Synthesis of ADP-Ribosylated Histones Reveals Site-Specific Impacts on Chromatin Structure and Function

Article abstract of DOI:10.1021/jacs.1c05429

ADP-ribosylation of nuclear proteins is a critical feature of various DNA damage repair pathways. Histones, particularly H3 and H2B, are major targets of ADP-ribosylation and are primarily modified on serine with a single ADP-ribose unit following DNA damage. While the overall impact of PARP1-dependent poly-ADP-ribosylation is heavily investigated, very little is known about the specific roles of histone ADP-ribosylation. Here, we report the development of an efficient and modular semisynthetic route to full-length ADP-ribosylated histones H3 and H2B, chemically installed at specific serine residues. The modified histones were used to generate various chemically defined ADP-ribosylated chromatin substrates, which were employed in biophysical assays. These studies revealed that ADP-ribosylation of serine-6 of histone H2B (H2BS6ADPr) inhibits chromatin folding and higher-order organization; notably, this effect was enhanced by ADP-ribosylation of H3S10. In addition, ADP-ribosylated nucleosomes were utilized in biochemical experiments employing a panel of lysine methyltransferase enzymes, revealing a context-dependent inhibition of histone H3K9 methylation. The availability of designer ADP-ribosylated chromatin described here is expected to facilitate further biochemical and structural studies regarding the roles of histone ADP-ribosylation in the DNA damage response.

Full text of DOI:10.1021/jacs.1c05429

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Synthesis of ADP-Ribosylated Histones Reveals Site-Specific Impacts
on Chromatin Structure and Function
Nir Hananya, Sara K. Daley, John D. Bagert, and Tom W. Muir*
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ABSTRACT: ADP-ribosylation of nuclear proteins is a critical feature of various DNA damage repair pathways. Histones,
particularly H3 and H2B, are major targets of ADP-ribosylation and are primarily modified on serine with a single ADP-ribose unit
following DNA damage. While the overall impact of PARP1-dependent poly-ADP-ribosylation is heavily investigated, very little is
known about the specific roles of histone ADP-ribosylation. Here, we report the development of an efficient and modular
semisynthetic route to full-length ADP-ribosylated histones H3 and H2B, chemically installed at specific serine residues. The
modified histones were used to generate various chemically defined ADP-ribosylated chromatin substrates, which were employed in
biophysical assays. These studies revealed that ADP-ribosylation of serine-6 of histone H2B (H2BS6ADPr) inhibits chromatin
folding and higher-order organization; notably, this effect was enhanced by ADP-ribosylation of H3S10. In addition, ADP-
ribosylated nucleosomes were utilized in biochemical experiments employing a panel of lysine methyltransferase enzymes, revealing a
context-dependent inhibition of histone H3K9 methylation. The availability of designer ADP-ribosylated chromatin described here is
expected to facilitate further biochemical and structural studies regarding the roles of histone ADP-ribosylation in the DNA damage
response.
ells are constantly exposed to exogenous and endogenous
genotoxic agents which induce various DNA lesions,
study biochemical effects (Figure 1). In contrast to most other
known histone post-translational modifications, currently there
exists no in vitro strategy for the preparation of chemically
defined chromatin containing a native ADP-ribose (ADPr)
modification. This is particularly striking considering the
enormous value of such “designer” chromatin substrates for
studying attendant biochemistry.13−15 This problem motivated
us to develop a method for generating chemically defined
ADP-ribosylated histones, which were further used to assemble
designer ADP-ribosylated chromatin substrates. These reagents
were employed in various biophysical and biochemical assays
to explore the effects of histone ADP-ribosylation.
Inspired by recent progress on the generation of O-linked
ADP-ribosylated peptides using synthetic16−19 or enzymatic11
means, we imagined that native full-length ADP-ribosylated
histones could be accessible through a semisynthetic
approach,20 that is, by traceless native chemical ligation of an
ADP-ribosylated peptide α-thioester with a recombinant
fragment containing an N-terminal cysteine. H2B contains an
alanine at position 17, suggesting that native H2BS6ADPr
could be generated by ligation of an H2B(1−16)S6ADPr
synthetic peptide α-thioester with a recombinant H2B(17−125)
containing an A17C mutation, followed by desulfurization to
convert the cysteine ligation “scar” to a native alanine residue.
