Synthetic Posttranslational Modifications: Chemical Catalyst-Driven Regioselective Histone Acylation of Native Chromatin

Article abstract of DOI:10.1021/jacs.7b02138

Posttranslational modifications (PTMs) of histones play an important role in the complex regulatory mechanisms governing gene transcription, and their dysregulation can cause diseases such as cancer. The lack of methods for site-selectively modifying nati

Full text of DOI:10.1021/jacs.7b02138

Article
pubs.acs.org/JACS
Synthetic Posttranslational Modifications: Chemical Catalyst-Driven
Regioselective Histone Acylation of Native Chromatin
Yoshifumi Amamoto,†,‡,⊥ Yuki Aoi,†,‡,⊥ Nozomu Nagashima,† Hiroki Suto,†,‡ Daisuke Yoshidome,§
Yasuhiro Arimura,∥ Akihisa Osakabe,∥ Daiki Kato,∥ Hitoshi Kurumizaka,∥ Shigehiro A. Kawashima,*,†,‡
Kenzo Yamatsugu,*,†,‡ and Motomu Kanai*,†,‡
†Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
‡JST-ERATO, Kanai Life Science Catalysis Project, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
§Schrodinger K. K., 17F Marunouchi Trust Tower North, 1-8-1 Marunouchi Chiyoda-ku, Tokyo 100-0005, Japan
̈
∥Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho,
Shinjuku-ku, Tokyo 162-8480, Japan
S
* Supporting Information
ABSTRACT: Posttranslational modifications (PTMs) of histo-
nes play an important role in the complex regulatory
mechanisms governing gene transcription, and their dysregula-
tion can cause diseases such as cancer. The lack of methods for
site-selectively modifying native chromatin, however, limits our
understanding of the functional roles of a specific histone PTM,
not as a single mark, but in the intertwined PTM network. Here,
we report a synthetic catalyst DMAP-SH (DSH), which activates
chemically stable thioesters (including acetyl-CoA) under physiological conditions and transfers various acyl groups to the
proximate amino groups. Our data suggest that DSH, conjugated with a nucleosome ligand, such as pyrrole-imidazole-polyamide
and LANA (latency-associated nuclear antigen)-peptide, promotes both natural (including acetylation, butyrylation,
malonylation, and ubiquitination) and non-natural (azido- and phosphoryl labeling) PTMs on histones in recombinant
nucleosomes and/or in native chromatin, at lysine residues close to the DSH moiety. To investigate the validity of our method,
we used LANA-DSH to promote histone H2B lysine-120 (K120) acylation, the function of which is largely unknown. H2BK120
acetylation and malonylation modulated higher-order chromatin structures by reducing internucleosomal interactions, and this
modulation was further enhanced by histone tail acetylation. This approach, therefore, may have versatile applications for
dissecting the regulatory mechanisms underlying chromatin function.
tion of a PTM in recombinant nucleosomes using these
techniques enabled us to explore how the PTM modulates the
biochemical function of the nucleosomes.20 In addition,
because a specific histone PTM often functions in a dynamic
and cooperative network with other PTMs, examining a PTM
in native chromatin that contains diverse histone PTMs helps
better understand how the biological properties of native
chromatin are regulated by the PTM network.21 For such a
purpose, protein semisynthesis has been extended to histone
ubiquitination within native chromatin.22 However, this
technique is restricted to histone tail regions and requires
high-level expression of histone mutant proteins, which would
potentially disturb the network of histone PTMs in chromatin.
Protein synthesis by genetic code expansion has allowed site-
specific incorporation of histone acetylation in human cells.23
However, an amber codon suppression strategy of this
technique provokes genome-wide alteration of translation
termination sites, preventing simple understanding of the
INTRODUCTION
■
Posttranslational modifications (PTMs) of histones play an
important role in the complex regulatory mechanisms
governing gene transcription. Dysregulation of histone PTMs
can result in the development of diseases such as cancer.1
Lysine acylation on histones, which is a representative PTM in
chromatin, affects gene transcription and chromatin structure.
For example, acetylation, butyrylation, and crotonylation on
histone tails have been shown to directly stimulate tran-
scription.2−4 Ubiquitination of H2A and H2B influences gene
transcription and chromatin structure.5−7 Although other types
of lysine acylation, such as malonylation,8 have been recently
identified at various sites on histones,9 their functions are
largely unknown.
