Recognition of a Mononucleosomal
Histone Modification Pattern
by BPTF via Multivalent Interactions
Alexander J. Ruthenburg,1,7Haitao Li,5,8Thomas A. Milne,1,9Scott Dewell,2Robert K. McGinty,3Melanie Yuen,3
Beatrix Ueberheide,4Yali Dou,6Tom W. Muir,3Dinshaw J. Patel,5and C. David Allis1,*
1Laboratory of Chromatin Biology and Epigenetics
2Genomics Resource Center
3Laboratory of Synthetic Protein Chemistry
4Laboratory of Mass Spectrometry and Gaseous Ion Chemistry
The Rockefeller University, 1230 York Avenue, New York, NY, 10065, USA
5Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, 10065, USA
6Department of Pathology, University of Michigan Medical School, 1301 Catherine Street, Ann Arbor, MI, 48109, USA
7Present address: Department of Molecular Genetics and Cell Biology, The University of Chicago, 920 East 58th Street, Chicago,
IL 60637, USA
8Present address: School of Medicine, Tsinghua University, Beijing 100084, People’s Republic of China
9Present address: MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Headington,
Oxford OX3 9DS, UK
Little is known about how combinations of histone
marks are interpreted at the level of nucleosomes.
The second PHD finger of human BPTF is known to
specifically recognize histone H3 when methylated
on lysine 4 (H3K4me2/3). Here, we examine how
additional heterotypic modifications influence BPTF
binding. Using peptide surrogates, three acetyllysine
ligands are indentified for a PHD-adjacent bromodo-
main in BPTF via systematic screening and biophys-
ical characterization. Although the bromodomain
displays limited discrimination among the three
possible acetyllysines at the peptide level, marked
selectivity is observed for only one of these sites,
nucleosome level. In support, these two histone
marks constitute aunique trans-histone modification
pattern that unambiguously resides within a single
nucleosomal unit in human cells, and this module
colocalizes with these marks in the genome.
Together, our data call attention to nucleosomal
The mechanism bywhich covalent modifications of histones and
DNA contribute to the chromatin structural states that govern all
DNA-templated processes is a central question to under-
standing genome management and its dysregulation in human
disease. Advances in understanding the role of chromatin
modifications may be divided into two separate veins: (1)
enumerating and characterizing chromatin modification-effector
pairs (Taverna et al., 2007) and (2) discerning relative modifica-
tion patterns at the genome level and correlating these patterns
to function (Bernstein et al., 2005; Guenther et al., 2007; Wang
et al., 2008). However, the convergence of these two areas—
remains poorly understood.
We have recently proposed that multivalent engagement of
nucleosomal units bearing distinct epigenetic signatures by
chromatin modification complexes may be involved in many
chromatin transactions (Ruthenburg et al., 2007b). Though
compelling tests of the ‘‘multivalency hypothesis’’ have yet to
occur, earlier studies have provided hints that this phenomenon
may be more general than currently appreciated: (1) Greater net
binding affinity and substrate specificity beyond the sum of
constituent parts may arise in the binding of two proximal acetyl-
lysines in the H4 tail by the bromodomain proteins hTaf1 and
Brdt (Jacobson et al., 2000; Morinie `re et al., 2009); (2) Multiple
contact surfaces distributed over a number of subunits (some
of which appear to be histone modification dependent) are
required for Rpd3S histone deacetylase complex binding to a
single nucleosome (Li et al., 2007). However, the interplay
between discrete histone modification-dependent interactions
has not been well studied in a nucleosomal context, nor is there
a clear example of a protein complex or single polypeptide that
simultaneously engages two or more histone modifications on
a nucleosome for which the discrete constituent interactions
are clearly defined. Thus, several key questions posed in the
histone code hypothesis (Strahl and Allis, 2000) still remain
unresolved: how are combinations of histone modifications
692 Cell 145, 692–706, May 27, 2011 ª2011 Elsevier Inc.
interpreted at the molecular level, are there units of recognition
beyond single tails, and what are the functional consequences?
Here, we sought to address how chromatin modification
patterns may be simultaneously engaged on the nucleosome
level using a PHD finger and adjacent bromodomain of the
NURF chromatin remodeling complex subunit BPTF as a para-
digm to provide insights into the above questions. We examine
the biochemical, structural, and functional properties endowed
by a bivalent configuration of these linked effector domains,
the simplest case of multivalent histone modification-dependent
nucleosomal engagement (Ruthenburg et al., 2007b).
BPTF in the context of the NURF complex is an essential regu-
lator of chromatin structure in development (Badenhorst et al.,
2002; Landry et al., 2008; Wysocka et al., 2006), bringing about
transcriptional activation or repression in a locus-specific
manner (Bai et al., 2007; Kwon et al., 2008) by virtue of the
complex’s chromatin remodeling activity (Hamiche et al., 1999;
Tsukiyama and Wu, 1995). The second PHD finger of BPTF,
implicated in recruitment or stabilization of the NURF complex
to active homeotic genes as a consequence of MLL1-mediated
H3K4 trimethylation, is followed closely by a bromodomain
whose mechanistic role is obscure (Wysocka et al., 2006). The
spatial coupling of these two domains is sufficiently tight to
permit determination of the structure of the terminal PHD-bro-
modomain module spanned by an apparently rigid linker a helix
(Li et al., 2006). Though the molecular details of H3K4me3
binding by the PHD finger are known, the ligand for the associ-
ated bromodomain remains unclear. Given that the bromodo-
main is a well-established histone acetyllysine recognition
domain (Dhalluin et al., 1999; Mujtaba et al., 2007), we envi-
sioned that, together with the PHD finger, this bivalent structural
element may bind two different classes of histone modifications
generally associated with euchromatin and transcription initia-
tion (Guenther et al., 2007; Ruthenburg et al., 2007a; Shogren-
Knaak et al., 2006). In support, deletion of both the PHD finger
and the adjacent bromodomain rescued BPTF knockdown in
Xenopus less efficiently than a PHD finger mutation that com-
pletely abolishes H3K4me3 interactions (Wysocka et al., 2006).
To examine the nature of this putative bivalent nucleosomal
recognition by the PHD-bromo module (Figure 1A), we first
sought to identify and characterize potential bromodomain
The BPTF Bromodomain Binds Three Different
Acetylated H4 Peptides
Using SPOT blotting (Nady et al., 2008), we screened a spatially
arrayed library of all known core histone acetylation marks (Basu
et al., 2009). This analysis revealed two acetylation marks that
specifically bound recombinant BPTF bromodomain (Figure 1B).
