Towards elucidating the stability, dynamics andarchitecture of the nucleosome remodeling anddeacetylase complex by using quantitative interactionproteomicsSusan L. Kloet1*, H. Irem Baymaz1*, Matthew Makowski1*, Vincent Groenewold2, Pascal W. T. C.Jansen1, Madeleine Berendsen1, Hassin Niazi1, Geert J. Kops2,3 and Michiel Vermeulen1,2,3
1 Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen,
the Netherlands
2 Department of Molecular Cancer Research, UMC Utrecht, the Netherlands
3 Cancer GenomiCs Netherlands, the Netherlands
Keywords
chromatin; mass spectrometry; NuRD;
protein-protein interactions; quantitative
proteomics
Correspondence
M. Vermeulen, Department of Molecular
Biology, M850/3.79 259 RIMLS, P.O. Box
9101 6500 HB Nijmegen, The Netherlands
Tel: +31 024 3616848
E-mail: [email protected]
*These authors contributed equally to this
work.
(Received 15 July 2014, revised 7 August
2014, accepted 12 August 2014)
doi:10.1111/febs.12972
The nucleosome remodeling and deacetylase (NuRD) complex is an evolu-
tionarily conserved chromatin-associated protein complex. Although the sub-
unit composition of the mammalian complex is fairly well characterized, less
is known about the stability and dynamics of these interactions. Furthermore,
detailed information regarding protein–protein interaction surfaces within the
complex is still largely lacking. Here, we show that the NuRD complex inter-
acts with a number of substoichiometric zinc finger-containing proteins. Some
of these interactions are salt-sensitive (ZNF512B and SALL4), whereas others
(ZMYND8) are not. The stoichiometry of the core subunits is not affected
by high salt concentrations, indicating that the core complex is stabilized by
hydrophobic interactions. Interestingly, the RBBP4 and RBBP7 proteins are
sensitive to high nonionic detergent concentrations during affinity purifica-
tion. In a subunit exchange assay with stable isotope labeling by amino acids
in cell culture (SILAC)-treated nuclear extracts, RBBP4 and RBBP7 were
identified as dynamic core subunits of the NuRD complex, consistent with
their proposed role as histone chaperones. Finally, using cross-linking MS,
we have uncovered novel features of NuRD molecular architecture that com-
plement our affinity purification-MS/MS data. Altogether, these findings
extend our understanding of MBD3–NuRD structure and stability.
Structured digital abstract
� MBD3 physically interacts with ZNF512B, HDAC1, ZMYND8, GATAD2B, SALL4,
GATAD2A, ZNF592, MTA3, ZNF687, CDK2AP1, CHD3, ZNF532, HDAC2, MTA2,
CHD4, MTA1, KPNA2, CHD5, RBBP4 and RBBP7 by pull down (View interaction)
� CDK2AP1 physically interacts with MBD3, MTA3, HDAC2, GATAD2A, CHD4,
CDK2AP1, MTA2, HDAC1, MTA1, CHD3, GATAD2B, MBD2,
RBBP4 and RBBP7 by pull down (View interaction)
� MBD3 physically interacts with MTA2, MTA3, RBBP4, RBBP7, HDAC2, HDAC1,
CHD4, CHD3 and MTA1 by cross-linking study (View interaction)
Abbreviations
AP, affinity purification; FDR, false discovery rate; GFP, green fluorescent protein; HDAC, histone deacetylase; iBAQ, intensity-based
absolute quantification; NuRD, nucleosome remodeling and deacetylase; PDB, Protein Data Bank; SAS, solvent-accessible surface; SILAC,
stable isotope labeling by amino acids in cell culture; TAP, tandem affinity purification; TEV, tobacco etch virus.
1FEBS Journal (2014) ª 2014 FEBS
Introduction
The nucleosome remodeling and deacetylase (NuRD)
complex is a conserved chromatin-associated protein
complex that was first purified and characterized from
Xenopus laevis and human cells in the late 1990s [1–3].As the name implies, the NuRD complex has both
histone deacetylase (HDAC) and ATP-dependent
chromatin remodeling activity. In the human complex,
HDAC activity is catalyzed by HDAC1 and HDAC2.