C
including DNA breaks.1 Since single- or double-strand DNA
breaks pose a threat to genome integrity and cell viability, cells
have evolved various repair pathways to correct such
damage.2,3 One of the first responders to broken DNA is
poly(ADP-ribose)polymerase 1 (PARP1)this enzyme is
activated upon binding to DNA breaks and, subsequently,
catalyzes the mono- and poly-ADP-ribosylation of a multitude
of nuclear proteins.4,5 The physiological roles of ADP-
ribosylation include modulation of chromatin structure and
recruitment of proteins that participate in DNA repair.6,7 The
importance of protein ADP-ribosylation in various DNA repair
pathways makes PARP1 an important target for cancer
therapy.8 Because of the biological and clinical significance of
ADP-ribosylation, it is essential to understand the cellular
pathways involved in its regulation and delineate its function
within the DNA repair process.
Histone proteins are major targets of ADP-ribosylation, and
several serine residues, H2BS6, H3S10, and to a lesser extent
H3S28, are the most abundant ADP-ribosylation sites within
core histones.4,9,10 While for many years it has been assumed
that the major final output of PARP1 signaling is poly-ADP-
ribosylation, recent studies show that for nuclear proteins in
general, and histone proteins in particular, a dedicated
glycohydrolase rapidly trims this initial polymeric modification
back to the mono-ADP-ribosyl unit and that this is thought to
be the predominant species upon DNA damage.11,12
Importantly, while the overall impact of PARP1 activation
upon DNA damage is the subject of intensive investigation,
very little is known about the specific roles of histone ADP-
ribosylation, in large part due to the lack of molecular tools to
Received: May 26, 2021
Published: July 15, 2021
https://doi.org/10.1021/jacs.1c05429
J. Am. Chem. Soc. 2021, 143, 10847−10852
© 2021 American Chemical Society
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Figure 1. Histones become mono-ADP-ribosylated in response to DNA damage. Binding of histone PARylation factor 1 (HPF1) to PARP1 is
required for ADP-ribosylation of serine residues on histones H3 and H2B. The action of poly(ADP-ribose) glycohydrolase (PARG) rapidly trims
initial poly-ADP-ribosylated products back to mono-ADP-ribosylated histones (inset). The impact of this modification on chromatin structure and
function is poorly understood.
Figure 2. Synthesis of ADP-ribosylated “designer” chromatin. (a) Scheme showing the chemical synthesis of ADP-ribosylated synthetic peptide α-
thioesters (H31−14 sequence is depicted). Key building blocks 1 and 2 are shown in the inset. (b) Generation of ADP-ribosylated histones by
traceless native chemical ligation. (c, d) ESI-MS characterization of H2BS6ADPr (c) and H3S10ADPr (d). Inset: Deconvoluted MS. (e) Native
PAGE of indicated reconstituted MNs. Treatment with the ADP-ribosylhydrolase, ARH3, efficiently removes the modification as indicated by a
change in migration on the gel. (f) Left: Analysis of the ADP-ribosylated 12-mer arrays by native gel electrophoresis on an agarose-acrylamide
composite gel. Right: Native PAGE of the same arrays following restriction enzyme digestion to MNs and treatment with ARH3. UM: Unmodified.
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Similarly, native H3S10ADPr could be generated by ligation of
an H3(1−14)S10ADPr synthetic peptide α-thioester with a
recombinant H3(15−135) containing an A15C mutation,
followed by desulfurization.
a faster-migrating species that corresponds to the unmodified
MN (Figure 2e), which indicates that the ADP-ribosylated
MNs contain the native α-anomeric serine-ADP-ribose link-
age.17 In addition, all MNs were tested in a fluorescence-based
thermal stability assay (Figure S4).27 The ADP-ribosylated
MNs exhibited a melting temperature similar to that of the
unmodified MN, further indicating that the presence of the
ADP-ribose moiety does not affect proper nucleosome
assembly or thermal stability.