Peptides representing histone H4 acetylated lysines 16 and 20
(H4K16ac and H4K20ac) consistently displayed the strongest
interaction with the BPTF bromodomain in this assay, whereas
the unmodified counterpart displayed no detectable signal
above background (Figure 1B and Figure S1C available online).
Affinities for these two peptides and set of control peptides
were validated by peptide pull-down and then examined more
carefully via fluorescence polarization anisotropy (FPA) and
isothermal titration calorimetry (ITC) (Figures 1C–1E). These
additional experiments revealed affinity for H4K12ac, perhaps
not apparent with SPOT analysis because the array peptide
only included three residues N terminal to the K12ac mark.
specificity for these three acetylation sites in the H4 tail, whereas
other acetylated H4 peptides and unmodified H4 counterparts
displayed only weak affinity outside of the experimentally quan-
S1H). These measurements are consistent with the promiscuity
and affinity (H4K12ac [Kd, ITC= 69 ± 1 mM], H4K16ac [Kd, ITC=
99 ± 7 mM), and H4K20ac [Kd, ITC= 130 ± 10 mM]) reported for
other bromodomains (Dhalluin et al., 1999; Mujtaba et al.,
2007; Zeng et al., 2008).
Properties of BPTF PHD-Bromo Binding at the Peptide
versus Nucleosome Level
combined with the well-established PHD finger affinity for
H3K4me2/3 (Li et al., 2006; Wysocka et al., 2006), we sought
to examine the properties of bivalent ligand binding by these
two linked binding domains. Simultaneous binding of the BPTF
PHD-bromo cassette could have two nonexclusive conse-
quences: allosteric cooperativity wherein the binding of a given
peptide in one of the two modules may influence the binding of
cognate peptide to the other by a conformational shift
(Changeux and Edelstein, 2005) or a multivalent interaction
wherein two coupled entities may bind with greater net affinity
and specificity than their discrete constituent binding equilibria,
largely as an entropic effect (Krishnamurthy et al., 2006; Ruthen-
burg et al., 2007b). We used peptide-level binding experiments
with the PHD-bromo module exploiting the heterotypic ligand
binding properties of each domain with one ligand at saturating
concentration to query for possible allosteric enhancement. For
simplicity, we restricted our initial experiments to H4K16ac in
combination with H3K4me3. The binding of the PHD-bromo
unit to a fluorescein-labeled H3K4me3 peptide was assessed
by FPA in the presence of unlabeled H4K16ac peptide at a
concentration 5-fold greater than its bromodomain binding Kd.
This titration did not reveal any significant displacement of the
binding curve relative to similar titrations with excess unmodified
H4 peptide or without any H4 peptide (Figure 2A). Moreover, the
reciprocal experiment did not detect cooperativity (Figure 2B).
Thus, we conclude that, at the peptide level, each binding event
is free from detectable allostery.
If the binding of both peptides is effectively bimolecular, as
might be anticipated for the BPTF PHD-bromodomain engaging
both marks within a nucleosome, a free energy enhancement in
binding might occur due to multivalency (Krishnamurthy et al.,
2006; Ruthenburg et al., 2007b). To test this possibility, we
developed a novel biophysical assay with histone peptides
attached to a rigid DNA duplex to assess the spatial require-
ments of cooperative and simultaneous histone tail binding.
When the spacing between these two short peptides along
relative to DNA-peptide conjugates bearing each single peptide
Cell 145, 692–706, May 27, 2011 ª2011 Elsevier Inc. 693
annealed to an unmodified complementary DNA strand (Fig-
ure 2C). Given the distance spanned by the two peptide-binding
pockets (?70 A˚), this result suggests simultaneous binding by
both modules.ThattheH3K4me3peptide-duplex conjugatedis-
played little binding affinity was unexpected because the Kdfor
the PHD finger binding this peptide is ?100-fold tighter than
that of the bromodomain binding H4K16ac (Li et al., 2006). Our
interpretation of this result is that the H3K4me3 peptide is
static repulsion from the DNA conjugate upon binding, sup-
ported by the observation that this short peptide is bound effec-
tively in the absence of DNA (Figure S2D). Remarkably, this
impairment of PHD finger binding can be partially overcome by
H4K16ac into WT bromo
H4 unmod. into WT bromo
s / l a
) . t p
e l o
/ l a
0 10 203040 5060 70
H4K20ac into WT bromo
) . t p
e l o
/ l a
s / l a
H4K12ac into WT bromo
s / l a
) . t p
e l o
/ l a
Figure 1. Systematic Characterization of Preferred Acetylated Histone Ligands for the BPTF Bromodomain
(A) Schematic representation of a putative bivalent nucleosomal interaction with the BPTF PHD-bromo module. The known point of contact (Li et al., 2006;
Wysocka et al., 2006) is illustrated between the PHD finger (red) and H3K4me3 (red circle atop green histone tail), whereas the bromodomain (blue) interacts with
an unknown acetylation site (flags on histone tails).
(B) A SPOT blot of an array containing all known human core histone acetylation sites on a modified cellulose scaffold probed with GST-tagged bromodomain.
A representativeSPOT blot(controls and remainingreplicates,Figures S1A–S1C)displaying reproducible staining for H4peptides(residues11–25)acetylated on
the 3 amines of lysines 16 and 20, respectively (H4K16ac and H4K20ac). The staining of H2BK85ac (red asterisk) appears to be a peptide-HRP interaction, and
there is no detectable binding between the bromodomain and this peptide in solution (Figure S1E).
(C) Peptide pull-down experiments with GST and GST-BPTF bromodomain (GST-bromo) against three peptide series indicated. Full gels with GST controls are
available in Figure S1H.
(D) Fluorescence polarization anisotropy-based titration of the BPTF bromodomain against each of the indicated peptides (data are represented as mean ± SD).
(E) Isothermal titration calorimetry-based binding curves; the indicated H4 peptides are titrated into a solution of BPTF bromodomain (see Figure S1F for
See also Figure S1.
694 Cell 145, 692–706, May 27, 2011 ª2011 Elsevier Inc.
Encouraged that simultaneous binding of the PHD-bromodo-
main modules may provide an affinity enhancement, we sought
to study these multivalent interactions on a more physiologically
relevant substrate, the nucleosome.
