangeably incorporated into multiprotein complexes under endogenous conditions. Because the transient transfections of HEK293F cells with HDAC1 plasmid leads to massive overexpression of HDAC1 relative to endogenous levels of HDAC1 or HDAC2, we think it is highly likely that the vast majority of HDAC isoform in these complexes is HDAC1. Prior published structural studies of these complexes from the Schwabe lab and mass spectroscopic analyses of tryptic digests are also consistent with this idea. Having said that, because of their very high sequence homology, we believe it is likely that HDAC1 and HDAC2 will behave in similar ways in terms of the nucleosome deacetylation assays carried out here. We have summarized these HDAC2 related points in the revised manuscript.

6) The authors extend their discovery to the discussion that H3K14ac and H3K4me2 may co-exist at enhancers to prevent CoREST activity. This may be the case, however other enzymes such as KDM4 (Jumonji-domain containing lysine specific demethylase 4) are equally able to demethylate H3K4me2. There is no evidence that these KDMs are sensitive to inhibition by H3K14ac, or that they are excluded from enhancers while CoREST is recruited to enhancers. Therefore, the presented argument may need some additional discussion. Could the presence of H3K14ac at high basal levels equally likely reflect higher degrees of basal HAT activity at this site?

It is certainly possible that high HAT activity could also drive elevated H3K14ac as suggested by the reviewers. We believe that the reviewers may be referring to KDM5 rather than KDM4 as an JMJ family H3K4me demethylase (KDM4 is better known at targeting other H3 sites). Interestingly, there is a report that the JMJ H3K4me demethylase paralog in yeast appears to be more weakly able to demethylate in the context of H3K14ac (Maltby et al., 2012). We have emphasized these points more clearly in the revised manuscript.

https://doi.org/10.7554/eLife.57663.sa2

Article and author information

Author details

  1. Zhipeng A Wang

    1. Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Boston, United States
    2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States
    Contribution
    Conceptualization, Data curation, Investigation, Methodology, Writing - original draft, Writing - review and editing
    Contributed equally with
    Christopher J Millard and Chia-Liang Lin
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5693-7359
  2. Christopher J Millard

    Leicester Institute of Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, Leicester, United Kingdom
    Contribution
    Conceptualization, Resources, Investigation, Methodology, Writing - review and editing
    Contributed equally with
    Zhipeng A Wang and Chia-Liang Lin
    Competing interests
    No competing interests declared
  3. Chia-Liang Lin

    Leicester Institute of Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, Leicester, United Kingdom
    Contribution
    Conceptualization, Resources, Investigation, Methodology, Writing - review and editing
    Contributed equally with
    Zhipeng A Wang and Christopher J Millard
    Competing interests
    No competing interests declared
  4. Jennifer E Gurnett

    Leicester Institute of Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, Leicester, United Kingdom
    Contribution
    Conceptualization, Resources, Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
  5. Mingxuan Wu

    1. Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Boston, United States
    2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States
    Contribution
    Conceptualization, Formal analysis, Investigation, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
  6. Kwangwoon Lee

    1. Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Boston, United States
    2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States
    Contribution
    Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2021-5186
  7. Louise Fairall

    Leicester Institute of Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, Leicester, United Kingdom
    Contribution
    Conceptualization, Resources, Supervision, Project administration, Writing - review and editing
    Competing interests
    No competing interests declared
  8. John WR Schwabe

    Leicester Institute of Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, Leicester, United Kingdom
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Methodology, Project administration, Writing - review and editing
    For correspondence
    john.schwabe@le.ac.uk
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2865-4383
  9. Philip A Cole

    1. Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Boston, United States
    2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    pacole@bwh.harvard.edu
    Competing interests
    Senior editor, eLife
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6873-7824

Funding

NIH (GM62437)

  • Philip A Cole

Leukemia and Lymphoma Society (SCOR)

  • Philip A Cole

Wellcome Trust (100237/Z/12/Z)

  • John W R Schwabe

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors would like to thank the PROTEX cloning service, University of Leicester, for producing the constructs used to make HDAC complexes. The authors thank James Lee (Molecular Biology Core Facilities of Dana Farber Cancer Institute) for the assistance of MALDI, Yi Zheng for illustrations, Ben Martin and Sam Whedon for advice, and Taylor Kay for technical support. The authors acknowledge NIH, LLS, and the Wellcome Trust for financial support.

Senior Editor

  1. John Kuriyan, University of California, Berkeley, United States

Reviewing Editor

  1. Wilfred A van der Donk, University of Illinois at Urbana-Champaign, United States

Publication history

  1. Received: April 9, 2020
  2. Accepted: June 4, 2020
  3. Accepted Manuscript published: June 5, 2020 (version 1)
  4. Version of Record published: June 25, 2020 (version 2)

Copyright

© 2020, Wang et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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