To prepare the requisite ADP-ribosylated peptide α-
thioesters, we designed a modified ‘on-resin pyrophosphate
formation’ strategy,16,17 as depicted in Figure 2a. A hydrazine
linker, a thioester surrogate, was loaded on a trityl resin.21
Standard Fmoc-based SPPS was used to synthesize the peptide
sequence, in which the phosphoribosylated serine derivative 1
(see Supporting Information for synthetic details) was
incorporated at the appropriate position (S6 for H2B, S10
for H3). The phosphate group in compound 1 is protected
with allyl moieties, which enabled Pd-catalyzed selective
deprotection of the phosphate following peptide chain
synthesis. Subsequently, the pyrophosphate bond was formed
on-resin through a reaction between the free phosphate and
the Boc-protected adenosine-phosphoramidite 2 (see Support-
ing Information for synthetic details), followed by phosphite
oxidation. The 2-cyanoethyl group was removed by DBU, and
subsequent cleavage and side-chain deprotection with TFA
yielded the ADP-ribosylated peptide hydrazide. Hydrazides are
often converted to thioesters via an acyl azide intermediate
through treatment with nitrous acid;21 however, the adenine
moiety contains an aromatic amine and is not compatible with
such oxidizing conditions. Therefore, we employed a recent,
milder method in which the peptide hydrazide is reacted with
acetylacetone (acac) to provide an acyl pyrazole intermedi-
ate.22 This active species further reacts with 4-mercaptophenyl-
acetic acid (MPAA), generating the corresponding thioester.
The overall synthetic workflow provided highly pure ADP-
ribosylated peptide α-thioesters: H2B(1−16)S6ADPr and
H3(1−14)S10ADPr (Figure S1), in 26% and 12% yield,
respectively.
Following successful synthesis of the ADP-ribosylated
peptide α-thioesters, we turned to the preparation of full-
length ADP-ribosylated histones. The N-terminal cysteine-
containing fragments H2B(17−125)A17C and H3(15−135)A15C
were expressed in Escherichia coli as reported previously.23,24
Native chemical ligation of the peptide thioesters with the
corresponding recombinant fragments, followed by free-
radical-mediated desulfurization of the cysteine ligation “scar”
proceeded smoothly, affording the native full-length ADP-
ribosylated proteinsH2BS6ADPr and H3S10ADPr (Figures
2b−-d and S2).
Next, we utilized the modified histone proteins for the
reconstitution of ADP-ribosylated chromatin substrates,
starting with mononucleosomes (MNs). To this end, we
refolded three different ADP-ribosylated histone octamer
complexes, which contained either ADP-ribosylated H2B,
ADP-ribosylated H3, or both, in addition to a control octamer
complex in which all histones are unmodified. The various
octamers were mixed with the “Widom 601” nucleosome-
positioning DNA sequence25 and salt gradient dialysis was
employed to reconstitute MNs. Analysis by native poly-
acrylamide gel electrophoresis (PAGE) indicated that the
bulky, anionic ADP-ribose moiety did not impede efficient MN
formation, although, as expected, the modified MNs migrate
slightly slower compared to the unmodified MN (Figures 2e
and S3). To evaluate the authenticity of the ADP-ribosylated
MNs, we treated them with the ADP-ribosylhydrolase, ARH3,
which removes the ADP-ribose mark from serine residues.26
Treatment with ARH3 converted all ADP-ribosylated MNs to
In addition to MNs, the modified histones were utilized to
prepare ADP-ribosylated 12-mer nucleosome arrays; such
reagents are useful for gathering information about the
regulation of chromatin folding and compaction.28,29 To this
end, the various histone octamer complexes were mixed with a
DNA template that harbors 12 copies of the “Widom 601”
sequence, separated by a 35-bp linker DNA. Salt gradient
dialysis and subsequent Mg2+-induced precipitation provided
pure 12-mer arrays (Figure 2f). Digestion of the arrays to MNs
with BstXI restriction enzyme yielded the expected ADP-
ribosylated MNs, as indicated by native PAGE (Figure 2f).
With the designer chromatin substrates in hand, we set out
to explore the biophysical and biochemical effects of histone
ADP-ribosylation. The first question we aimed to address is
whether the modification alters chromatin higher-order
structure. It is well established that PARP1-mediated poly-
ADP-ribosylation induces chromatin relaxation,30 thereby
facilitating the accessibility of DNA repair factors to the
damage site.31 However, it is unknown whether chromatin
relaxation requires long poly(ADP-ribose) chains or whether
histone mono-ADP-ribosylation is sufficient to impede
chromatin compaction. This question is of particular
importance since mono-ADP-ribosylation is the primary form
of histone ADP-ribosylation in PARP1-dependent DNA
damage signaling.11 Furthermore, it is not clear whether the
site-specificity of histone ADP-ribosylation is important in
determining its potential impact on chromatin decompaction.
