Poly-acetylated chromatin signatures are preferred epitopes for site-specific histone H4 acetyl antibodies.
ABSTRACT Antibodies specific for histone post-translational modifications (PTMs) have been central to our understanding of chromatin biology. Here, we describe an unexpected and novel property of histone H4 site-specific acetyl antibodies in that they prefer poly-acetylated histone substrates. By all current criteria, these antibodies have passed specificity standards. However, we find these site-specific histone antibodies preferentially recognize chromatin signatures containing two or more adjacent acetylated lysines. Significantly, we find that the poly-acetylated epitopes these antibodies prefer are evolutionarily conserved and are present at levels that compete for these antibodies over the intended individual acetylation sites. This alarming property of acetyl-specific antibodies has far-reaching implications for data interpretation and may present a challenge for the future study of acetylated histone and non-histone proteins.
- SourceAvailable from: Brian D Strahl[show abstract] [hide abstract]
ABSTRACT: Histones, the fundamental packaging elements of eukaryotic DNA, are highly decorated with a diverse set of post-translational modifications (PTMs) that are recognized to govern the structure and function of chromatin. Ten years ago, we put forward the histone code hypothesis, which provided a model to explain how single and/or combinatorial PTMs on histones regulate the diverse activities associated with chromatin (e.g., gene transcription). At that time, there was a limited understanding of both the number of PTMs that occur on histones and the proteins that place, remove, and interpret them. Since the conception of this hypothesis, the field has witnessed an unprecedented advance in our understanding of the enzymes that contribute to the establishment of histone PTMs, as well as the diverse effector proteins that bind them. While debate continues as to whether histone PTMs truly constitute a strict "code," it is becoming clear that PTMs on histone proteins function in elaborate combinations to regulate the many activities associated with chromatin. In this special issue, we celebrate the 50th anniversary of the landmark publication of the lac operon with a review that provides a current view of the histone code hypothesis, the lessons we have learned over the last decade, and the technologies that will drive our understanding of histone PTMs forward in the future.Journal of Molecular Biology 01/2011; 409(1):36-46. · 3.91 Impact Factor
Article: Histone modification: cause or cog?[show abstract] [hide abstract]
ABSTRACT: Histone modifications are key components of chromatin packaging but whether they constitute a 'code' has been contested. We believe that the central issue is causality: are histone modifications responsible for differences between chromatin states, or are differences in modifications mostly consequences of dynamic processes, such as transcription and nucleosome remodeling? We find that inferences of causality are often based on correlation and that patterns of some key histone modifications are more easily explained as consequences of nucleosome disruption in the presence of histone modifying enzymes. We suggest that the 35-year-old DNA accessibility paradigm provides a mechanistically sound basis for understanding the role of nucleosomes in gene regulation and epigenetic inheritance. Based on this view, histone modifications and variants contribute to diversification of a chromatin landscape shaped by dynamic processes that are driven primarily by transcription and nucleosome remodeling.Trends in Genetics 07/2011; 27(10):389-96. · 9.77 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Histone acetylation regulates many cellular processes, and specific acetylation marks, either singly or in combination, produce distinct outcomes. For example, the acetylation pattern on newly synthesized histones is important for their assembly into nucleosomes by histone chaperones. Additionally, the degree of chromatin compaction and folding may be regulated by acetylation of histone H4 at lysine 16. Histone acetylation also regulates the formation of heterochromatin; deacetylation of H4 lysine 16 is important for spreading of heterochromatin components, whereas acetylation of this site serves as a barrier to this spreading. Finally, histone acetylation is critical for gene transcription, but recent results suggest that deacetylation of certain sites also plays an important role. There are many histone acetyltransferases (HATs) and deacetylases, with differing preferences for the various histone proteins and for specific sites on individual histones. Determining how these enzymes create distinct acetylation patterns and regulate the functional outcome is an important challenge.Annual Review of Biochemistry 02/2007; 76:75-100. · 27.68 Impact Factor
Poly-acetylated chromatin signatures are
preferred epitopes for site-specific
histone H4 acetyl antibodies
Scott B. Rothbart1,2, Shu Lin3, Laura-Mae Britton3, Krzysztof Krajewski1, Michael-C Keogh4,
Benjamin A. Garcia3& Brian D. Strahl1,2
1Department of Biochemistry & Biophysics,2Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill,
Chapel Hill, NC27599,USA,3Departmentof Molecular Biology, Princeton University, Princeton, NJ 08544, USA,4Department of
Cell Biology, Albert Einstein College of Medicine, New York, NY 10461, USA.
