Dynamic acetylation of all lysine-4 trimethylated histone H3 is evolutionarily conserved and mediated by p300/CBP.

Abstract

Histone modifications are reported to show different behaviors, associations, and functions in different genomic niches and organisms. We show here that rapid, continuous turnover of acetylation specifically targeted to all K4-trimethylated H3 tails (H3K4me3), but not to bulk histone H3 or H3 carrying other methylated lysines, is a common uniform characteristic of chromatin biology in higher eukaryotes, being precisely conserved in human, mouse, and Drosophila. Furthermore, dynamic acetylation targeted to H3K4me3 is mediated by the same lysine acetyltransferase, p300/cAMP response element binding (CREB)-binding protein (CBP), in both mouse and fly cells. RNA interference or chemical inhibition of p300/CBP using a newly discovered small molecule inhibitor, C646, blocks dynamic acetylation of H3K4me3 globally in mouse and fly cells, and locally across the promoter and start-site of inducible genes in the mouse, thereby disrupting RNA polymerase II association and the activation of these genes. Thus, rapid dynamic acetylation of all H3K4me3 mediated by p300/CBP is a general, evolutionarily conserved phenomenon playing an essential role in the induction of immediate-early (IE) genes. These studies indicate a more global function of p300/CBP in mediating rapid turnover of acetylation of all H3K4me3 across the nuclei of higher eukaryotes, rather than a tight promoter-restricted function targeted by complex formation with specific transcription factors.

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Dynamic acetylation of all lysine-4 trimethylated
histone H3 is evolutionarily conserved and
mediated by p300/CBP
Nicholas T. Crumpa, Catherine A. Hazzalina, Erin M. Bowersb, Rhoda M. Alanib,1, Philip A. Coleb,
and Louis C. Mahadevana,2
aNuclear Signalling Laboratory, Department of Biochemistry, Oxford University, Oxford OX1 3QU, United Kingdom; andbDepartment of Pharmacology and
Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205
Edited* by Gary Felsenfeld, National Institutes of Health, Bethesda, MD, and approved March 31, 2011 (received for review January 7, 2011)
Histone modifications are reported to show different behaviors,
associations, and functions in different genomic niches and organ-
isms. We show here that rapid, continuous turnover of acetylation
specificallytargetedtoallK4-trimethylatedH3tails(H3K4me3),but
not to bulk histone H3 or H3 carrying other methylated lysines, is
a common uniform characteristic of chromatin biology in higher
eukaryotes, being precisely conserved in human, mouse, and Dro-
sophila. Furthermore, dynamic acetylation targeted to H3K4me3
is mediated by the same lysine acetyltransferase, p300/cAMP re-
sponseelementbinding(CREB)-bindingprotein(CBP),inbothmouse
and fly cells. RNA interference or chemical inhibition of p300/CBP
using a newly discovered small molecule inhibitor, C646, blocks dy-
namic acetylation of H3K4me3 globally in mouse and fly cells, and
locally across the promoter and start-site of inducible genes in the
mouse, thereby disrupting RNA polymerase II association and the
activation of these genes. Thus, rapid dynamic acetylation of all
H3K4me3 mediated by p300/CBP is a general, evolutionarily con-
served phenomenon playing an essential role in the induction of
immediate-early (IE) genes. These studies indicate a more global
function of p300/CBP in mediating rapid turnover of acetylation of
all H3K4me3 across the nuclei of higher eukaryotes, rather than
atightpromoter-restrictedfunctiontargetedbycomplexformation
with specific transcription factors.
histone acetylation turnover|Trichostatin A|p300/CBP inhibitor
H
specifically with active genes and a more open chromatin
structure (1–3). Charge neutralization of lysines reduces the af-
finity of acetylated histone tails for DNA, potentially creating
a more open state for transcription (3), and specific acetyllysines
can form binding sites to recruit other effectors (4), suggesting
distinct roles for distinct lysine residues. Most current analytical
techniques provide a “snapshot” of acetylation frozen by fixation
or crosslinking, with no information about the stability of modi-
fication (discussed in ref. 5). However, radiolabeling studies (6, 7)
and genetic experiments in yeast (8, 9) show acetylation is highly
dynamic and heterogeneous, with different populations of histo-
nes characterized by distinct rates of acetylation and deacetyla-
tion (5, 10). We have shown that all detectable K4-trimethylated
histone H3 (H3K4me3) across murine nuclei, irrespective of lo-
cation or association, is subject to dynamic acetylation at multiple
lysine residues, becoming maximally acetylated within 10–30 min
of treatment with trichostatin A (TSA) (11). Continuous turn-
over, by opposing lysine (K) acetyltransferase and histone
deacetylase (KAT and HDAC; Fig. 1A) activities, is extremely
rapid (11). The relevant KAT/HDAC enzyme system must
therefore be tightly coupled to H3K4me3, and on these tails it is
not restricted to any specific lysine but acetylates multiple resi-
dues (excluding the trimethylated lysine 4) to rapidly produce
tetra- and pentaacetylated histones (11). Such exquisite targeting
is remarkable, given the massive excess of other nucleosomes
and histones available to KATs and HDACs in the nucleus.