To assess the consequences of bivalent nucleosome engage-
ment, we sought to construct nucleosomes bearing the desired
combinations of posttranslational modifications. To this end, we
employed a histone semisynthesis approach of expressed
protein ligation (EPL) (Muir, 2003; Shogren-Knaak and Peterson,
2004) to afford homogenously modified histones that could be
reconstituted with recombinant human core histones into oc-
tamers and then mononucleosomes on a strong positioning
sequence (Figure S3 and Figure S4). GST-tagged BPTF PHD-
bromodomain was immobilized on a glutathione resin and
interrogated for binding to these radiolabeled mononucleo-
somes. A 2- to 3-fold enhancement of nucleosomal binding
affinity was reproducibly observed for nucleosomes bearing
both H4K4me3 and H4K16ac over mononucleosomes with
only H3K4me3, whereas no binding was observed for the corre-
sponding unmodified species (Figure 3A). Importantly, there was
little detectable binding in this assay for nucleosomes acetylated
only at H4K16—as might be anticipated from the ?100-fold Kd
difference of the two discrete interactions extrapolated to an
off-rate dominated binding measurement. To exclude GST-tag
dimerization artifacts and surface effects, we performed a recip-
rocal experiment wherein nucleosomes are immobilized to a
solid support and protein without the GST-tag is queried for
nucleosome interaction by pull-down. The results are similar
(Figure 3B and Figures S3K and S3L). We interpret the enhanced
binding of doubly modified mononucleosomes to suggest that
both marks may play a role in nucleosome-level binding, yet
the PHD-H3K4me3 interaction is dominant.
Using this same assay, we revisited the question of specificity
ously identified in peptide-binding assays. Although selectivity
between H4K12ac, H4K16ac, and H4K20ac peptides is limited,
we wondered whether there might be additional binding con-
straints imposed by these marks when presented in a nucleo-
somal context. Though this assay is not sufficiently sensitive to
detect bromodomain engagement of the acetylation marks in
the absence of H3K4me3 binding (Figure 3C), again we observe
a binding enhancement attributable to bivalent engagement only
for H3K4me3 in combination with H4K16ac. Surprisingly, the
other two acetylation marks (H4K12ac and H4K20ac), when
paired with H3K4me3, do not display any binding enhancement
beyond that due to H3K4me3 binding alone (Figure 3D). Despite
similar peptide-level binding by the bromodomain, there is clear
binding specificity at the mononucleosome level.
Does the BPTF PHD-bromo module preferentially bind to
mononucleosomes, or do higher-order chromatin structures
present the respective tails in a more productive spatial disposi-
tion for engagement? To begin to address this question, we
constructed a series of dinucleosomal species via heteromeric
DNA ligation of two mononucleosomes (McGinty et al., 2008;
pull-down experiments indicate that the dinucleosome com-
posed of a nucleosome bearing both H3K4me3 and H4K16ac
in position A and an unmodified nucleosome in position B is
a modestly preferred binding partner of the PHD-bromodomain
over the same two marks, each in adjacent nucleosomes
assorted into either configuration (lane 6 versus 4 and 8 in Fig-
100 200300 400
H3K4me3 + H4K16ac
0.01 0.11 10 100
– H4 peptide
+ H4K16ac (unlab)
+ H4 unmod (unlab)
Fraction H3K4me3 bound
– H3 peptide
+ H3K4me3 (unlab)
+ H3 unmod (unlab)
Fraction H4K16ac bound
0.11 10100 1000
Figure 2. Is There Allostery or Bivalent Cooperativity in Simulta-
neous Ligand Binding by the BPTF PHD-Bromodomain?
(A) The possible allosteric cooperative binding at the peptide level is examined
by fluorescence polarization anisotropy using 100 nM fluorescein-labeled
H3K4me3 peptide, with no additional peptide, excess unlabeled H4K16ac
(B) The converse experiment with respect to (A). With unlabeled H3K4me3 in
excess (20 mM, ?10-fold above PHD-H3K4me3 Kd) and fluorescein-H4K16ac
(150 nM), protein is titrated and resulting fluorescence polarization anisotropy
measured (expressed here as fraction bound).
(C) A dsDNA scaffold, selected for rigidity while retaining little predicted
bending, was used to covalently install H3K4me3 (H3K4me3[1–8], green),
H4K16ac (H4K16ac [12–20], red), or both peptides (blue) at specific positions
by disulfide formation with cystamine-derivatized convertible dC nucleosides
(Figure S2). All of these DNA-protein conjugates were immobilized in different
flow channels via a single 30-biotin linkage to a streptavidin-coated surface
plasmon resonance chip at low density; untagged PHD-bromo was applied,
and background binding was subtracted from an empty flow cell.
Data are represented as mean ± SD. See also Figure S2.
Cell 145, 692–706, May 27, 2011 ª2011 Elsevier Inc. 695
H4K12ac + Me
H4K16ac + Me
H4K20ac + Me
H4K12ac + Me
H4K16ac + Me
H4K20ac + Me
Figure 3. The BPTF PHD-Bromo Module Simultaneously Engages Two Heterotypic trans-Histone Marks in Nucleosomal Contexts
(A)GSTpull-downofmodifiednucleosomes withsemisynthetichistonesproduced byEPL.Nucleosomes(unmod,WTrecombinanthistones;H4K16acmodified;
H3K4me3 modified; and dual, H4K16ac and H3K4me3 modified) pulled down with resin-bound GST or GST-BPTF PHD-bromo module protein are detected
by autoradiography after native gel electrophoresis or scintillation counting normalized to indicated % input. Rel. CPM, yellow bars, represent mean ± SD.
(B) The reciprocal experiment relative to Figure 3A. A western blot of HA-tagged PHD-bromodomain (without GST-tag) retained on streptavidin-immobilized
mononucleosomes following extensive washing relative to 1% input.
(C) The nucleosomes bearing acetylated H4 alone do not display significant binding in the same GST-pull-down experimental format as described in (A).
An additional control nucleosome species with H3T32C and H4R23C histones (unmod-C) serves as an unmodified nucleosome control that retains the
cysteine ligation scars.
(D) In this experiment, all three H4 acetylation marks that are bound at the peptide level (Figure 1) are examined in combination with H3K4me3 at the nucleosome
level as in (A). Data are represented as mean ± SD.
696 Cell 145, 692–706, May 27, 2011 ª2011 Elsevier Inc.
binding to H3K4me3 and either of the other candidate H4 acetyl
marks across two nucleosomes. Though this experiment does
not exclude the possibility that higher-order nucleosome arrays
or different spacing of linker DNA between nucleosomes A and
B could produce more favorable tail orientations, our data
suggest that the PHD-bromo module engages chromatin in an
intra- rather than internucleosomal binding mode under the
To more precisely examine the intranucleosomal binding
properties of the PHD-bromodomain, we next explored a panel
of mutations that either abrogate the capacity of each domain
to bind their respective substrates or perturb the spatial appo-
sition of these two domains at the nucleosome level. An inser-
tion of two amino acids (+QS) into the apparently rigid helix
that links the PHD and bromodomains should rotate the two
domains ?200?out of phase, assuming that the helix remains
intact, changing the relative orientation of histone binding
pockets (Figure 4A, inset). Neither perturbation of this helix
nor mutations in the other domain substantially impact a given
domain’s intrinsic peptide binding capacity by FPA (Figure 4B).