Antibodies specific for histone post-translational modifications (PTMs) have been central to our
understanding of chromatin biology. Here, we describe an unexpected and novel property of histone H4
site-specific acetyl antibodies in that they prefer poly-acetylated histone substrates. By all current criteria,
these antibodies would have normally passed specificity standards. However, we find these site-specific
histone antibodies preferentially recognize chromatin signatures containing two or more adjacent
acetylated lysines. Significantly, we find that the poly-acetylated epitopes these antibodies prefer are
evolutionarily conserved and are present at levels that compete for these antibodies over the intended
individual acetylation sites. This alarming property of acetyl-specific antibodies has far-reaching
implications for data interpretation and may present a challenge for the future study of acetylated histone
and non-histone proteins.
side chains, thus making local chromatin structure more permissive to specific protein machineries5. Lysine
acetylation can also function by serving as a docking site for bromodomain-containing proteins, often found as
subunits of histone acetyltransferases (HATs), ATP-dependent chromatin remodelers, and transcriptional coac-
tivators6,7. Significantly, recent studies show that bromodomain-containing proteins preferentially recognize
poly-acetylated chromatin signatures7–9. These studies lend further support to the ‘histone code’ hypothesis,
which suggests that histone PTMs function in a combinatorial fashion to regulate chromatin architecture and
DNA-templated cellular processes10,11.
Direct investigations of biological functions associated with specific histone PTMs have been facilitated by
genetic and biochemical methods, and often depend on antibodies to monitor these PTMs. Furthermore, large
scale epigenomics efforts, like the ENCODE and modENCODE projects, rely on these antibodies to map the
genomic distribution of chromatin signatures12–14. Therefore, antibody specificity is of utmost importance for
accurate data interpretation. The standard criteria for characterizing antibody specificity typically involves
primary reactivity with a single species from cell extracts by immunoblotting that is diminished in the absence
involve characterizing the ability of antibodies to perform in biological assays, like chromatin immunoprecipita-
tion (ChIP), immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and immunoblots.
Recent studies from our lab and others demonstrate that neighboring PTMs often enhance or perturb the
ability of histone antibodies to recognize their intended target9,15,16. Furthermore, these studies have found that
histone antibodies often have specific difficulties in recognizing the appropriate epitopes, either due to the
inability to distinguish methyl-lysine states (mono-, di-, and tri-methylation) or to recognize off-target PTMs.
fail basic quality control measures17. Here, we uncover a novel property of histone H4 antibody-antigen recog-
he identification and biological characterization of histone post-translational modifications (PTMs) has
been the subject of intense recent investigation1–3. One of the most studied histone PTMs is lysine acetyla-
tion, which typically occurs on the N-terminal ‘‘tails’’ and globular domains of histones and can influence
SCIENTIFIC REPORTS SREP-12-01796.3d25/6/12 18:39:12
21 May 2012
22 May 2012
3 July 2012
requests for materials
should be addressed to
SCIENTIFIC REPORTS | 2 : 489 | DOI: 10.1038/srep00489
use of these reagents. Our findings caution interpreting results to
date that employ these site-specific acetyl antibodies and suggest
more thorough validation of antibodies is needed before they can
be labeled as specific.
Site-specific H4 acetyl antibodies prefer poly-acetylated sub-
strates. To interrogate the interactions of chromatin-associated
proteins and antibodies with combinatorial histone PTMs, we
recently developed a peptide microarray platform where . 250
unique biotinylated histone peptides, containing 0–8 possible
PTMs, were immobilized on streptavidin-coated glass slides
(Supplemental Table 1)9,16. These peptide arrays were probed with
a number of commonly used commercial histone acetyl-specific
antibodies (Supplemental Table 2) to discern their specificities. We
found that acetyl-specific antibodies directed against H3 lysines 9
and 14 (H3K9ac and H3K14ac) generally performed as expected,
in that they showed no discernable interaction with unmodified
histones, and detected their intended PTM alone and in the
context of adjacent acetylation events with similar signal intensity
(Fig. 1a and Supplemental Fig. 1). Of note, H3S10 phosphorylation
(H3S10p) perturbed the recognition of H3K9ac (see peptides 37, 41,
recognition. H3S10p, enriched on mitotic chromatin18, has been
shown to exist on the same histone tail as H3K9ac in cells19–21. Our
array analysis therefore suggests this H3S10 phosphorylated
population of H3K9 acetylated histone tails may be underre-
presented in biological assays using this antibody. We also detected
peptides (Supplemental Fig. 1).