In high-resolution quantitative studies of c-fos and c-jun, his-
tone H3 lysine-9 acetylation (H3K9ac) peaks at the promoter
istone acetylation is widely associated with gene regulation,
and 5′ end with a gap at the start site, most likely due to the
absence of a nucleosome, and decreases before the end of the
gene (12). This distribution precisely mirrors that of H3K4me3
across these genes (12), also shown, albeit at lower resolution,
in several chromosome-wide mapping studies in yeast (13, 14),
Drosophila (15), mouse (16), and human (17, 18). In TSA-treated
quiescent cells, H3K4me3 across this region of c-fos and c-jun
becomes rapidly hyperacetylated (11) even though the genes
remain inactive. In fact, hyperacetylation inhibits physiological
gene induction, challenging the link between state of acetylation
and transcription and suggesting that turnover is the important
factor. Consistent with this, genome-wide mapping of KATs and
HDACs places these enzymes together at many gene loci (18),
and a requirement for HDAC activity in gene expression has
been reported (reviewed in ref. 19).
WeshowherethatdynamicacetylationtargetedtoH3K4me3is
conserved in human and Drosophila as well as mouse cells. RNA
interference studies in Drosophila indicate that depletion of any
single HDAC does not abolish TSA-sensitive acetylation of
H3K4me3. By contrast, knockdown of a single KAT, dCBP, se-
verely reduced dynamic acetylation of H3K4me3. A new small-
molecule p300/cAMP response element binding (CREB)-binding
protein (CBP) inhibitor, C646 (20), was used to confirm its role
mediatingdynamicH3K4me3acetylationinDrosophilaandmouse
andtostudyitsfunctionininduciblegeneactivation.Weconclude
thatdynamicacetylationtargetedtoallH3K4me3isevolutionarily
conserved, mediated by p300/CBP, and essential for RNA poly-
meraseIIassociationandprotooncogeneinduction.Thesestudies
throw light on the role that p300/CBP plays in gene regulation,
indicating a more dynamic, global function across all H3K4me3-
containing promoters in human, mouse, and Drosophila.
Results
All K4-Trimethylated Histone H3 in Mouse, Human, and Drosophila
Cells Is Subject to Dynamic Acetylation. All H3K4me3, but not
H3 methylated at lysine 9 or bulk H3, in murine nuclei is TSA
hypersensitive (11). This is visualized by Western blots of histone
H3laddersonacid–urea(AU)gels(Fig.1B).TSAhypersensitivity
is defined by the rapid appearance of tetra- and pentaacetylated
histones (labeled bands 4 and 5) and complete loss of less acety-
lated forms (bands 1 and 2). Total conversion from lower to
hyperacetylated forms occurs within 15 min, even at very low
concentrations (33 nM) of inhibitor (Fig. 1B, H3K4me3, compare
Author contributions: N.T.C., C.A.H., E.M.B., R.M.A., P.A.C., and L.C.M. designed research;
N.T.C., C.A.H., E.M.B., and R.M.A. performed research; N.T.C., C.A.H., E.M.B., R.M.A., P.A.C.,
and L.C.M. contributed new reagents/analytic tools; N.T.C., C.A.H., E.M.B., R.M.A., P.A.C.,
and L.C.M. analyzed data; and N.T.C., C.A.H., E.M.B., R.M.A., P.A.C., and L.C.M. wrote the
paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1Present address: Department of Dermatology, Boston University School of Medicine, 609
Albany Street, J-507, Boston, Massachusetts 02118-2515.
2To whom correspondence should be addressed. E-mail: louis.mahadevan@bioch.ox.ac.uk.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1100099108/-/DCSupplemental.
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lanes 7 and 8). Thus, rapid turnover of acetylation occurs at mul-
tiple residues on H3K4me3, excluding the trimethylated lysine 4.
Turnover is mediated exclusively by TSA-sensitive HDACs, be-
cause nicotinamide, which inhibits sirtuin-like HDACs, does not
elicit this effect (Fig. 1B, lanes 7, 11, and 12). Further, hypersen-
sitivityisspecifictoH3K4me3,asmono-anddimethylatedlysine4
do not show the same rapid response to TSA (11). Recognition of
the K4me3 epitope by this antibody is not affected by acetylation
at lysine 9 (Fig. S1 A and B), indicating that these observations,
particularly the complete loss of lower bands, are not an artifact
of antibody specificity or occlusion. Histone H3K79me2 and
H3K36me3,whichareenrichedinthetranscribedregionsofmany
genes (reviewed in ref. 21), become acetylated much more slowly
than H3K4me3 (Fig. 1B, lanes 7–10). Notably, even after 2-h
HDAC inhibition, lower acetylated forms comparable to those
seen in untreated cells remain and no maximally hyperacetylated
form is detectable (Fig. 1B, compare lanes 7 and 10).