In the GST pull-down experiment, the W32E mutant most
severely impaired BPTF association, whereas the F154A mutant
displayed similar binding to the WT protein engaging the
H3K4me3-modified nucleosome (Figure 4C). (For compact
notation, all amino acid numbering here is relative to the start
of the PHD finger at amino acid 2717 in the full protein.) Interest-
ingly, a helix insertion mutant (+QS) is no longer able to bind
doubly modified nucleosomes with both binding modules
concomitantly reducing the amount of nucleosome retained to
levels that are commensurate to the WT protein-binding nucle-
osomes bearing only H3K4me3. We further explored the role of
this linker with extensive mutagenesis depicted schematically in
Figure 4A insets. Perturbations of the linker were designed to
extend the helix or introduce flexibility in this linkage with
canonical helix-forming or helix-breaking residues (Chou and
Fasman, 1978). Insertion near or replacement of several resi-
dues in the center of the helix uniformly impaired apparent biva-
lent interaction (Figure 4D). Taken together, these results
suggest that the precise relative orientation of the two domains
is a critical determinant of bivalent mononucleosome binding
Structural Analysis Reveals Two Distinct Acetyl-Histone
As the mechanism of the composite selectivity in bivalent
binding at the nucleosome level remained elusive, we wondered
whether the molecular basis of H4ac peptide binding might
provide meaningful insight. To this end, we solved high-resolu-
tion crystal structures of two different crystal forms of the
BPTF bromodomain in complex with H4K16ac peptide—the
preferred ligand of the PHD-bromodomain when combined
with H3K4me3—and one structure of PHD-bromodomain in
complex with H4K12ac peptide, representing a binding partner
that is not selected for in bivalent nucleosome binding, as a point
of comparison (Figure 5 and Table S1). All of these data sets
flanking residues (Figures S5D–S5F). Beyond the well-docu-
mented bromodomain-acetyllysine contacts that are nearly
identical in all three structures (Dhalluin et al., 1999; Mujtaba
et al., 2007; Zeng et al., 2008), specific contacts are apparent
in each structure that account for sequence context specificity.
The crystal form I H4K16ac-bromodomain complex reveals
interactions that are analogous to those observed with the
GCN5 bromodomain bound to the same mark (Ca rmsd =
0.95 A˚; Figure S5C). Remarkably, the H4 peptide orientation
(N to C terminus) is inverted in our second structure (form II) rela-
tive to the first structure (Figures 5A, 5B, and 5F), despite nearly
identical bromodomain conformation (Ca rmsd = 0.52 A˚). A
similar reversal of peptide binding orientation has been previ-
ously noted with the PCAF bromodomain, albeit with two
different H3 acetylation sites (Zeng et al., 2008). As a point of
comparison to the two H4K16ac structures, we examined a
PHD-bromodomain complex with ligand that does not con-
tribute to bivalent binding of the PHD-bromodomain in combina-
tion with H3K4me3. The structure of the PHD-bromodomain in
complex with H4K12ac displays a ‘‘reversed’’ peptide binding
orientation similar to that observed in the form II complex
(Figures 5B and 5C). However, the ordered region of the
H4K12ac peptide is located atop the aB helix, more reminiscent
of the peptide positioning in the form I H4K16ac complex
(Figures 5A and 5F; for further details, see Supplemental
Do both of these binding orientations contribute to the net
affinity of the bromodomain for H4K16ac peptide? ITC was per-
formed with bromodomain mutants designed to specifically per-
mode intact (Figures 5D and 5E). Although the binding of these
mutant proteins to H4K16ac peptide was not strong enough to
reliably quantify dissociation constants, residual binding affinity
is apparent in the solution ITC measurements (compare ITC in
Figures 5D and 5E with the H4 unmodified peptide binding in
Figure 1E). Drastic alterations of contacts proper to the form II
structure via point mutation of Trp 91 or Asp101 to alanine
both severely erode but do not completely destroy binding (Fig-
ure 5E and Figures S5G and S5H). Selective disruption of inter-
actions found only in the form I structure proved challenging,
yielding conservative mutations that are more modest in their
efficacy, V108A and Y147F. Nevertheless, these mutants
demonstrate the solution binding relevance of this binding
mode (Figure 5D and Figures S5G and S5I). From these data,
we conclude that both bromodomain binding modes may play
roles in the net affinity for H4K16ac peptide binding in solution.
How the distinct molecular interactions in both the H4K12ac
and H4K16ac complexes provide a plausible mechanism for
nucleosomal H4 acetyl selectivity during bivalent binding of the
nucleosome is addressed below (see Discussion).
(E) GST pull-down of hetero-dinucleosomes composed of two mononucleosomes indicated in positions A and B ligated together; each lane is labeled below
numerically. The input dinucleosomes for this experiment are presented in Figure S3N.
See also Figure S3.
Cell 145, 692–706, May 27, 2011 ª2011 Elsevier Inc. 697
BPTF Colocalizes with Doubly Modified Nucleosomes
in the Nucleus
The localization of BPTF to the HOXA9 gene locus that is contin-
gent upon H3K4 methylation mediated by the MLL complex has
previously been established in HEK293 cells (Wysocka et al.,
2006). Furthermore, MOF-mediated H4K16ac is highly enriched
at the HOXA9 locus in these cells (Dou et al., 2005). Native
chromatin immunoprecipitation (ChIP) followed by qPCR on
mononucleosome biased fragments recapitulates these trends
and affirms more than an order of magnitude signal difference
for amplicons along this locus with the H4K12ac and H4K16ac
antibodies (Figure 6A). To critically assess the relative import of
each element in the BPTF PHD-bromo cassette for the localiza-
tion of the full-length BPTF polypeptide to the HOXA9 locus, we
established HEK293 cell lines with stable and equivalent full
BPTF expression (WT and several of the mutants described
above; Figures S6A and S6B). The association of ectopically
tagged BPTF protein with regions of the HOXA9 locus, as as-
sessed by ChIP (Figure 6B), largely recapitulates our in vitro find-
ings within the modest dynamic range of the experiment. All
WTFA dblWE +QSInWTFA dblWE +QS
WTFA dblWE +QSWTFA dblWE +QS
+2 helix insertions
(TED→GGG, TED→GGSGG, TED→GGSGGSG)
“helix breaking” mutations
DDT PHD PHD bromo
Fraction H4K16ac bound
+QS (helix insertion)
1 10 100 1000
0.11 10 100
Fraction H3K4me3 bound
+QS (helix insertion)
Figure 4. Querying the Roles of the PHD Finger, the Helical Linker, and the Bromodomain in Bivalent Nucleosome Binding via Mutagenesis
(A) A ribbon representation of the composite structure of the BPTF PHD-bromodomain module derived from superposition of the previously determined
PHD-bromo in complex with H3K4me3 (Li et al., 2006) with the form I BPTF bromodomain H4K16ac complex (Figure 5A). The predicted domain structure of
human BPTF shows the position of this module within the whole protein, colored as in Figure 1A, and mutations are indicated in gray. Inset panels schematically
depict the anticipated consequences of a series of linker helix mutations.