To address these questions, we performed sedimentation
velocity analysis of the various 12-mer nucleosome arrays in
the absence or presence of 1 mM Mg2+ ions. Without Mg2+
supplementation, the various nucleosome arrays adopt an
extended structure and sediment similarly at around 32 S
(Figure 3a). As expected,32 the addition of 1 mM Mg2+ to the
unmodified array induced the formation of a more compact
structure that sediments at 45 S (Figure 3a). Remarkably,
whereas the H3ADPr array behaves similarly to the unmodified
control, the H2BADPr array sediments significantly slower (41
S) upon Mg2+ addition (Figure 3a). The dual-modified ADPr
array is even less compacted and sediments at 36 S, a surprising
result given that the H3ADPr modification on its own has no
obvious effect. Notably, pretreatment of the same batch of
modified arrays with ARH3 resulted in sedimentation behavior
similar to the unmodified control (Figure S5).
To further explore the impact of ADP ribosylation on
chromatin structure, we measured the effect of the
modification on Mg2+-mediated array self-association, as
indicated by the decrease in the material that remained in
the supernatant upon centrifugation.33 As before, we observed
differential behavior depending on which histone the ADP-
ribose mark was located; H3ADPr arrays again behaved
similarly to the unmodified control, whereas attachment of the
modification to H2B inhibited array self-association as
reflected by the higher Mg2+ concentration needed to
precipitate the sample (Figure 3b). Also consistent with the
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trend observed in sedimentation velocity analysis, an even
bigger inhibitory effect was observed for arrays containing the
modification on both H2B and H3. Thus, using two quite
different measures of chromatin compaction, we observe that
the inhibitory effect of ADP ribosylation is enhanced when it is
present on the two histone tails, even though as a single
modification an effect is only observed when attached to H2B.
The physical basis of this apparent nonadditive behavior
warrants further study, including whether the presence of the
H3ADPr modification somehow enhances the existing
inhibitory effect of H2BADPr or conversely whether the latter
somehow turns on an otherwise latent property of the former.
Regardless, our data clearly suggest that the mono-ADP-
ribosylation of histones is sufficient to inhibit chromatin fiber
compaction and interarray interactions. These findings imply a
role of histone mono-ADP-ribosylation in chromatin relaxation
in the context of DNA repair.
Our next goal was to explore the interplay between histone
ADP-ribosylation and other histone post-translational mod-
ifications. Specifically, we asked how the ADP-ribosylation of
H3S10 affects lysine methylation by different lysine methyl-
transferases (KMTs) that modify the H3 tail. We, therefore,
employed the ADP-ribosylated MNs in histone methyltransfer-
ase experiments with the following KMTs: (i) MLL1 (H3K4
KMT), (ii) G9a (H3K9 KMT), (iii) PRC2 (H3K27 KMT),
and (iv) NSD2 (H3K36 KMT). Methylation of H3K4,
H3K27, and H3K36 was detected by Western blot, as the
antibodies against these methylation sites are insensitive to
H3S10 ADP-ribosylation (Figure S6). H3K9 methylation was
detected by autoradiography since the ADPr moiety on H3S10
is expected to interfere with antigen recognition by the
H3K9me antibody. ADP-ribosylation of H3S10 inhibits G9a-
mediated H3K9 methylation; a decrease of 85% in the
autoradiography signal is observed with H3ADPr- and
Figure 3. Effect of ADP ribosylation on higher-order chromatin
structure. (a) Integrated sedimentation coefficient distributions of
indicated 12-mer nucleosome arrays in the presence (solid lines) or
absence (dashed lines) of 1 mM Mg2+, as determined by
sedimentation velocity experiments and van Holde-Weischet analysis.
(b) Mg2+-mediated self-association of the various arrays, determined
by the percentage of material remaining in solution following
centrifugation. Error = S.D., n = 3.