To investigate whether dynamic acetylation of H3K4me3 is
evolutionarily conserved, histones from human WI 38 fibroblasts
(Fig. 1B, lanes 1–6) and Drosophila S2 cells (Fig. 1B, lanes 13–18)
were analyzed. Specificity of rapid hyperacetylation for H3K4me3,
compared with other active (H3K79me2 and H3K36me3) and
silent (H3K9me2 and H3K27me3) modifications, is absolutely
maintained in all three organisms. Apart from H3K9me2, which
in Drosophila strikingly appears as a single band resistant to
acetylation and unresponsive to TSA after a 2-h treatment, all
modifications show virtually identical responses between mouse,
human, and fly (Fig. 1B, compare lanes 1–4, 7–10, and 13–16).
As in the mouse, nicotinamide has no effect (Fig. 1B, lanes 1, 5,
and 6 and 13, 17, and 18). Continuous turnover of acetylation
targeted to H3K4me3 is therefore evolutionarily conserved in
higher eukaryotes.
H3K4me3 Is Dynamically Acetylated on Lysine 9, 14, and 18. Sequential
ChIP studies have shown H3K4me3 and H3K9ac on the same
nucleosomes at the promoters of a number of genes, including c-
fos and c-jun (11, 22). To investigate coexistence of modifications
on individual histone molecules rather than nucleosomes, we de-
veloped a protocol to immunodeplete free histones from mouse
fibroblasts using antibodies against H3K4me3. Unbound material
was analyzed on SDS (Fig. 2A) or AU (Fig. 2B) gels to monitor
any corresponding loss of acetylation and blots quantified by
scanning (Fig. 2 A, ii and B, ii). Depletion of H3K4me3 has very
little effect on the total amount of H3K9ac left in the unbound
fraction (Fig. 2A, compare lanes 1 and 4 with 2 and 5), suggesting
that the majority of K9ac occurs on H3 that is not K4 trimethy-
lated. However, reciprocal depletion using anti-H3K9ac anti-
bodies dramatically decreases the amount of H3K4me3 left
unbound to approximately 20% of input (Fig. 2A, compare lanes 1
and 4 with 3 and 6). Taken together, these data show that much of
the H3K4me3 population is acetylated at lysine 9, but the vast
Fig. 1.
yotes. (A) Dynamic histone acetylation is mediated by lysine acetyl-
transferases (KATs) and histone deacetylases (HDACs). TSA inhibits classes I,
II, and IV HDACs to promote hyperacetylation of H3K4me3. (B) Human WI 38
fibroblasts (lanes 1–6), murine C3H 10T1/2 fibroblasts (lanes 7–12), or Dro-
sophila S2 cells (lanes 13–18) were treated with trichostatin A (TSA) (33 nM;
lanes 2–4, 8–10, and 14–16) or nicotinamide (NAM) (20 μM; lanes 5, 6, 11, 12,
17, and 18) as indicated. Histones were separated on acid–urea (AU) gels on
which acetylation incrementally retards migration to produce the “ladder”
of bands seen. Numbers indicate extent of acetylation, 0 referring to non-
acetylated H3. Different exposures are shown for H3K4me3 in mouse and
Drosophila (Top) due to the higher proportion of this modification in the fly
(Fig. S1C for comparison). The H3K9me2 (fourth panel down) signal in mouse
has been overexposed to allow detection of low levels of this modification in
Drosophila. (Lanes 1, 7, and 13: control untreated cells.)
Dynamic acetylation of all H3K4me3 is conserved in higher eukar-
Fig. 2.
methylated at lysine 4. (A, i) Purified free histones from untreated (lanes 1–
3) or TSA-treated (33 nM, 30 min; lanes 4–6) C3H 10T1/2 cells were immu-
nodepleted with antibodies against H3K4me3 (lanes 2 and 5) or H3K9ac
(lanes 3 and 6). Unbound material was resolved by SDS/PAGE and subjected
to Western blotting with antibodies against total H3 (Bottom), H3K9ac
(Middle), or H3K4me3 (Top). (Lanes 1 and 4: input material before immu-
nodepletion.) (A, ii) Blots from A, i were quantified using ImageJ and nor-
malized to total H3. Data (mean of three biological replicates, plotted ±
SEM) are presented relative to input under untreated or TSA-treated con-
ditions (lanes 1 and 4 from A, i). (B, i) Purified free histones from control
(lanes 1 and 2) or TSA-treated (33 nM, 30 min; lanes 3 and 4) C3H 10T1/2 cells
were immunodepleted with anti-H3K4me3 antibody, resolved on acid–urea
gels, and subjected to Western blotting using total H3, anti-H3K4me3, or
anti-acetyl H3 antibodies as indicated on the Left of each panel. (Lanes 1 and
3: input material; lanes 2 and 4: immunodepleted fraction.) (B, ii) Total
staining for each modification in blots from B, i was quantified using ImageJ,
with normalization to total H3 levels. Data (mean of three biological repli-
cates, plotted ± SEM) are presented relative to input under untreated or
TSA-treated conditions (lanes 1 and 3 from B, i).