In sharp contrast to above, antibodies designed to recognize
H4K5ac, H4K8ac, and H4K12ac preferentially bound H4 peptides
harboring two or more adjacent acetylation events. Importantly,
acetyl recognition was often dependent on the single acetylation
event that was intended to be recognized by the antibody. For
example, the H4K8ac antibody bound to H4K8ac/K12ac and
H4K8ac/K16ac, but not to the H4K12ac/K16ac peptide (Fig. 1b).
Similar observations were observed for the H4K5ac and H4K12ac
ferred substrates on our arrays for the H4 acetyl antibodies tested,
closer examination of the individually acetylated peptides revealed
bodies do indeed recognize their intended PTM preferentially to
other single acetylation events on the H4 tail (Fig. 1c). By these
criteria, these antibodies would likely be labeled as specific.
However, strong preference for poly-acetylated peptides (4- to 20-
fold; Table 1) suggests an added layer of complexity to the specificity
Table 1 | Antibody preference for tetra-acetyl epitopes
Antibodytetra-acetyl/mono-acetyl (6 s.e.m.)
1.8 6 0.3
1.1 6 0.2
4.0 6 0.3
21.9 6 5.0
18.6 6 2.1
1.7 6 0.3
H4K8ac AbH4K12ac Ab H4K16ac AbH4 tetra-ac Ab
H4K5ac AbH4K8ac Ab
H4 tetra-ac Ab
H4K12ac,16ac + K20me3
H4K12ac,16ac + K20me2
H4R3me1 + K5ac,8ac,12ac,16ac,20ac
H4R3me2s + K5ac,8ac,12ac,16ac,20ac
H4R3me2a + K5ac,8ac,12ac,16ac,20ac
H4S1p + K5ac,8ac,12ac,16ac
H4K12ac + K5Q,8Q,16Q
Figure 1 | (a-c) Heat maps summarizing peptide array results for H3 and H4 acetyl antibodies. For each array, the most intense series of peptide spots
(12 individual spots per peptide) is assigned a value of 1 (blue), and all values are normalized to this peptide. Values $ 0.1 are colored red in panel C to
enable interpretation of low signal intensities. Each interaction is presented as an averaged normalized intensity from at least two independent arrays
(r2. 0.9). See Rothbart et al16for details of the array methodology. Antibody information can be found in Supplemental Table 2. (d) Normalized array
signal intensities for H4K12ac antibody binding to the indicated peptides. Values are presented as an average of 24 individual spots (2 arrays) 6 s.e.m.
SCIENTIFIC REPORTS | 2 : 489 | DOI: 10.1038/srep00489
criteria and implies these antibodies may have difficulty distinguish-
ing singly modified epitopes in vivo. Importantly, the antibody we
tested that recognizes H416ac was not dramatically influenced by
poly-acetylated H4. However, unlike the other site-specific H4 anti-
bodies examined, the H4K16ac antibody cross-reacted with acety-
lated H3 and H2A peptides (Supplemental Fig. 2).
As acetylation masks the positive charge on lysine e-amines, we
next wondered if poly-acetyl recognition might be a consequence of
‘‘charge masking.’’ To test this idea, we synthesized an H4K12ac
peptide in which lysines 5, 8, and 16 were mutated to glutamine, a
commonly used acetyl mimic. Importantly, H4K12ac antibody
recognition of this peptide did not mimic that seen with a tetra-
acetylated peptide (Fig. 1d). These results strongly suggest poly-
acetyl antibody recognition is not solely a property of charge
Poly-acetylated chromatin signatures are evolutionarily con-
served. Our array results suggest that poly-acetylated H4 chro-
matin signatures could be problematic for antibody-based
detection of single H4 acetylation events in cells. However, the
extent to which poly-acetylated H4 chromatin signatures exist in
relation single acetylation events in cells is unknown. We therefore
sought to determine and compare the abundance of histone H4
acetylation signatures by mass spectrometry in budding yeast,
mouse embryonic stem cells (mESCs), mouse embryonic fib-
roblasts (MEFs), and the HeLa human cervical carcinoma cell line
H4 tail (residues 4–17) is unmodified, while H4K16ac marks 20–
40%. H4K12ac is the next most abundant single mark (5–15%) in
all cells analyzed, followed by H4K5ac (,5%) and the H4K8ac
(,2%). Importantly, the presence of poly-acetylation events (i.e.
two or more) on the H4 tail is detectable in all cells examined, and
this signature is often present at levels comparable to both H4K5ac
and H4K8ac as single acetylation events (Fig 2a).