Several powerful techniques that allow site-selective incor-
poration of noncanonical amino acids in recombinant proteins
have furthered our understanding of the function of histone
PTMs. These include protein semisyntheses using native
chemical ligation,10−14 ribosome-mediated protein syntheses
using expanded genetic code,15−17 and recently reported
chemistry-coupled posttranslational mutagenesis.18,19 Installa-
Received: March 2, 2017
© XXXX American Chemical Society
A
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Figure 1. Development of a DMAP-SH (DSH) catalyst for regioselective acetylation. (a) Ligand-directed catalysis to achieve lysine residue-
selectivity in native chromatin. A catalyst motif conjugated with a ligand that binds to a specific region in a nucleosome enables selective acetylation
of a proximal lysine residue using an acetyl donor, acetyl-CoA, leading to an epigenetic output. (b) Peptide model studies for identifying an efficient
lysine acetylation catalyst moiety. Peptides (0.1 mM) containing a catalyst moiety and a substrate lysine were mixed with acetyl-CoA (1 mM) under
aqueous buffered conditions (pH 7.5) at room temperature (rt) for 8 h, and acetylation yields were determined by HPLC measurements. (A) No
catalyst, (B) DMAP, (C) thiosalicylamide, (D) DSH, and (E) DSH and lysine in a different peptide. Detailed structures of the peptides are shown in
Figure S1. ND denotes not detected. Error bars represent standard deviations of three independent experiments. (c) A plausible mechanism for
lysine acetylation by DSH (1). Ac, acetyl.
effects of the incorporated histone PTMs. This technique also
depends on the availability of an orthogonal tRNA synthetase−
tRNA pair, which limits the scope of PTMs. Therefore, a
versatile method that allows site-selective introduction of a
specific histone PTM, such as lysine acylation, in recombinant
nucleosomes as well as in native chromatin without genetic
manipulations is required for a better understanding of how the
PTM functions in the intertwined PTM network in chromatin.
Herein, we report a synthetic catalyst DMAP-SH (DSH),
which activates chemically stable thioesters including acetyl-
CoA under physiological conditions. Our data suggest that
DSH promotes a variety of regioselective histone acylations
both in recombinant nucleosomes and in native chromatin,
when conjugated with a proper ligand. To investigate the
validity of our method, we used DSH to promote histone H2B
lysine-120 (K120) acylation, the function of which is largely
unknown, and revealed that H2BK120 acetylation and
malonylation modulate higher-order chromatin structures by
reducing internucleosomal interactions. Thus, this approach
may have versatile applications for dissecting regulatory
mechanisms underlying chromatin functions.
RESULTS
■
Development of a Catalyst Moiety Activating Acetyl-
CoA. In living cells, histone acetyltransferases (HATs) catalyze
acetyl transfer reactions from acetyl-CoA to specific lysine
B
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Figure 2. Regioselective histone acetylation in recombinant nucleosomes by ligand-conjugated DSH. (a) Chemical structures of LANA-DSH (4)
and PIP-DSH (5). (b) An X-ray crystal structure of the LANA-DSH−nucleosome complex. Because the electron density of the DSH moiety was not
observed, its estimated position is depicted with a dashed circle (PDB: 5GTC). (c) Positional relationships between PIP-DSH-binding DNA
sequences and proximal lysine residues in nucleosomes 1, 2, and 3, which are highlighted in the same color, and mapped in a single nucleosome
structure (PDB: 3LZ0; magenta, nucleosome 1; green, nucleosome 2; cyan, nucleosome 3). (d) DNA sequence-dependency of histone acetylation
by LANA-DSH and PIP-DSH using the nucleosomes 1, 2, and 3. Each nucleosome (33 ng/μL DNA, 0.37 μM) was reacted with LANA-DSH (2
μM) or PIP-DSH (5 μM) in the presence of acetyl-CoA (1 mM) and Tris(2-carboxyethyl)phosphine (TCEP, 0.1 mM) at 37 °C for 8 h, and
acetylated lysines were detected by immunoblotting using residue-specific antiacetyl lysine antibodies. Protein amount is indicated by Coomassie
Brilliant Blue (CBB) staining. (e) LC−MS/MS analysis of acetylation yield of each lysine residue on a nucleosome (33 ng/μL DNA, 0.37 μM)
treated with acetyl-CoA (1 mM) and TCEP (0.1 mM) with and without LANA-DSH (2 μM) at 37 °C for 2 h. Each number on the x-axis indicates
the position of a lysine residue on histones. The list of chemical yields is shown in Table S2. A representative LC−MS/MS data set is shown in Table
S4. Error bars represent standard deviations of three independent experiments. Ac, acetyl.