The most highly acetylated histone H3 in TSA-treated cells is tri-
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majority of H3K9ac in the mouse nucleus occurs on H3 tails that
are not trimethylated at lysine 4. Although genome-wide studies
show colocalization of H3K4me3 and H3K9ac (15, 16, 18), these
peaks constitute an exceedingly minute fraction of the genome. A
relatively low level of H3K9 acetylation at other locations, not
detectable as major peaks, can account for the majority of K9ac
that does not occur on K4-trimethylated H3 tails.
Analysis of H3K4me3-immunodepleted fractions (Fig. 2A) on
AU gels showed that depletion of H3K4me3 has no obvious
effect on H3K9ac in untreated cells (Fig. 2B, i, compare lanes 1
and 2, and Fig. 2B, ii), but a striking effect on the most highly
acetylated H3K9ac bands following acute HDAC inhibition (Fig.
2B, i, compare lanes 3 and 4 and bands 4 and 5). These bands
(tetra- and pentaacetylated H3) are preferentially removed by
H3K4me3 depletion (Fig. 2B, i, lanes 3 and 4; quantified as
79.5% (± 11.1%) reduction in band 5). Thus, the most highly and
dynamically acetylated histone H3 at lysine 9 is distinguished by
trimethylation at lysine 4.
H3K18-acetylated histones do not become noticeably hyper-
acetylated upon TSA treatment, but the appearance of a faint
band above bulk H3K18ac following TSA treatment, which is
lost following H3K4me3 depletion (Fig. 2B, compare lanes 1, 3,
and 4) suggests that the small proportion of the K18ac pop-
ulation that is marked by K4me3 is targeted for dynamic acety-
lation. In contrast, depletion of H3K4me3 reduces the H3K14ac
population to less than 40% input (Fig. 2B), suggesting that
a significant proportion of histone H3 acetylated at lysine 14 is
also trimethylated at lysine 4. These data demonstrate that
hyperacetylated H3K4me3 molecules are acetylated at K9, K14,
and K18, suggesting that these three lysines are dynamically
acetylated on K4-trimethylated histone H3.
Analysis of HDACs and KATs Shows That p300/CBP Mediates Acetyla-
tion of H3K4me3 in Drosophila and Mouse. The Drosophila genome
encodes five potentially TSA-sensitive HDACs—dHDACs 1
(also known as dRpd3), 3, 4, 6 (also known as dHDAC2), and X
(23). We found redundancy among these enzymes in mediating
deacetylation of histone H3K4me3 (Fig. S2). dsRNA-mediated
knockdown of dHDAC1 produced some increased basal acety-
lation of H3K4me3 in control cells, but none of the individual
HDAC knockdowns affected the TSA-induced hyperacetylation
of H3K4me3 (Fig. S2). Even allowing for the incomplete nature
of dsRNA-mediated knockdown (Fig. S2C), these studies are in
agreement with a previous study (23) showing that deacetylation
in Drosophila is mediated redundantly by multiple HDACs.
By contrast, our studies on KATs identified a single enzyme
responsible for dynamic acetylation of H3K4me3. We again used
Drosophila cells in which KAT enzyme families are smaller;
dCBP (dKAT3) is homologous to mammalian CBP (KAT3A)
and p300 (KAT3B), and dGCN5 (dKAT2) to GCN5 (KAT2A)
and p300/CBP-associated factor (PCAF) (KAT2B) in mammals.
Specific knockdown of these two transcripts was verified by qRT-
PCR (Fig. S3A).
Knockdown of dGCN5 has little effect on the basal acetylation
of H3K4me3 compared with the mock knockdown targeting lacZ
(Fig. 3A, compare lanes 1 and 5). Further, TSA-induced hyper-
acetylation of H3K4me3 is unimpaired in the dGCN5 knock-
down (Fig. 3A, compare lanes 2 and 6). In contrast, knockdown
of dCBP reveals a key role for this enzyme in dynamic histone
H3K4me3 acetylation. Loss of dCBP has little effect on the basal
level of H3K4me3 acetylation (Fig. 3A, compare lanes 3 and 5).
However, TSA-induced hyperacetylation is severely reduced,
especially the appearance of the most acetylated forms (bands 4
and 5), leaving a predominance of triacetylated H3K4me3 in the
dCBP knockdown, compared with tetra- and pentaacetylated
forms in both dGCN5 and lacZ knockdowns (Fig. 3A, compare
lanes 2, 4, and 6). Although the response to TSA is clearly di-
minished after dCBP knockdown, some H3K4me3 TSA sensi-
tivity is detectable in these cells, which could come from residual
dCBP remaining after knockdown (Fig.S3A) or from other en-
zymes involved in targeting H3K4me3.