Site-specific H4 acetyl antibodies preferentially recognize poly-
acetylation signatures in bulk chromatin. The biological iden-
tification of appreciable poly-acetylated H4 finally led us to
determine whether site-specific H4 acetyl antibodies preferentially
recognize this poly-acetylated chromatin signature in cells. Using
peptide competition assays, we first examined the H4K12ac
antibody, which showed a strong (20-fold) preference for poly-
acetylated peptides over the single mark by array (Table 1), but
conversely was under-represented as a poly-acetylated chromatin
signature in vivo in comparison to the single mark by mass
spectrometry (Fig 2a). Importantly, a tetra-acetylated H4 peptide
was able to compete the H4K12ac antibody at a concentration 10-
fold lower than an H4K12ac peptide (Fig 2b). Similar results were
seen with an H4K5ac antibody (Fig 2c). Collectively, these results
demonstrate that poly-acetylation signatures are prevalent in cells
and are the preferred epitope for these site-specific acetyl antibodies.
Selectivity issues related to antibodies, especially those targeting his-
tone PTMs, is by no means a new problem. Common concerns
include cross-reactivity with other PTMs or states of methyl-lysine,
influence antibody recognition in a similar manner to effector pro-
tein binding9,15,16,22. With so many biological methods relying on
antibodies for detection and enrichment, thorough characterization
uncharacterized property of histone H4 site-specific acetyl antibod-
ies, an inherent preference for poly-acetylated chromatin signatures.
By standard criteria, these antibodies would be judged selective for
single acetylpoly acetyl
S. cerevisaeS. cerevr
H4K12ac AbH4K5ac Ab
Figure 2 | (a) Quantitative mass spectrometry to determine the distribution of single- and poly-acetylation of the indicated H4 peptide across species.
Poly-ac is represented as a summation of 2 or more acetylations in the context of the single mark. A complete analysis is shown in Supplemental Fig. 3.
(b-c) Western blots of HeLa chromatin extracts following antibody incubation with the indicated concentrations of competing peptide.
SCIENTIFIC REPORTS | 2 : 489 | DOI: 10.1038/srep00489
their intended PTM. They all recognize a single protein band by
immunoblot, can be competed with a peptide antigen, do not
cross-react with other singly acetylated epitopes, and even have been
shown to lose reactivity when probing for histones mutated at the
asanew criterionforantibodycharacterization, our findingssuggest
H4K5ac, H4K8ac and H4K12ac antibodies may not recognize their
intended target, but instead, all recognize the same poly-acetylated
Our unexpected finding regarding the property of H4 acetyl-spe-
cific antibodies raises new questions with the interpretation of
genome-wide mapping studies of H4 acetylation events across spe-
cies and cell lines. In general, genomic studies find ahigh correlation
for the positioning of H4K5ac, K8ac, and K12ac across genomes23–25.
However, it is entirely possible that these findings represent the
preference of these antibodies for H4 poly-acetylation – a chromatin
signature that is present in vivo and at levels that these antibodies
would compete for over their individually acetylated counterparts.
Given the problematic nature of these antibodies, the true genomic
locations and relative distributions of the individual H4K45ac,
spectrometry results show high species conservation and significant
abundance of singly acetylated H4 tails, we suggest that the indi-
vidual H4K5, H4K8 and H4K12 acetylation events might have
non-overlapping genomic distributions and functions. Future stud-
ies involving improved site-specific H4 acetyl antibodies will be
needed to test this hypothesis.