residues in target proteins.24 HATs accelerate lysine acetylation
by enhancing the reactivity of the amino groups of lysine
residues.24 We conceived that activation of the acetyl donor
side, acetyl-CoA, would be more feasible for an artificial small-
molecule catalyst. We reasoned that regioselectivity of lysine
acetylation could be achieved by employing a ligand-directed
approach,25,26 which allows delivery of a catalyst to a target site
(Figure 1a). To develop a catalyst that can activate acetyl-CoA
and exclusively acetylate a lysine residue proximal to the
catalyst, we undertook a quantitative investigation using a
peptide containing both a catalyst moiety and a lysine residue
as a model of ligand-directed acetylation of a protein (Figure
1b). Lysine acetylation of peptides lacking any catalyst moiety
or containing a 4-(dimethylamino)pyridine (DMAP) moiety,
which is a well-known nucleophilic acyl transfer catalyst,27
barely proceeded (Figure 1b, conditions A and B, and Figure
S1a,b).
thiosalicylamide moiety, which is reported to transfer an acetyl
group from cellular metabolites, presumably acetyl-CoA, to a
target protein,30,31 was tried, but the lysine acetylation did not
proceed efficiently (Figure 1b, condition C, and Figure S1c).
This would be due to high acidity of the thiol moiety in the
thiosalicylamide, which shifts an equilibrium of the thiol-
thioester exchange to the side of acetyl-CoA. We, therefore,
designed DMAP-SH (DSH, 1), in which a mercaptomethyl
group (less acidic than a thiophenol) was conjugated to the 2-
position of DMAP (Figure 1b,c). We postulated facile thiol−
thioester exchange between the aliphatic thiol moiety and
acetyl-CoA, and subsequent intramolecular acetylpyridinium
formation through nucleophilic catalysis. HPLC measurements
supported the notion that the thiol−thioester exchange
between DSH and acetyl-CoA proceeded and generated
DMAP-SAc (DSAc, 2, Figure 1c) under neutral conditions
(Figure S2). Remarkably, acetylation of the lysine residue on
the model peptide with DSH as a catalyst moiety proceeded
efficiently under physiological conditions (Figure 1b, condition
D, and Figure S1d). Importantly, intermolecular acetyl transfer
Therefore, to more efficiently activate acetyl-CoA, we
conceived the use of a thiol-thioester exchange28,29 to receive
the acetyl group from acetyl-CoA into the catalyst moiety. A
C
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Figure 3. Regioselective introduction of various histone acylations in recombinant nucleosomes by LANA-DSH. (a) Scope and chemical yields of
H2BK120 acylations. A recombinant nucleosome (33 ng/μL DNA, 0.37 μM) was incubated with LANA-DSH and an acyl donor in the presence of
TCEP (0.1 mM), and the acylation yield at H2BK120 was determined by LC−MS/MS analysis. Detailed reaction conditions are summarized in the
Supporting Information. The mean chemical yields are shown with standard deviations of three independent experiments. The acetylation yield
shown is the same as that reported in Figure 2e. The structure of ubiquitin is obtained from PDB (1UBQ). (b) Histone- and H2BK120-selective
acetylation and malonylation of recombinant nucleosomes by LANA-DSH. A mixture of wild-type (WT) or H2BK120R (KR) mutant nucleosomes
(33 ng/μL DNA, 0.37 μM) and HeLa cell extract (to evaluate protein selectivity) was incubated with LANA-DSH and acyl-CoAs in the presence of
TCEP (0.1 mM), and acylated lysines were detected by immunoblotting using the indicated antibodies. Protein amount is indicated by CBB staining.
Other reaction conditions are the same as in (a). For histone-selectivity in other acylations promoted by LANA-DSH, see Figure S7. N3-Ac,
azidoacetyl; Phospho-Ac, phosphoryloxyacetyl; Ac, acetyl; Mal, malonyl.
from DSAc to a lysine residue in a different peptide did not
proceed, suggesting that proximity effects dominated the
reactions (Figure 1b, condition E, and Figure S1e). This
feature is advantageous for avoiding undesired acetylation of
off-target lysines. The DMAP moiety is important for efficient
acetyl transfer, as was evident from the results that lysine
acetylation of a peptide with a simple cysteine residue or a
simple benzyl thiol as a catalyst moiety proceeded less
efficiently (Figure S1f−h).