We recently reported the discovery and characterization of
C646 (Fig. 3B), a small molecule inhibitor of p300/CBP identi-
fied by virtual ligand screening of a library of compounds using
the crystal structure of the bisubstrate inhibitor Lys-CoA bound
to p300 (20). C646 produced 86% inhibition of p300 in vitro at
10 μM, and less than 10% inhibition of the KATs PCAF, GCN5,
Sas, and Moz (20). It was used in parallel with a control com-
pound, C37 (Fig. 3B), chemically similar to C646 but inac-
tive against p300/CBP (20). Consistent with dCBP knockdown,
pretreatment of S2 cells with C646 completely abrogates TSA-
induced hyperacetylation of H3K4me3 histones (Fig. 3C, com-
pare lanes 3 and 4 with 1 and 2 and 5 and 6), confirming that this
enzyme mediates dynamic H3K4me3 acetylation. Note that ad-
dition of dCBP inhibitor after treatment with TSA to enhance
acetylation produces no loss of hyperacetylated H3K4me3 (Fig.
S3B, compare lanes 2 and 3), so loss of the TSA-sensitive effect
cannot be explained by C646-induced deacetylation. Further-
more, this effect is conserved in higher eukaryotes, as pretreating
mouse fibroblasts with p300/CBP inhibitor completely abolishes
TSA-induced hyperacetylation of H3K4me3 (Fig. 3D). The fact
that RNA interference and chemical inhibition, two widely dif-
ferent approaches to inhibit p300/CBP, efficiently block dynamic
acetylation of H3K4me3 in mouse and fly proves that this en-
zyme is responsible and the phenotype is conserved.
p300/CBP Is Required for TSA- and Stimulus-Induced Acetylation
Across the Promoters of c-fos and c-jun. We have previously de-
scribed quantitative distributions of histone modifications, in-
cluding H3K4me3 and H3K9ac, across c-fos and c-jun in mouse
fibroblasts (12). We used quantitative ChIP to map p300/CBP
KAT activity, defined by sensitivity of histone acetylation to in-
hibition by C646, across these genes, with gapdh and β-globinmaj
(hbb) used to represent constitutively active and transcriptionally
silent genes (Fig. 4). As reported previously (11), treatment with
TSA for 30 min strongly enhances H3K9ac at c-fos, c-jun, and
gapdh (Fig. 4A, dark blue bars). These increases are greatest at
the promoter (c-fos −260, c-jun −966) and 5′ end (c-fos +444,
c-jun +1,119) of these genes, identifying continuous KAT and
Fig. 3.
(A) Drosophila S2 cells were treated with dsRNA targeting dGCN5 (lanes 1
and 2), dCBP (lanes 3 and 4), or lacZ (nontargeting control; lanes 5 and 6) as
described. Histones from untreated (lanes 1, 3, and 5) or TSA-treated (33 nM,
30 min; lanes 2, 4, and 6) cells were resolved on acid–urea gels and probed
using antibodies against H3K4me3 (Upper) or total H3 (Lower). Blots are
representative of three replicate experiments. (B) Structures of p300/CBP
inhibitor (C646) and control compound (C37). (C) Quiescent S2 cells were
treated for 60 min with p300/CBP inhibitor (C646, 30 μM; lanes 3 and 4) or
control compound (C37, 30 μM; lanes 5 and 6) with TSA (33 nM; lanes 2, 4,
and 6) added where indicated for the final 30 min. Histones were resolved
on acid–urea gels and probed for H3K4me3 (Upper) or total H3 (Lower).
(Lanes 1 and 2: control and TSA-treated cells.) (D) Quiescent C3H 10T1/2 cells
were treated and analyzed exactly as described above for Drosophila S2 cells
in Fig. 3C.
p300/CBP is required for TSA-induced hyperacetylation of H3K4me3.
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HDAC activity at these nucleosomes. Dynamic acetylation is
independent of transcription, as c-fos and c-jun are not expressed
under these conditions and pretreatment with the transcriptional
inhibitor DRB (Fig. 4A, purple bars) has no effect on TSA-
stimulated H3K9ac. TSA-stimulated acetylation is completely
lost in cells pretreated with C646 (Fig. 4A, cream-colored bars)
showing that this is mediated by p300/CBP. This is not a sec-
ondary effect of transcriptional inhibition induced by the com-
pound (Fig. 5) as pretreatment with DRB has no effect on
acetylation (Fig. 4A, purple bars). Note that ChIP using anti-
p300 antibody shows that C646 treatment does not disrupt its
localization at c-fos or c-jun (Fig. S4A).