(B) Mutations of the PHD-bromo module used to interrogate multivalent binding suggested by the structure assessed by FPA. (Top) A W32E mutation abolishes
H3K4me3 binding without disrupting the PHD finger fold (Li et al., 2006; Pen ˜a et al., 2006; Ruthenburg et al., 2007a), whereas a two amino acid linker helix
insertion (labeled +QS) leaves the H3K4me3 binding capacity intact. (Bottom) A F154A mutation, designed by analogy to previous bromodomain mutagenesis
(Dhalluin etal.,1999),abolishes bromodomain bindingofH4K16ac,whereasneitherthe+QSnortheW32Emutantsdisrupt binding. SeeFigureS1FforKdvalues.
(C) Comparison of the WT GST-PHD-bromodomain (WT) to the series of mutant proteins in the same GST pull-down format as Figure 3. Mutants are labeled WE
(W32E), FA (F154A), dbl (W32E + F154A), and +QS (a QS insertion after S58 in the bridging helix between the PHD) as depicted in (A), and 5% input is loaded for
(D) Mutations designed to break the a helix (residues 59–61 all mutated to glycine, TED/ GGG; the same residues mutated to glycines in combination with ‘‘SG’’
or SGGS insertions, TED/ GGSGG and TED/ GGSGGSGG, respectively) are compared to mutations intended to extend the helix and thereby rotate the two
domains out of phase (+QS or +AA inserted between and S58 and T59) in the same experimental format as (C). Mutations that effectively insert two amino acids
into the linker helix are indicated in red.
Data are represented as mean ± SD. See also Figure S4.
698 Cell 145, 692–706, May 27, 2011 ª2011 Elsevier Inc.
mutations diminish binding, suggesting that a minimally bivalent
mode of nucleosomal engagement is important for BPTF recruit-
ment or stabilization at this locus. Importantly, the +QS mutation
disrupts localization to HOXA9. Thus, even in the context of full-
length BPTF and presumably other NURF complex members,
the precise orientation of the PHD finger and the bromodomain
appears to be crucial for full binding both in vitro and in vivo.
Given that the cell lines were constructed by stable integration
of tagged BPTF into a genome with native BPTF expressed
and that the NURF complex is dimeric in this subunit (Barak
et al., 2003), complete loss of localization is not expected. In
order to bypass this complication, we then restricted our ChIP-
seq experiments to the BPTF PHD-bromodomain module.
Native ChIP followed by Illumina sequencing was performed
H3K4me3, H4K12ac, and H4K16ac marks in this cell line and
correlate them with the PHD-bromodomain localization (Figures
6C–6E). The latter data set was gathered via cross-linking ChIP
from a tagged PHD-bromo expressing HEK293 cell line (Fig-
ure S6A). We detect numerous gene-proximal chromatin
domains that bear significant peaks for H3K4me3 and each of
the two acetylation marks examined; two examples are depicted
in Figures 6C and 6D. Significant overlap between peaks of
H3K4me3 and the tagged PHD-bromodomain was observed in
a global sense. Although the number of clear peaks for the
PHD-bromo is a much smaller set than the H3K4me3, 84% of
these PHD-bromo peaks appear within 500 bp of an H3K4me3
peak (Figure S6C). As previously observed (Wang et al., 2008),
the acetyl-specific ChIP-seq tracks are qualitatively more diffuse
and less peak-like than the H3K4me3 signal, and consequently,
peak calling relative to input was more challenging. Even so, the
average tag densities differ in a locus-specific manner biased
toward euchromatic regions, suggesting that the acetyl histone
signal is meaningful (Figure S6D). Given that the PHD-bromo
module colocalizes with a subset of H3K4me3 peaks (28%)
and the broader distribution of acetyl marks, we would not
expect very significant overlap in a global metagene analysis.
Indeed, as depicted in Figure S6E, there is some overlap in the
PHD-bromo and H3K4me3 average signal plotted for all genes
normalized to 3 kb and very modest correlation with acetyl
mark signal (Shin et al., 2009).
Although the PHD-bromo is not present at every H3K4me3
site, its presence appears to be more correlated with loci that
also bear histone H4 acetylation. Plotting the average PHD-
bromo tag count over the peak regions in the H4K16ac and
H3K4me3 data sets, as well as where these two peaks intersect
(within 150 bp), the apparent tag density is much higher for the
PHD-bromo when H3K4me3 is combined with H4K16ac relative
to either mark in isolation (Figure 6E). However, this also
appears to be the case for the PHD-bromodomain plotted on
intervals that are called peaks for both H4K12ac and
H3K4me3. Taken together, these data suggest that the PHD-
bromodomain tends to colocalize with H3K4me3 in regions
that appear to have a reasonably high density of both H4K12
and H4K16 acetylation marks, particularly near TSS elements.