We examined the reaction profiles of lysine acetylation
promoted by DSH. The reaction rates increased with
temperature, but there was no obvious peptide concentration
dependency (Figure S3a,b). One DSH moiety promoted more
than two lysine acetylations, indicating that DSH undergoes
catalytic turnover (Figure S3c). Sufficient lysine selectivity was
observed, while cysteine was acetylated without DSH via direct
thiol−thioester exchange from acetyl-CoA (Figure S4). The
cysteine acetylation would not be a critical side reaction,
however, because most cysteine residues are buried inside
proteins by the formation of disulfide bonds, and also the
acetylation is reversible as long as there are no proximal
nucleophiles. A hypothetical reaction mechanism (Figure 1c) of
DSH-mediated lysine acetylation is (1) dynamic thiol−
thioester exchange between acetyl-CoA and the mercapto-
methyl group, generating DSAc (2); (2) enhanced acetylation
activity by the generation of acetylpyridinium intermediate 3
through acetyl group migration; and (3) transfer of the
activated acetyl group to the amino group of a lysine, producing
N-acetyl lysine. Density functional theory (DFT) calculations
suggested that conjugation of the mercaptomethyl group to
DMAP markedly decreases the activation energy to form
acetylpyridinium ion from methyl thioacetate (the highest
activation energies of 33.5 kcal/mol vs 19.0 kcal/mol for direct
and stepwise activation by DMAP and DSH, respectively;
Figure S5), supporting the postulated reaction mechanism in
Figure 1c.
Regioselective Histone Acetylation in Recombinant
Nucleosomes by DSH. We then synthesized DSH derivatives
containing ligands that bind to specific nucleosome regions.
Because DSH selectively acetylates proximal lysine residues
(Figure 1b), we expected that a limited number of lysine
residues located near the ligand-binding site would be
selectively acetylated when DSH is conjugated with ligands.
Kaposi’s sarcoma-associated herpesvirus LANA (latency-
associated nuclear antigen) is an 1162-amino acid viral protein
interacting with an acidic patch of H2A-H2B via its N-
terminus.32 Thus, we synthesized LANA-DSH (4, Figure 2a) by
conjugating DSH with the N-terminus of LANA (residues 5−
D
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15), the minimum chromosome-binding region of LANA.32 X-
ray crystallography analysis confirmed that LANA-DSH binds
to the acidic patch (Figure 2b and Table S1).32 Although the
electron density of the DSH moiety was not determined, the
structural information suggests that the DSH moiety of LANA-
DSH is proximally located to H2BK120 on the disk surface of
the nucleosome (Figure 2b). We used pyrrole-imidazole
polyamide (PIP)-DSH (5) as an alternative catalyst containing
a PIP ligand moiety that binds to DNA with a 5′-WGWWWW-
3′ (where W is A or T) sequence33 (Figure 2a). To evaluate the
DNA sequence-dependency of lysine residue selectivity, we
prepared three kinds of recombinant nucleosomes in which the
recognition DNA sequence by PIP-DSH was proximal to three
different lysine residues on the lateral surface of the nucleosome
(H4K77, H3K56, and H3K122 in nucleosomes 1, 2, and 3,
respectively, Figure 2c; for DNA sequences, see the Supporting
Information).34
Western blot analysis using residue-specific antiacetyl lysine
antibodies indicated that acetyl-CoA with LANA-DSH (2 μM)
acetylated H2BK120 regardless of the DNA sequence in the
nucleosomes (0.37 μM, Figure 2d). On the other hand, acetyl-
CoA with PIP-DSH (5 μM) acetylated lysine residues proximal
to its recognition DNA sequence; H4K77, H3K56, and
H3K122 of nucleosomes 1, 2, and 3 (0.37 μM) were selectively
acetylated, respectively (Figure 2d). Liquid chromatography
tandem mass spectroscopy (LC−MS/MS) analysis revealed
that acetyl-CoA with LANA-DSH acetylated H2BK120 in high
yield and selectivity (69 6%: Figure 2e, red bars), while no
significant acetylation of any lysine residue proceeded in the
absence of LANA-DSH (Figure 2e, black bars; see Table S2 for
each value). Acetylation at H2BK116 was detected to a minor
3a and Figure S7c,d). Our results indicate that ligand-
conjugated DSH can introduce various types of biologically
relevant acylations at predictable lysine residues, illustrating its
utility for regioselective acylations of histones.