Stimulation with EGF to activate expression of c-fos and c-jun
(11) produces an increase in H3K9ac in a distribution similar
to that seen with TSA (Fig. 4A, compare dark blue bars). Pre-
treatment with p300/CBP inhibitor before stimulation with EGF
abrogates any increase in H3K9ac (Fig. 4A, cream-colored bars),
confirming that p300/CBP KAT activity is also responsible for
stimulus-induced acetylation at c-fos and c-jun. Interestingly,
whereas EGF-induced H3K9 acetylation at c-fos is unaffected by
DRB pretreatment, the increase is lost at c-jun (Fig. 4A, purple
bars), suggesting that stimulus-induced acetylation is dependent
on transcription at this gene. EGF treatment has no effect on
H3K9ac levels at the nonresponsive gapdh or hbb genes, demon-
stratingthatacetylationatc-fosandc-junisspecificallytargetedto
these genes by EGF-stimulated signaling mechanisms. In agree-
ment with previous work at c-fos and c-jun (11, 12) and genome-
wideanalyses (13,15–18),highlevels ofH3K4me3 colocalize with
peaks of H3K9ac induction, and where levels of H3K4me3 are
lower, there is reduced basal and inducible H3K9ac (Fig. S4B).
p300/CBP Acetyltransferase Activity Is Required for Immediate-Early
(IE) Gene Expression. To ask whether p300/CBP KAT activity is
required for gene expression, the effect of C646 on c-fos and
c-jun induction was analyzed. Inhibition of p300/CBP produces
complete loss of induction of both genes (Fig. 5A, compare lanes
1–3 with 4–6), whereas the control compound C37 has no effect.
The inhibitor does not act by interfering with signaling to these
genes, as ERKs are phosphorylated efficiently in the presence of
C646 (Fig. S5A, compare lanes 3 and 4 with 5 and 6). Further,
inhibition of gene induction is virtually instantaneous and ob-
served when the compound is added simultaneously with, or
even soon after, stimulation (Fig. S5B, compare lanes 1–3 with
4–16). Specificity of inhibition is confirmed by the fact that the
compound does not affect global transcription, as assessed by
bromouridine labeling (Fig. S6).
To understand how p300/CBP inhibition ablates c-fos and
c-jun induction, we analyzed the distribution of RNA polymerase
II (Pol II) at these genes (Fig. 5B, dark blue bars). Highest levels
of Pol II are recovered at the transcriptional start site of c-fos
(−79) and c-jun (−57), with levels decreasing downstream (c-fos
Fig. 4.
H3K9 acetylation at c-fos and c-jun independent of transcription. Control
C3H 10T1/2 cells (dark blue bars) or cells pretreated with p300/CBP inhibitor
(C646, 30 μM, 30 min; cream-colored bars) or DRB (20 μg/mL, 10 min; purple
bars), were incubated for an additional 30 min with TSA (33 nM) or EGF
(50 ng/mL). Crosslinked, MNase-released nucleosomes were immunopreci-
pitated using antibodies against H3K9ac. Bound DNA analyzed by real-time
PCR using primers to c-fos, c-jun, gapdh, and hbb (primer positions indicated
above each panel) and compared with input. Data are the mean of at least
three biological replicates, plotted ± SEM. Schematic representations of these
genes are shown, indicating the locations of PCR primers used in Figs. 4 and 5B
and Figs. S4 and S7. Boxes are exons; closed boxes are coding regions; open
boxes are 5′ and 3′ untranslated regions; circles are polyadenylation sites.
p300/CBP KAT activity is required for stimulus- and TSA-induced
Fig. 5.
gene induction. (A) Control C3H 10T1/2 cells (lanes 1–3) or cells pretreated for
30 min with p300/CBP inhibitor (C646, 30 μM; lanes 4–6), control compound
(C37, 30 μM; lanes 7–9), or TSA (33 nM; lanes 10–12), or pretreated for 30 min
with TSA, then C646 added with EGF (lanes 13–15). After pretreatment, cells
were stimulated with EGF (50 ng/mL) for a further 30 (lanes 2, 5, 8, 11, and 14)
or60min(lanes3,6,9,12,and15).TotalRNAwasprobedbyNorthenblotting
for expression of c-fos (Top), c-jun (Middle) and gapdh (Bottom). (Lanes 1, 4,
7, 10, and 13: non-EGF treated controls.) (B) Control C3H 10T1/2 cells (dark
blue bars) or cells pretreated with p300/CBP inhibitor (C646, 30 μM, 30 min;
cream-colored bars), TSA (33 nM, 30 min; red bars), or TSA then C646 (30 min
TSA alone, followed by C646 for a further 30 min; purple bars), were
incubated for an additional 15 min with EGF (50 ng/mL). Crosslinked, MNase-
released nucleosomes were immunoprecipitated using antibodies against
RNA polymerase II. Bound DNA was analyzed by real-time PCR using primers
to specific positions along c-fos, c-jun, gapdh, and hbb (positions indicated
above each panel; shown schematically in Fig. 4) and compared with their
representation in the input DNA before immunoprecipitation. Data are the
mean of two biological replicates, plotted ± SEM.