The ChIP-seq data are consistent with the proposed role of
both H3K4me3 and H4 acetylation in PHD-bromo recruitment,
but there seems to be little distinction between the two H4ac
marks when examined by this method. This raises an important
question regarding overlapping ChIP-seq peaks: do they actu-
ally represent coexistence of two given marks within a single
In order for the simultaneous bivalent binding of NURF
described in vitro to be meaningful, mononucleosomes bearing
both H3K4me3 and H4K16ac marks must exist in cells. To
address this issue, we isolated high-purity mononucleosomes
by sucrose gradient fractionation of MNase fragmented chro-
matin derived from HEK293 nuclei (Figure 7A and Figures S7A–
S7F) (Mizzen et al., 1999). We then examined these purified
nucleosomes for the coexistence of H3K4me3 with H4K16ac
marks by coimmunoprecipitation relative to additional modifica-
tions and variants. There is a substantial pool of mononucleo-
somes bearing both modifications implicated in BPTF binding,
H3K4me2/3 and H4K16ac (Figure 7B), whereas neither of these
marks was found to reside in the same mononucleosomes as
canonical repressive marks (H3K9me3 and H3K27me3). To
exclude the possibility of off-target antibody recognition bias,
we performed the reciprocal immunoprecipitation experiments,
employing an a-H3K4me3 antibody for IP followed by staining
for H4K16ac (Figure 7B, right). H3K4me3 antibodies did not
robustly IP H4K12ac—another mark that the bromodomain is
capable of binding at the peptide level (Figure 1) —or any other
H4 acetylation mark. This finding suggests another possible
source of selectivity: H4K12ac/H3K4me3 doubly modified
mononucleosomes do not appear to be present in a detectable
population (antibodies are not available to H4K20ac, the other
at the peptide level; see Figure 1). However, we observed
substantial co-occupancy of H3K4me3 and H4K20me2, consis-
tent with the observation that > 80% of H4K20 is dimethylated at
any given time, whereas the corresponding acetylation is not
very abundant (Pesavento et al., 2008).
The analogous pull-down from the same highly purified
mononucleosomal pools employing GST-tagged BPTF in place
of antibodies again demonstrates preferential binding of the
PHD-bromo module to H3K4me3- and H4K16ac-bearing nucle-
marks resembled the pattern observed in the mononucleosomal
IPs. Consistent with our semisynthetic nucleosome experi-
ments, the PHD-bromo module bound a greater quantity of
H4K16ac mononucleosomes as compared to the bromodomain
alone (Figure 7D), with similar resin loading levels (Figure S7F).
mononucleosomes under these conditions. This could reflect
lower abundance of intranucleosomal H3K4me3 but may also
be a function of reduced BPTF binding. In order to distinguish
these two possibilities, we cultured cells in trichostatin A,
a potent HDAC inhibitor that serves to enrich otherwise transient
acetylation marks like H4K12ac (Pesavento et al., 2006); and
despite substantial enrichment of the mark, we observed
minimal binding to the PHD-bromo module (Figure S7H).
One explicit prediction of the histone code hypothesis is the
combinatorial readout of multiple histone marks (Strahl and Allis,
Cell 145, 692–706, May 27, 2011 ª2011 Elsevier Inc. 699
form IIform II
010 2030 4050 6070
0 102030 4050 6070
H4K16ac into D101A bromo
Kd > 600 µM
H4K16ac into W91A bromo
Kd > 700 µM
s / l a
s / l a
Kd > 200 µM
H4K16ac into Y147F bromo
s / l a
Kd > 500 µM
H4K16ac into V108A bromo
s / l a
Figure 5. Structural Analysis of the BPTF Bromodomain Peptide Complexes
(A) The model derived from crystal form I (bromodomain in blue) with the apical binding site for the H4K16ac peptide (green) rendered in ribbons and sticks.
Hydrogen bonds are displayed as yellow dashed lines.
(B) Interactions in crystal form II. The bromodomain is colored purple, and the bound H4K16ac peptide is depicted in gray.
(C) Interaction of the PHD-bromodomain in complex with H4K12ac peptide (peptide, orange; bromodomain, cyan).
700 Cell 145, 692–706, May 27, 2011 ª2011 Elsevier Inc.
2000), although experimental support for this prediction is lack-
typic histone modifications simultaneously in a binding event
that spans two different histone tails to establish nucleosome-
level engagement contingent upon two discrete modifications.
Of the three acetylated peptides with measureable bromodo-
main affinity, H4K16ac is an intuitively attractive BPTF binding
partner. This mark is installed by the MOF acetyltransferase
that may reside within the same MLL1 complex (Dou et al.,
2005) that methylates H3K4 (Milne et al., 2002), the preferred
binding partner of the proximal PHD finger (Li et al., 2006;
Wysocka et al., 2006). The aggregate affinity of the PHD-bromo
module for H3K4me3 and H4K16ac doubly modified nucleo-
somes is greater than that of the PHD and bromodomains alone
yet more modest in magnitude than perhaps one would antici-
pate from discrete module-mark dissociation constants. Impor-
tantly, the bivalent nature of this interaction appears to effec-
tively enhance the specificity of the bromodomain. Tethering of
the PHD-finger to nucleosomal H3K4me3 appears to constrain
the bromodomain to bind only H4K16ac, although at the peptide
level, there is not much distinction in binding affinities for this
mark relative to the two flanking H4 acetyl marks.
What is the molecular mechanism for this composite speci-
ficity? Notwithstanding the molecular basis of each domain’s
interactions with cognate peptides and the clear importance of
the linker in permitting bivalent interactions, the precise molec-
ularmechanismof thisselectivity remainselusive.Our combined
structural and mutagenesis studies with the BPTF bromodomain
provide one potential explanation: the H4K16ac peptide has
access to two alternate binding modes, whereas the H4K12ac
may have access to only one of these peptide binding orienta-
tions. Modeling suggests that only the peptide orientation in
(Figure S5J), whereas the binding mode in the crystal form II
structure does not seem to be compatible with bivalent binding
(Figure S5K). Using similar constraints, we are unable to model
the PHD-bromodomain spanning H4K12ac and H3K4me3 that
is consistent with binding orientations observed in the struc-
tures. Literal interpretation of this bivalent model brings the
BPTF PHD-bromo module in close contact with DNA. Yet,
inspection of protein surface electrostatics suggest that the
association may not be this intimate; H3 tail flexibility could
enable bivalent binding without engendering significant electro-
static repulsion from the nucleosomal DNA (Figure S5J, red
arrow displays potential rigid body movement of PHD-bromo
module away from the nucleosome). This model is consistent
with the hydroxyl-radical footprint of the Drosophila NURF
complex bound to a strongly positioned nucleosome; the region
of DNA near the pseudo-dyad at the duplex entrance/exit where
the N-terminal H3 and H4 tails emerge from the octamer core is
protected from cleavage (Schwanbeck et al., 2004). However,
unambiguous delineation of the mechanism of H4 acetyl mark
discrimination awaits the structural elucidation of the doubly
How might this modest affinity gain be functionally important
for the full NURF complex? It appears that every element of
the bivalent nucleosome-binding interface described here is
requisite for proper localization of the NURF complex to devel-
opmentally important loci in human cells. Yet in Drosophila,
genetic deletions of a large portion of the C terminus of BPTF
(including the PHD-bromodomain) present no developmental
defects outside of gametogenesis (Kwon et al., 2009), and there
are known DNA sequence-specific factors involved in NURF
recruitment (Badenhorst et al., 2005; Tsukiyama and Wu, 1995;
Xiao et al., 2001). In vertebrates, there are no analogous factors
that are known to be involved in NURF recruitment, nor have
clear DNA sequence elements related to NURF recruitment
been identified. Here, the role of histone modifications in the
recruitment or stabilization of NURF complex at target loci
module results in severe homeotic, hematopoietic, and gut
abnormalities that are not found in flies (Wysocka et al., 2006).