Biochemical Properties of H2BK120-Acylated Nucle-
osomes. H2BK120 acetylation and other acylations, such as
ubiquitination and malonylation, are observed in higher
eukaryotes.9 H2BK120 ubiquitination is known to promote
gene transcription through structural changes of chromatin5,35
and/or downstream histone lysine methylations.7,13 While
acetylation of H2BK120 is enriched in core promoter regions of
actively transcribed genes,36,37 it remains to be elucidated how
this acetylation modulates the properties of chromatin. The
functional roles of H2BK120 malonylation38 are not known. As
LANA binds not only to recombinant nucleosomes but also
endogenous chromatin32 containing polynucleosomes with
various native PTMs, we envisioned that acylation by LANA-
DSH could be used as a versatile approach to study the
function of H2BK120 acylations.
We first produced H2BK120-acetylated (69
6%) and
-malonylated (65 5%) nucleosomes by treating recombinant
nucleosomes with LANA-DSH and the corresponding acyl-
CoAs. A nucleosome thermal stability assay39 indicated that
neither acetylation nor malonylation of H2BK120 affected
nucleosome stability (Figure 4a). On the other hand, Mg2+-
promoted nucleosome self-association40 was significantly
inhibited by LANA-DSH-mediated acetylation or malonylation
of H2BK120 (Figure 4b). The effect of malonylation was
greater than that of acetylation (Figure 4b). The inhibitory
effects on nucleosome self-association were significantly
suppressed by H2BK120R mutation (Figure S8), indicating
that H2BK120 acylation was responsible for these effects.
These results suggest that the H2BK120 acylation attenuates
internucleosomal interactions without affecting the stability of
individual nucleosomes.
Regioselective Histone Acylation in Native Chroma-
tin. Next, to evaluate the effects of H2BK120 acetylation and
malonylation on native chromatin, we treated purified nuclei
with LANA-DSH and the corresponding acyl-CoAs (Figure
5a). We initially found that addition of only acetyl-CoA
promoted histone tail acetylation (Figure 5b and Figure S9a),
probably mediated by endogenous HATs in the purified nuclei.
Several types of HAT recognize acetyl-CoA through the CoA
moiety,24 and therefore we hypothesized that endogenous
HAT-mediated acetylation could be suppressed when thioesters
lacking the CoA moiety were used as an acetyl donor. As
expected, p300 HAT hardly promoted histone acetylation of
recombinant nucleosomes using TEG-Ac (6, Figure 5c and
Figure S9b). In the purified nuclei, the increase of histone tail
acetylation by TEG-Ac was also barely detectable (Figure 5b
and Figure S9a). LANA-DSH, however, activated TEG-Ac,
promoting selective acetylation of H2BK120 in the presence of
other nucleoplasmic proteins (Figure 5b, acetic anhydride was
used as a nonselective acetylation control).
extent (15.1
0.3% yield) under acetyl-CoA + LANA-DSH
conditions due to its proximity to H2BK120 (Figure 2b). LC−
MS/MS analyses of the nucleosomes treated with acetyl-CoA +
PIP-DSH indicated that PIP-DSH promoted the target
acetylation with a moderate regioselectivity (Figure S6, Table
S3). This moderate regioselectivity could be due to the
nonoptimized PIP-sequence and linker length. These findings
indicate that DSH promotes acetylation of lysine residues at
predictable sites in nucleosomes by conjugating with suitable
ligands.
Expansion of Histone Acylation Types. Other acylation
types, such as malonylation, are observed in cellular
chromatins.9 DSH activated acyl-CoAs containing a variety of
acyl groups and promoted the corresponding acylations in a
peptide-model substrate (Figure S3d). Moreover, treatment of
a recombinant nucleosome with LANA-DSH and acyl-CoAs led
to acylations (e.g., formylation, butyrylation, biotinylation, 2-
hydroxyisobutyrylation, malonylation, and glutarylation) at
H2BK120 (Figure 3a). Those acylations were significantly
suppressed in the H2BK120R mutant nucleosome, in which
H2BK120 was replaced with arginine (R), suggesting that the
acylations were H2BK120-selective (Figure 3b and Figure S7a).