p300/CBP acetyltransferase activity is required for immediate-early
Crump et al. PNAS
| May 10, 2011
| vol. 108
| no. 19
| 7817
CELL BIOLOGY

Page 5

+119, +1,443; c-jun +153, +1,119). Gene induction with EGF
increases Pol II levels at all sites to high levels, reflecting an
extremely active but short-lived burst of transcription, in contrast
to lower levels recovered at gapdh. Treatment with C646 strongly
disrupts Pol II localization at c-fos and c-jun irrespective of
transcriptional induction (Fig. 5B, cream-colored bars), although
it does not affect the presence of p300 at these genes (Fig. S4A).
This suggests that p300/CBP KAT activity is required for Pol II
occupancy at these genes.
One model for the role of p300/CBP in gene expression is that
it acetylates nucleosomes to provide a more “open” template
through which transcription can proceed (3). Although TSA
treatment produces highly acetylated histones at c-fos and c-jun
(Fig. 4A), it interferes with gene expression (11). However, this is
not as severe as the complete loss of expression caused by in-
hibition of p300/CBP (Fig. 5A, compare lanes 1–3 with 4–6 and
10–12). Noting that addition of C646 after TSA treatment does
not reduce the high levels of histone acetylation generated, ei-
ther globally (Fig. S3B, lane 3) or at c-fos, c-jun, and gapdh (Fig.
S7), we asked whether preacetylation of histones with TSA could
rescue gene induction abrogated by p300/CBP inhibition. In fact,
forced hyperacetylation by TSA does not restore the loss of gene
induction upon p300/CBP inhibition (Fig. 5A, compare lanes 4–6
with 13–15), nor does it prevent the loss of Pol II at c-fos and
c-jun (Fig. 5B, purple bars). Taken together, these findings sug-
gest that dynamic modification is more important than static
acetylation for efficient gene induction.
Discussion
We show here that dynamic acetylation of all H3K4me3 mediated
by CBP is evolutionarily conserved, observed before the diver-
gence of the single Drosophila enzyme dCBP into the paralogs p300
and CBP in mammals. CBP was discovered as a transcriptional
coactivator that binds to CREB (24, 25) and p300 complements
this activity (26). Whereas p300 and CBP are often considered
functionally redundant, some studies support unique roles
(reviewedinref.27).Themajorityofgenesboundbyonealsoshow
high levels of the other (18), suggesting common targeting. They
interact with many transcription factors and coactivators, initially
suggesting a structural role in promoter complexes (reviewedin
ref. 28); p300 and CBP have been localized to c-fos and c-jun by
ChIP (18, 29) and through interactions with other proteins (30–
32).A purely structural role waschallenged following discovery of
their acetyltransferase activity (33, 34) with the catalytic domain
required for transcription from chromatinized promoter con-
structs in vitro and in vivo (35, 36). Recent in vitro studies with
reconstituted nucleosome arrays show a requirement for p300
KAT activity to allow decompaction of 30 nm chromatin, nucle-
osome remodelling, and transcription factor binding (37–39).
p300/CBP, Dynamic Histone Acetylation, and Gene Induction. Con-
sistentwithpreviousmassspectrometry(40)andbiochemical(41)
work,weshowthatH3K4me3tailsaredynamicallymodifiedupto
thepentaacetylatedstate,includingatlysines9,14,and18(Fig.2),
This suggests that the enzyme responsible, p300/CBP, targets
specific H3 tails but no specific lysine. In p300/CBP double-
knockout mouse fibroblasts, forskolin-induced acetylation of ly-
sine 5, 8, 12, and 16 of histone H4 at c-fos is inhibited (29), further
suggesting no specific targeting of residues. A high level of acety-
lationisinsufficientforefficientgeneexpressioninvivo;treatment
of cells with TSA enhances acetylation (Fig. 1) but interferes with
c-fosandc-juninduction(11).Further,lossofgeneexpressionand
Pol II localization caused by p300/CBP inhibition cannot be re-
lieved by preacetylating nucleosomes before inhibition (Fig. 5).
This indicates a more dynamic role for acetylation in gene ex-
pression, suggesting that turnover may be the important factor.
Analyses of quiescent cells in which c-fos and c-jun are poised but
inactive and inhibition of transcription with DRB (Fig. 4A) both
indicate that transcription isnot required for dynamicacetylation.
Cotargeting of H3K4 Trimethylation and Dynamic Acetylation to the
Promoter and 5′ End of Genes. Thefinding that dynamicacetylation
and H3K4me3 (Figs. 1 and 2) colocalize on the same nucleosomes
across the promoter and start site of c-fos and c-jun (11) raises
the question of their cotargeting. Even when the KAT–HDAC en-
zyme balance is drastically forced in favor of acetylation by HDAC
inhibitors, strict targeting to H3K4me3 does not break down. Nu-
merous ChIP studies have established the presence of H3K4me3,
H3K9ac, p300/CBP, and HDACs at the promoter and 5′ end of
many genes (17, 18, 22), suggesting widespread colocalization.