Figure 4. Effect of ADP ribosylation on histone methyl transferase activity (a) Autoradiogram showing G9a-mediated H3K9 methylation of the
various MNs. Coomassie stain of core histones was used as a loading control for the MNs. (b) Quantification of the autoradiogram in panel a: error
= S.D., n = 3. (c−e) Representative Western blots showing the impact of ADP ribosylation on the methyltransferase activity of MLL1 (c), PRC2
(d), and NSD2 (e). In each case, H4 was used as a loading control.
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H3ADPr/H2BADPr-MNs (Figure 4a and b). By contrast,
H2BS6 ADP-ribosylation on its own does not affect G9a
activity. This suggests that the inhibitory effect of H3S10 ADP-
ribosylation on G9a activity occurs in cis and is presumably
due to an inability of G9a to engage its target lysine (which is
the preceding amino acid in the substrate sequence). Previous
work employing an ADP-ribosylated peptide, showed that
H3S10 ADP-ribosylation interferes with H3K9 methylation
carried out by the fungal KMT, Dim5.34 Our data extends this
to the human H3K9 KMT, G9a, using a physiologically
relevant substrate, an ADP-ribosylated MN. Notably, we and
others have previously shown that modifications on H3K9
inhibit H3S10 ADP-ribosylation by the PARP1/HPF1
complex.24,34 Thus, there exists a reciprocal negative crosstalk
between modifications on these adjacent amino acids. In
contrast to G9a-mediated H3K9 methylation, the methylation
of H3K4 and H3K27 is moderately increased by H3S10 ADP-
ribosylation (Figures 4c,d and S7). This effect may be related
to modulation of the H3-tail dynamics by ADP-ribosylation, as
H3-tail modifications that weaken its binding to nucleosomal
DNA increase accessibility to tail-interacting proteins.35
Consistent with this idea, we did not observe a significant
effect of histone ADP-ribosylation on the methylation of
H3K36 (Figures 4e and S7), which we note resides at the base
of the H3 N-terminal tail and hence might be expected to be
less affected by changes in dynamics. Further research is
required to explore the possible effect of ADP-ribosylation on
histone tail dynamics and how this might modulate the
interaction of the tail with chromatin modifiers including those
relevant to DNA repair pathways.
In conclusion, we developed an efficient synthesis of ADP-
ribosylated peptide α-thioesters, which enabled production of
native full-length ADP-ribosylated histones through protein
semisynthesis. ADP-ribosylated H2B and H3 were utilized to
generate chemically defined ADP-ribosylated mononucleo-
somes and nucleosome arrays, which were employed in various
biophysical and biochemical experiments. These investigations
revealed that (i) histone mono-ADP-ribosylation is sufficient
to impede chromatin compaction and histone PTM deposition
and (ii) the specific site of ADP-ribosylation is critical in
determining these effects. This work sheds light on a role of
histone mono-ADP-ribosylation in DNA repair and also
highlights the value of designer ADP-ribosylated chromatin
substrates in exploring the effects of this unique modification.
More generally, we imagine that by extending the scope of
protein semisynthesis to ADP-ribosylated targets, the proce-
dures described herein will help illuminate the roles of this
fascinating and poorly understood post-translational modifica-
tion.
orcid.org/0000-0001-9635-0344; Email: muir@
princeton.edu
Authors
Nir Hananya − Department of Chemistry, Princeton
University, Princeton, New Jersey 08544, United States
Sara K. Daley − Department of Chemistry, Princeton
University, Princeton, New Jersey 08544, United States
John D. Bagert − Department of Chemistry, Princeton
University, Princeton, New Jersey 08544, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c05429
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Merck Research Laboratories for financial support
(LKR 179713). N.H. is a Robert Black Fellow of the Damon
Runyon Cancer Research Foundation, DRG-2425-21. We
thank E. Ge for providing PRC2, and other members of the
Muir lab for helpful discussions.
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ASSOCIATED CONTENT
■
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* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c05429.
Experimental details, supplementary figures, synthetic
protocols, and NMR spectra (PDF)
2021.01.26.428255, January 27, 2021.
2021.01.26.428255 (accessed 2021-05-26).
DOI: 10.1101/
AUTHOR INFORMATION
■
Corresponding Author
Tom W. Muir − Department of Chemistry, Princeton
University, Princeton, New Jersey 08544, United States;
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