It is likely that the interplay of histone modification-specific
the ultimate energetics and specificity in binding that culminates
in genomic localization.Indeed,
described here could be tetravalent in the context of the NURF
complex, as there are two copies of BPTF in the NURF complex
(Barak et al., 2003). Beyond the established nucleosomal
contacts made by other NURF complex subunits—SNF2L likely
has at least three DNA contacts (by analogy to ISWI) (Gru ¨ne
et al., 2003; Schwanbeck et al., 2004), and RbAp46/48 binds
the first helix of H4 (Verreault et al., 1998) —Drosophila BPTF
(NURF301) bears three distinct nucleosomal interaction regions,
only one of which may be attributed to the PHD-bromo module
(Xiao et al., 2001). Specific interactions with the H4 tail, proximal
to K16 and K20, seem to be essential for remodeling activity of
NURF and other ISWI family complexes (Clapier et al., 2001; Ha-
miche et al., 2001; Shogren-Knaak et al., 2006).
Genome-level ChIP experiments have yielded a wealth of
information about the localization of the H3K4me3 and
H4K16ac histone marks; there are domains of significant over-
is little data available that suggest what the absolute modifica-
tion densities are at a given locus, so it is unclear to what extent
the bivalent interaction
patterns described by
(D) ITC with BPTF bromodomain mutants designed to disrupt the form I binding mode (V108A and Y147F). Each of these mutations displays modest binding
deficits comporting with their modest roles in the form I interface. Dissociation constants were outside of the accurately measurable range, so a lower limit of
possible Kdis provided for qualitative comparison.
(E) Mutations to disrupt the form II binding interactions while leaving the form I binding mode intact. More substantial mutations, W91A and D101A, produce
a more severe loss of affinity. For convenience of comparison, all DP scales are identical in scale.
(F) Binding conformations of peptides (colored as in previous panels) from each of the structures are compared by Ca-superposition of their respective
See also Figure S5.
Cell 145, 692–706, May 27, 2011 ª2011 Elsevier Inc. 701
P2P3 P4(-) control region
W32E + F154A
P1 P2 P3
P1 P2 P3
P1 P2 P3
Relative Distance from the Center (bp)
−1000 −5000 5001000
Figure 6. Bivalent BPTF Binding Is Important for Localization of the BPTF PHD-Bromodomain and Full NURF Complex
(A) Native ChIP comparison of H3K4me3, H4K12ac, and H4K16ac marks. Four primer sets were employed to interrogate the HOXA9 locus (P1–P3, dark and light
gray bars represent HOXA9 exons, and an untranslated region, respectively) and a distal intergenic site ([?] control region). An average of three real-time PCR
replicates of a representative experiment is displayed as a function of % input signal, with error bars reflecting ± SD of PCR product threshold error among the
replicates. For simplicity of HOXA9 display, the gene structure annotation represents the Crick strand sense of the genome in this as well as (B) and (C).
702 Cell 145, 692–706, May 27, 2011 ª2011 Elsevier Inc.
ChIP-seq represent modifications that synchronously reside
within a given nucleosome. By restricting the native chromatin
queried to highly purified mononucleosomes, we provide
compelling evidence for robust H4K4me3 and H4K16ac coexist-
ing within a single nucleosome. In contrast, only modest coexis-
tence of H3K4me3 and H4K12ac is detected by this method.
This underscores a potential pitfall in interpreting ChIP-seq
data: significant overlap of H3K4me3 with both of these acetyl
marks, on average, may be observed across large genomic
regions. Thus, apparent colocalization by this measure does
not necessarily mean that two such marks actually coexist in
the same mononucleosome.
The discovery of a significant pool of doubly modified
H3K4me3/H4K16ac mononucleosomes is consistent with the
biochemical identification of both enzymes that are responsible
for installing these marks associating within the MLL1 complex
inonepreparation (Douetal.,2005;Milne etal.,2002).Thecoex-
istence of these marks at MLL1-regulated loci by ChIP is also
consistent with the notion that this distinct population of doubly
modified mononucleosomes resides there. The clear coexis-
tence of H3K4me3 and H3K79me2 marks in mononucleosomes
was unanticipated, although others have noted this overlap by
ChIP-seq (Wang et al., 2008). Interestingly, both H3K4me3 and
H3K79me2 marks are downstream of H2B ubiquitylation (Briggs
etal., 2002), and recent biochemical studies suggest direct stim-
ulation of each responsible human methyltransferase by this
ubiquitylation (Kim et al., 2009; McGinty et al., 2008).
Here, we have provided evidence not only for simultaneous
recognition of two heterotypic histone marks in a binding event
but also potential resolution of a purported weakness in the
histone code hypothesis (Strahl and Allis, 2000). If more than
one module iscapable of binding a given mark and each of these
discrete modules resides in a different complex that transduces
different downstream functional consequences, how can that
mark itself have any unique information encoding potential
(Becker, 2006)? For example, the ‘‘split personality’’ of the
H3K4me3 mark may be engaged by Taf3 of the TFIID complex
to increase transcription (Vermeulen et al., 2007) or the ING2-
bearing mSin3a complex to silence certain genes upon DNA
damage (Shi et al., 2006). How can complex localization in these
diametrically opposed processes be governed by the same
mark? Our work suggests that both the PHD finger and bromo-
domain binding modules, as well as their relative orientation, are
important for the full BPTF binding and Hox gene localization of
the NURF complex. In this case, it is not the information content
of interpreting a single mark that matters; rather, it is the combi-
nation of engaging a pattern of marks and perhaps other local
chromatin features that ultimately dictates cellular localization.
Our findings call attention to the histone code being more
complex than the unique interpretation of a single mark and
provide support for multivalent recognition of the chromatin
In addition to BPTF, there are 22 other polypeptides in the
human proteome that display linked PHD fingers and bromodo-
mains, some of which are remarkably similar to the BPTF-PHD
module studied here (Ruthenburg et al., 2007b). Initial work
with several of these proteins suggests that each domain may
be important for chromatin association (Eberharter et al., 2004;
Ragvin et al., 2004; Tsai et al., 2010; Zhou and Grummt, 2005).