In addition, acylations of nonhistone proteins hardly proceeded
when using a mixture of a recombinant nucleosome and HeLa
cell extract as substrates, indicating high histone-selectivity
(Figure 3b and Figure S7a). LANA-DSH can also transfer non-
natural acyl groups (azidoacetyl and phosphoryloxyacetyl) from
the corresponding acyl-CoAs to the H2BK120 residue (Figure
3a). Further, using ubiquitin-MES (mercaptoethanesulfonate)
thioester as an acyl donor, LANA-DSH can efficiently promote
monoubiquitination at H2BK120 in a nucleosome, which is
genuinely the same structure as that observed in cells (Figure
We then treated the nuclei with micrococcal nuclease
(MNase) to isolate nucleosomal arrays, the average of whose
length was around 3-kb (Figure 5d,e). LC−MS/MS analysis
indicated that treatment with both LANA-DSH and TEG-Ac
significantly increased H2BK120 acetylation, but did not
significantly increase histone tail acetylation (Figure 5f, see
Figure S9c for other residues). Acetylation by LANA-DSH
reduced Mg2+-dependent sedimentation of the nucleosomal
arrays (Figure 5g). Taken together with the results using
E
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S10c). Similarly to acetylation, malonylation by LANA-DSH
also reduced the sedimentation of the nucleosomal arrays
isolated from sodium butyrate-untreated and -treated cells
(Figure S10c−e), suggesting that H2BK120 malonylation can
also attenuate self-association of the nucleosomal array. Taken
together, these results suggest that H2BK120 acylations
modulate higher-order structures of native chromatin, and
that the effect is likely cooperative with those of histone tail
acetylation.
DISCUSSION
■
In this study, we developed a novel synthetic acylation system
involving a DSH catalyst and an acyl-donor, as a useful method
to evaluate functions of divergent lysine acylations. This is the
first synthetic system to promote regioselective histone
acylations of native chromatin without genetic manipulations.
While both HATs and DSH use an acyl-CoA as an acyl source,
the mechanism of acyl-transfer and the types of acylations that
can be introduced differ. Mechanistic studies of some HATs
indicated that ammonium groups of lysine residues are
activated via deprotonation with a glutamate residue in the
reaction cavity of HATs, which triggers nucleophilic attack of
the lysine amino group to acyl-CoA.24 In contrast, DSH
activates the acyl-CoA side through a dynamic thiol−thioester
exchange, followed by presumable nucleophilic catalysis (Figure
1c). It is noteworthy that HATs catalyzing anionic acylations
have not yet been reported, suggesting that each HAT
promotes a narrow scope of acylations. In contrast, DSH is
versatile, and can promote a wider range of acylations, including
anionic acylations and even non-natural acylations. Importantly,
direct and regioselective introduction of the anionic acylations
on lysines in proteins, such as histones, has not yet been
reported.44 The modularity of our catalyst is advantageous. The
lysine residue-selectivity as well as protein selectivity of our
catalysts can be easily modulated in a predictable manner
simply by changing the ligand conjugated to the DSH moiety
(Figure 2 and Figure S11). Therefore, synthetic acylation
approaches using DSH catalysts targeting nucleosomes through
regions other than the acidic patch or DNA, such as histone
tails,45 or nonhistone proteins would also be versatile tools for
elucidating the roles of biologically relevant acylations of
histone as well as nonhistone proteins, when such a ligand is
available.
We demonstrated, for the first time, the effects of histone
lysine malonylation at a specific site in chromatin structures,
and provided a functional comparison between acetylation and
malonylation. Our data suggest that acetylation of H2BK120, in
concert with histone tail acetylation, attenuates the self-
association of nucleosomal arrays, consistent with the fact
that H2BK120 acetylation coexists with a series of histone tail
acetylations at active gene promoter regions.37 Interestingly,
malonylation of H2BK120 induced slightly stronger suppres-
sion of internucleosomal interactions than acetylation, suggest-
ing that H2BK120 malonylation might be also associated with
active gene transcription. The distribution of H2BK120
malonylation in chromatin needs to be determined for further
characterization. Because H2BK120 is located near the acidic
patch, changing the size of the lysine residue and/or its positive
charge to neutral or negative may inhibit interactions between a
neighboring H4 tail and the acidic patch, which is important for
internucleosomal interactions.46 Although LANA-DSH pro-
moted acetylation of H2BK116 to a slight extent and the
biochemical characterization is based on a mixture of
Figure 4. Biochemical properties of H2BK120-acylated nucleosomes.