There are two classes of model by which cotargeting of
H3K4me3 and rapid dynamic acetylation may occur. The first
involves independent targeting to the same loci and H3 tails, as
previously shown for serine-10 phosphorylation and lysine-9 acet-
ylation(42).Thissuggeststhattherelevantenzymesmaybepartof
a common process, and cotargeting may arise from independent
DNA sequence recognition or unique interactions with the ma-
chinery of signal transduction andtranscriptional regulation; p300
and CBP have been isolated in complexes containing TATA-
binding protein (TBP) (43, 44) and RNA polymerase II (45–47).
A second class of model is based on dependence of one
modification on the other. For example, p300/CBP may mediate
dynamic acetylation through direct or indirect recognition of
trimethylated lysine 4, which may provide a binding platform
or enhance KAT activity. In support of this mechanism WDR5
knockdown, which depletes H3K4 methylation (48), attenuates
the TSA-induced increase in H3K9ac levels at promoters (18).
Other KATs are known to be recruited to H3K4me3 to induce
histone acetylation; yeast Yng1 and Yng2, which recognize
H3K4me3 via their plant homeo domain (PHD) fingers (49, 50),
form part of the NuA3 and NuA4 KAT complexes, respectively
(51, 52), and mammalian ING4 links HBO1 acetyltransferase
activity to H3 lysine-4 trimethylated nucleosomes (53, 54).
A sequential targeting mechanism is conceivable. Unmethy-
lated CpG dinucleotides within CpG islands (reviewed in ref.
55) may primarily recruit CXXC motif-containing proteins, in-
cluding the H3K4 methyltransferase MLL1 (56) and CGBP/
Cfp1, which associates with H3K4 methyltransferases (57, 58).
DNA binding by Cfp1 has recently been shown to restrict Setd1A
and H3K4me3 to euchromatic nonmethylated CpG regions
(59). Similarly, ChIP-seq analysis has shown a tight association
between Cfp1 and H3K4me3 at CpG islands, and Cfp1 knockdown
depletes H3K4me3 levels at nonmethylated CpGs (60). This
provides a plausible mechanism to target H3K4me3 to these
regions, which could then recruit p300/CBP for dynamic
histone acetylation.
Materials and Methods
Cell Culture, Protein Extraction, and Analysis. C3H 10T1/2 mouse fibroblasts
grown in DMEM (10% FCS, 37 °C, 6% CO2) were quiesced (0.5% FCS, 18–20 h)
before treatment. Drosophila S2 cells were grown in Schneider’s medium
(10% FCS, 25 °C) and quiesced (0.1% FCS, 24 h) before treatment. Pre-
treatment refers to addition of inhibitor before stimulation, with inhibitor
remaining on cells during stimulation. Cells were treated with TSA [10 ng/mL
(33 nM); Sigma], nicotinamide (20 μM; Sigma), DRB (20 μg/mL; Sigma), EGF
(50 ng/mL; Promega), and TPA (100 nM; Sigma). Synthesis and specificity of
C646 (30 μM) and C37 (30 μM) are described elsewhere (20). Acid extraction
of histones, resolution on SDS and acid–urea gels, and Western blotting are
as described previously (12, 61). Western blots were quantified using ImageJ
1.44 (National Institutes of Health (http://rsb.info.nih.gov/ij/). Antibodies
used are described in SI Materials and Methods.
Histone Immunoprecipitation. Histones were isolated by acid extraction (61)
and resuspended in 8 M urea, 5% acetic acid, then dialyzed into 10 mM Tris-
HCl pH 8.0, and adjusted to RIPA buffer (61). Histones were incubated with
anti-H3K4me3 or -H3K9ac serum with rotation for 2 h at 4 °C, then protein A-
sepharose beads (Sigma) preblocked with BSA (0.02% final concentration
after addition of histones) added for a further 2 h. The unbound fraction
was dialyzed into 10 mM Tris-HCl pH 8.0. Protein was precipitated (25% TCA,
4 °C, 30 min), washed three times with ice-cold acetone, dried, and used for
gel electrophoresis.
RNA Interference. Fragments (400–600 bp) of target genes were amplified
from cDNA prepared from Drosophila S2 RNA, using primers incorporating
T7 RNA polymerase promoters. Primer sequences obtained from the dkfz
E-RNAi Web service (http://e-rnai.dkfz.de) were used as template for dsRNA
synthesis using the MEGAscript T7 kit (Ambion) and dsRNA was purified
7818
| www.pnas.org/cgi/doi/10.1073/pnas.1100099108Crump et al.