A counter argument to the concern of recognizing poly-acetyla-
tion signatures are classic genetic studies in budding yeast showing
that mutation of H4 lysines 5, 8, and 12 individually have indistin-
guishable changes in gene expression profiles or growth defects,
while only mutation of lysine 16 has a unique gene expression sig-
lative effect on gene expression and defective growth - suggesting
ant and cumulative. However, parallel studies have not yet been
performed in more complex organisms where individual acetylation
events might play a more significant role. With a new Drosophila
melanogaster histone replacement model now available, important
and interesting questions such as these can be tested27.
One question remaining is why might these antibodies strongly
detect poly-acetylated histones in the first place? The fact that H3
acetyl antibodies do not have strong poly-acetyl preference suggests
this problem is specific towards histone H4. A potential explanation
may lie in the repetitive sequence surrounding the K5, K8 and K12
acetylation sites on the H4 tail. The GKG motif that surrounds these
lysines is repeated on the histone H4 tail. Lysine 16, however, differs,
the least enhanced preference for poly-acetylated H4. Regardless of
the reasons, one would ideally want the H4 acetyl antibodies to
singly modified acetyl-lysines similarly to poly-acetylated epitopes.
Beyond histones, we note that high-resolution mass spectrometry
studies have recently identified thousands of acetylation events
across multiple species28–31. Site-specific acetyl antibodies will
undoubtedly be developed for studying biological functions assoc-
iated with these non-histone acetylation events. Importantly, the
same GKG consensus motif that may pose a problem for H4 acetyl
antibodies surrounds a number of identified acetylated lysines on
non-histone proteins32,33. It remains to be determined whether sim-
ilar antibody-based detection issues will apply for these non-histone
In conclusion, we describe a new and concerning property of site-
specific acetyl antibodies that has been previously missed in all other
forms of characterization. This re-defined property has implications
future studies. This paper therefore serves to encourage more thor-
ough validation of the next generation of acetyl antibodies for the
biological community at large.
2. HeLa cells were cultured in suspension between 5210 3 105cells/ml in minimum
essential Joklik modified media supplemented with 10% new-born calf serum, 2 mM
L-glutamine, 100 units/mL penicillin, and 0.1 mg/mL streptomycin34. mESCs were
2 mML-glutamine,0.1 mMnon-essential amino acids, 0.1 mM 2-mercaptoethanol,
100 units/mL penicillin, 0.1 mg/mL streptomycin/mL, and 1000 units/mL LIF/
ESGRO35. MEFs were derived from 14.5 d mouse embryos and cultured in DMEM
high glucose medium supplemented with 10% fetal bovine serum, 2 mM
L-Glutamine, 100 units/mL penicillin, and 0.1 mg/mL streptomycin35.
Peptide arrays. Peptide synthesis and validation, array fabrication, and antibody
analysis were performed essentially as described9,16.
Mass spectrometry. Sample collection, derivation, and mass spectrometry were
performed as previously described34with the following modifications: 60 to 120 mg
bulk histones were used for each chemical derivation and analysis; the m/z of double,
triple and quadruple acetylations on the H4 4–17 peptide were targeted in the MS
Peptide competitions. Bulk chromatin was isolated from asynchronously growing
HeLacells asdescribed36withthe followingmodifications:Pelletedcells werelysedin
cold buffer containing 10 mM PIPES, pH 7.0, 300 mM sucrose, 100 mM NaCl,
3 mM MgCl2, 1x EDTA-free protease inhibitor (Roche), 1x phosphatase inhibitor
cocktail (Sigma), and 0.1% Triton X-100. Chromatin fractions were treated with
benzonase to solubilize histones prior to SDS-PAGE.
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This work was supported in part by grants from the NIH (GM085394) and the North
Carolina Biotechnology Center (NCBC) to BDS. SBR was supported by postdoctoral grant
S.B.R. performed array and peptide competition experiments, analyzed the data, and
and validated all peptides for arrays and peptide competitions. M.C.K. provided key
reagents and discussed results. S.B.R. and B.D.S. designed the study and wrote the
manuscript. All authors participated manuscript review.
Supplementary information accompanies this paper at http://www.nature.com/
Competing financial interests: The authors declare no competing financial interests.
License: This work is licensed under a Creative Commons
Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this
license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/
How to cite this article: Rothbart, S.B. et al. Poly-acetylated chromatin signatures are
preferred epitopes for site-specific histone H4 acetyl antibodies. Sci. Rep. 2, 489;
SCIENTIFIC REPORTS | 2 : 489 | DOI: 10.1038/srep00489