(a) Thermal stability assay of H2BK120-acylated nucleosomes.
Relative fluorescence intensity was plotted for every 0.53 °C (from
65 to 95 °C). The concentration of nucleosomes in the assay is 300
ng/μL DNA (3.4 μM). The result using acetylated nucleosome
(H2BK120Ac: 69 6%) is shown in the upper panel, and that using
malonylated (H2BK120Mal: 65 5%) is shown in the lower panel.
NaCl (100 mM) was used as a positive control, and products of
reaction using CoA, instead of acyl-CoAs, were used as a negative
control. Error bars represent the range of two independent
experiments. (b) Mg2+-promoted sedimentation assay of H2BK120-
acylated nucleosomes. The concentration of nucleosomes in the assay
is 120 ng/μL DNA (1.3 μM). The amount of nucleosomes in the
supernatant after incubation with MgCl2 was analyzed. The results
using nucleosomes incubated without LANA-DSH (H2BK120
acylations were not detected) are shown in the upper panel, and
those using nucleosomes acylated by LANA-DSH (H2BK120Ac, 69
6%; H2BK120Mal, 65
5%) are shown in the lower panel. For
control experiments using H2BK120R mutant nucleosomes, see
Figure S8. Error bars represent standard deviations of three
independent experiments. Ac, acetyl; Mal, malonyl.
recombinant nucleosomes (Figure 4), it suggests that
H2BK120 acetylation can attenuate self-association of the
nucleosomal arrays.
Acetylation of H4K16 has been reported to modulate higher-
order chromatin structures, probably by disrupting specific
binding of the H4 tail to the pocket within the H2A−H2B
acidic patch.41,42 Consistent with this, treatment of HeLa cells
with sodium butyrate (a histone deacetylase inhibitor)
promoted H4K16 acetylation and reduced Mg2+-dependent
sedimentation of the nucleosomal arrays (Figure 5h−j, see CoA
+ LANA-DSH condition) as observed previously.43 Intrigu-
ingly, we found that acetylation by TEG-Ac and LANA-DSH
further reduced the sedimentation of the nucleosomal arrays
isolated from sodium butyrate-treated cells (Figure 5h−j and
Figure S9d), suggesting that the effect of H2BK120- and
histone tail-acetylation on the self-association of the nucleoso-
mal arrays could be cooperative.
When the purified nuclei were treated with malonyl-CoA and
LANA-DSH, a protein corresponding to histone H2B was
efficiently and selectively malonylated, while treatment with
only malonyl-CoA exhibited no detectable malonylation
(Figure S10a,b; compound 7 was used as a nonselective
malonylation control). LC−MS/MS analysis suggested that
malonylation by LANA-DSH was H2BK120-selective (Figure
F
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Figure 5. Regioselective histone acetylation in intact nuclei by LANA-DSH and biochemical properties of the acetylated native chromatin. (a)
Schematic for histone acylation in intact nuclei by LANA-DSH. (b) Histone-selective acetylation of intact nuclei by LANA-DSH and acetyl donors.
Purified nuclei were incubated with the indicated acetyl donor (2 mM) with and without LANA-DSH (0.1 mM) in the presence of TCEP (0.7 mM)
at 37 °C for 4 h, and acetylated lysines were detected by immunoblotting using indicated antibodies. Protein amount is indicated by Ponceau
staining. Acetic anhydride (0.5 mM) was used as a nonselective acetylation control. (c) The chemical structure of TEG-Ac (6). (d) Schematic for
Mg2+-promoted sedimentation assay of native chromatin acetylated by LANA-DSH. (e−j) Mg2+-promoted sedimentation assay of native chromatin
acetylated by LANA-DSH with (h−j) or without (e−g) pretreatment with sodium butyrate (5 mM) for 24 h before preparation of the nuclei.
Purified nuclei were acetylated as in (b). Agarose gel electrophoresis image of DNA purified from the nucleosomal arrays (e,h) and percentage of the
acetylated lysine in the sum of the acetylated and unmodified lysines at selected residues of the nucleosomal arrays determined by LC−MS/MS
analysis (error bars represent standard deviations of three independent experiments.) (f) and (i) are shown. DNA was stained with ethidium
bromide (EtBr). The amount of chromatin in the supernatant after incubation with MgCl2 was plotted as a function of the concentration of MgCl2
(error bars represent standard deviations of three A260 measurements.) (g,j) Ac, acetyl; LD, LANA-DSH; NaB, sodium butyrate.
heterogeneously modified nucleosomes, proper control experi-
ments using easily accessible H2BK120R mutant nucleosomes
enabled the functional studies of the target H2BK120
acylations.