Investigation of whether similar bivalent interactions play a role
in their nuclear function will be of interest. More generally, we
anticipate that other examples of such combinatorial patterns
being recognized by multivalent contacts at the level of single
nucleosomes, oligonucleosomes, and chromatin territories will
be important for numerous genomic transactions.
SPOTblots,peptide pull-downs,and fluorescence polarizationanisotropyand
ITC were performed essentially as described (Li et al., 2006; Nady et al., 2008;
Wysocka et al., 2006). Details of recombinant protein production, X-ray
crystallography, DNA-ruler assays, histone semisynthesis, and nucleosome
reconstitution are available in the Extended Experimental Procedures. In brief,
recombinant histones were reconstituted into octamers with semisynthetic
histones prepared via expressed protein ligation as well as complementary
recombinant histones (Muir, 2003; Shogren-Knaak and Peterson, 2004);
then, nucleosomes on a [32P] end-labeled strong positioning sequence.
Each nucleosome type was incubated with glutathione resin-immobilized
GST-BPTF and washed five times for 45 min. The retained nucleosomal
to native gels, and/or quantified by scintillation counting. In nucleo MNase
digestion was performed as a hybrid of previous conditions (Brand et al.,
2008; Mizzen et al., 1999; O’Neill and Turner, 2003), with some modifications
ChIP was performed according to established protocols or adaptations
thereof; see Extended Experimental Procedures for details.
Protein Data Bank model coordinates and structure factors have been depos-
ing the H4K12ac + PHD-bromo complex, the P21 crystal form of the
(B) xChIP of HA-tagged BPTF at the HOXA9 locusfrom HEK293 cell lines that exhibit commensurate expression levels of the ectopically tagged constructs. ChIP
signal (with primer sets P2–P4) of tagged WT protein expressing cell lines is compared to discrete cell lines bearing the tagged mutant proteins corresponding to
mutations depicted in Figure 4. Data are represented as mean ± SD.
(C) ChIP-seq data for the HOXA9 locus, with the three histone modification tracks derived from nChIP-sequencing rendered in yellow, green, and red as labeled
with input tag counts overlayed in gray on the same scale. On the same abscissal scale and register, the xChIP sequencing counts from the 3 3 FLAG-tagged
BPTFPHD-bromodomain aredepicted inblue,withattendantinputsuperimposed ingray.Alltagcountsarenormalizedbythefactor(23107/totalmappedtags
pertrack) andareunique.Belowthecontinuoustagcountgraph,MACSpeak-calledregions relativetoinputforeachsequencing trackaredepicted inrectangles
of the same color.
(D) Another example of histone modification patterns and PHD-bromodomain binding, displayed as in the previous panel.
(E) Plot of average profile of the PHD-bromodomain at peak regions in the other data sets or intersects thereof. The average PHD-bromo signal is contoured on
the regions that have MACS called peaks for each individual modification (colored as in C) or at loci with called peaks within the same 150 bp window for both
H3K4me3 and H4K12ac data sets (purple) or H3K4me3 and H4K12ac data sets (blue).
See also Figure S6.
Cell 145, 692–706, May 27, 2011 ª2011 Elsevier Inc. 703
H4K16ac + bromodomain complex, and the C2 crystal form of the H4K16ac +
bromodomain complex, respectively.
Supplemental Information includes Extended Experimental Procedures,
seven figures, and two tables and can be found with this article online at
We would like to thank the staff at beamline 24ID-C of the Advanced Photon
Source at the Argonne National Laboratory and the staff at beamline X29 of
the National Synchrotron Light Source at Brookhaven National Laboratory,
supported by the US Department of Energy, for assistance with data collec-
tion. This work is based upon research conducted at the Northeastern Collab-
In nucleo digest
input IP IgG
Figure 7. The BPTF PHD-Bromodomain Preferentially Engages a Native Population of H3K4me3 and H4K16ac Doubly Modified
(A) Schematic representation of the experiments. Mononucleosome pools were isolated from in nucleo MNase digests and sucrose gradient ultracentrifugation.
See Figure S5 for details.
(B) Coimmunoprecipitation and western blotting of highly purified mononucleosomes with the well-validated modification and sequence-specific histone
(C) Recombinantly produced GST-PHD-bromodomain (GST-PB) was used to pull down native mononucleosomes, and the associated material is compared to
5% of the input mononucleosome pool and GST alone for the ability to bind native nucleosomes contingent upon modification patterns.
(D) An additional pull-down with the GST-tagged bromodomain alone (GST-bromo) was performed alongside GST and GST-PB, and the bound material was
probed by western Blot against the four acetyl marks on the H4 tail for which there are antibodies available.
See also Figure S7.
704 Cell 145, 692–706, May 27, 2011 ª2011 Elsevier Inc.
the Macromolecular Crystallography Research Resource (PXRR) at the
National Synchrotron Light Source, which are supported by the National
Center for Research Resources at the National Institutes of Health. We would
like to thank L. Liang for assistance in protein production and crystallization of
BPTF H4 peptide complexes; H.A. Zebrowski for assistance in the preparation
of the SPOT membrane; S.S. Yi of the Microchemistry and Proteomics Core at
Memorial-Sloan Kettering Cancer Center for synthesis of peptides; Y. Wei of
the Rockefeller Chemical Biology Spectroscopy center for use of their Biacore
instrument; S. Yokoyama and H. Kurumizaka for an expression construct
bearing codon optimized histone H4; K. Chiang for his 3_601_3_x32 repeat
plasmid; M.Vila-Perello for HF cleavage of Boc-peptides; H. Dormann for
HP1-chromodomain recombinantly produced protein; H. Yu of the Rockefeller
Proteomics Core for MS assistance; C. Wu for the human BPTF cDNA; and
L. Baker, F. Casadio, J. Denu, P.W. Lewis, K-M. Noh, R.G. Roeder, R. Sadeh,
D. Shechter, T. Swigut, G.G. Wang, and J. Wysocka for valuable discussions
and scientific input. A.J.R. is supported by Irvington Institute Fellowship
Program of the Cancer Research Institute, and R.K.M. is supported by an
MSTP grant. This work was supported by a MERIT grant from the NIH and
funds from The Rockefeller University to C.D.A., as well as funds from the
Leukemia and Lymphoma Society and Starr Foundation to C.D.A. and
D.J.P. D.J.P. is supported by funds from the Abby Rockefeller Mauze Trust
and the Dewitt Wallace and Maloris Foundations, and T.W.M. is supported
by an award from the NIH. D.J.P. is a consultant in GlaxoSmithKline’s
Received: March 12, 2010
Revised: September 7, 2010
Accepted: March 30, 2011
Published online: May 19, 2011
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