DSH (Figure S4), could be used as an exogenous acetyl donor.
Because of the high affinity of LANA-peptide to the acidic
patch, LANA-DSH may remain bound to the acidic patch after
promoting the acyl transfer reaction and affect downstream
biological outputs. Future investigations will be directed at
improving the properties of the synthetic catalysts, such as cell
permeability, intracellular stability, and reversible histone
binding, toward developing a new method to dissect epigenetic
mechanisms in living systems. This direction of the research is
now undergoing in our laboratory. Additionally, such a method
may open new therapeutic strategies in which synthetic
acylation would be used to restore the correct network of
PTM, leading to cures for diseases caused by the dysregulation
of histone PTM.
In principle, our system for regioselective histone acylation
can be expanded to living cells or even organisms. We have
already confirmed that lysine acetylation of recombinant
nucleosomes by LANA-DSH proceeds even in the presence
of the physiological concentration of glutathione, which is a
cellular thiol nucleophile and can interfere with the thiol−
thioester exchange between DSH and the acetyl donor, albeit
with a decreased efficiency (Figure S12). Although acetyl-CoA
is not membrane-permeable, N,S-diacetylcysteamine, which is a
permeable,47 truncated acetyl-CoA surrogate and is activated by
G
DOI: 10.1021/jacs.7b02138
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.7b02138.
■
S
Synthetic protocols, spectroscopic characterizations,
experimental details, Figures S1−12, and Tables S1−S3
(PDF)
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Representative LC−MS/MS data (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*skawashima@mol.f.u-tokyo.ac.jp
*yamatsugu@mol.f.u-tokyo.ac.jp
*kanai@mol.f.u-tokyo.ac.jp
Wiewiora, R.; Ng, W.-L.; Raj, R.; Faulkner, S.; Vallee
́
, M. R. J.;
■
Phanumartwiwath, A.; Coleman, O. D.; Thezenas, M.-L.; Khan, M.;
́
́
Galan, S. R. G.; Lercher, L.; Schombs, M. W.; Gerstberger, S.; Palm-
Espling, M. E.; Baldwin, A. J.; Kessler, B. M.; Claridge, T. D. W.;
Mohammed, S.; Davis, B. G. Science 2016, 354, aag1465.
(19) Yang, A.; Ha, S.; Ahn, J.; Kim, R.; Kim, S.; Lee, Y.; Kim, J.; Soll,
̈
ORCID
D.; Lee, H.-Y.; Park, H.-S. Science 2016, 354, 623.
(20) Muller, M. M.; Muir, T. W. Chem. Rev. 2015, 115, 2296.
Hitoshi Kurumizaka: 0000-0001-7412-3722
Motomu Kanai: 0000-0003-1977-7648
̈
(21) Methods have been reported for the locus-specific acetylation of
endogenous chromatin using dCas9-p300 fusion: Hilton, I. B.;
D’Ippolito, A. M.; Vockley, C. M.; Thakore, P. I.; Crawford, G. E.;
Reddy, T. E.; Gersbach, C. A. Nat. Biotechnol. 2015, 33, 510.
(22) David, Y.; Vila-Perello, M.; Verma, S.; Muir, T. W. Nat. Chem.
2015, 7, 394.
Author Contributions
⊥Yo.Am. and Yu.Ao. contributed equally.
Notes
The authors declare no competing financial interest.
(23) Elsasser, S. J.; Ernst, R. J.; Walker, O. S.; Chin, J. W. Nat.
Methods 2016, 13, 158.
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ACKNOWLEDGMENTS
■
We thank Prof. Takeshi Kawamura and Dr. Kana Tanabe for
helpful comments on LC−MS/MS analysis. We thank Dr. Jiaan
Liu for the synthesis of nonselective malonyl donor 7. We
thank Hidetoshi Kajino for HRMS analysis of compound S24.
This work was supported by ERATO from JST Grant no.
JPMJER1103 (M.K.) and in part by JSPS KAKENHI Grant no.
JP25116002 (H.K.) and the Japan Agency for Medical Research
and Development (AMED) (H.K.). This manuscript is
dedicated to the memory of Professor Takao Ikariya.
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