The large CHD3 and CHD4 subunits have ATPase
activity, which is used to move the position of nucleo-
somes on DNA. In addition to these enzymatic activi-
ties, the NuRD complex contains a number of other
core subunits: MTA1–MTA3, GATAD2A, GA-
TAD2B, MBD2, MBD3, RBBP4, RBBP7, and
CDK2AP1 [4,5]. Some of these proteins, such as
MBD2 and MBD3, are mutually exclusive within the
NuRD complex. For GATAD2A, GATAD2B,
RBBP4, RBBP7, and MTA1–MTA3, it is currently
not known whether these proteins exclusively form
heterodimers/trimers or form mutually exclusive
homodimers/trimers. In addition to the well-described
core subunits, a large number of proteins have been
reported to interact with the NuRD complex. Exam-
ples include SALL4, FOG1, and ZMYND8 [6–8].These proteins may recruit the NuRD complex to its
target sites in the genome.
Owing to the presence of HDACs, which are gener-
ally associated with gene silencing, the NuRD complex
has long been thought of as a transcriptional corepres-
sor complex. Indeed, in luciferase assays, NuRD
subunits repress the transcription of reporter constructs
[4]. Furthermore, the methyl-CpG-binding protein
MBD2 links MBD2-containing NuRD to methylated
CpG islands, and induces gene silencing [9]. However,
recent genome-wide profiling studies have revealed that
NuRD complexes, such as MBD3–NuRD, are also
found at active enhancers and promoters, indicating
that NuRD-mediated regulation of transcription is
more diverse than previously thought [10,11]. Also,
functional experiments have revealed an important role
for the NuRD complex in regulating cell fate decisions
[12,13]. These different biological functions of the
NuRD complex may be driven by subtle changes in
subunit composition.
Recently, a number of MS-based methods were
developed that can be used not only to confidently
identify protein–protein interactions, but also to obtain
information about the stoichiometry, dynamics and
the interaction surfaces between subunits [14–16].Here, we applied these different methods to gain fur-
ther insights into the structure and dynamics of the
MBD3–NuRD complex in HeLa cells. We applied
label-free quantitative MS to investigate the stoichiom-
etry of the MBD3–NuRD complex, using different
buffer stringencies during affinity purification (AP). A
stable isotope labeling by amino acids in cell culture
(SILAC)-based subunit exchange assay was used to
show that RBBP4 and RBBP7 are dynamic core su-
bunits within the NuRD complex. Finally, cross-link-
ing MS was used to identify interprotein contacts
within the MBD3–NuRD complex. Altogether, these
experiments increase our understanding of the stabil-
ity, dynamics and architecture of the NuRD complex.
Results
RBBP4 and RBBP7 interactions with the NuRD
complex are salt-stable but detergent-sensitive
To facilitate purification of the MBD3–NuRD com-
plex, we created a stable cell line with doxycycline-
inducible green fluorescent protein (GFP)-tagged
MBD3. Cells were incubated for 16 h in the presence
or absence of doxycycline. Expression of GFP–MBD3
occurred only after addition of doxycycline to the
medium (Fig. 1A). Nuclear extracts from this cell line
were used for GFP AP combined with label-free LC-
MS/MS analysis. Briefly, triplicate pulldowns were
performed with GFP-Trap_A beads and GFP–MBD3-
containing nuclear extracts (+Dox). Simultaneously,
triplicate pulldowns were performed with GFP beads
on control nuclear extracts (�Dox). Proteins that bind
specifically to GFP–MBD3 will be enriched in the
GFP pulldowns and appear on the right side of the
Fig. 1. Label-free proteomics reveal that RBBP4 and RBBP7 interactions with NuRD are NP-40-sensitive. (A) An anti-MBD3 western blot of
GFP–MBD3 HeLa cells after 16 h of incubation with doxycycline (+Dox) or without doxycycline (�Dox). Nonspecific bands are marked with
an asterisk. (B–D) Volcano plots from label-free GFP pulldowns of GFP–MBD3 HeLa cell nuclear extracts with varying salt and NP-40
concentrations. Statistically enriched proteins in the GFP–MBD3 pulldown are identified by a permutation-based FDR-corrected t-test. The
label-free quantification (LFQ) intensity of the GFP pulldown relative to the control [fold change (FC), x-axis] is plotted against the – log10-
transformed P-value of the t-test (y-axis). The proteins in the upper right corner represent the bait and its interactors. (E) Stoichiometry of
NuRD core subunits. The iBAQ value of each protein group is divided by the iBAQ value of the GFP–MBD3 bait, which was set to 2. (F)
Stoichiometry of NuRD complex interactors relative to the GFP–MBD3 bait determined with the same data as in (E), but with a differently
scaled axis.
2 FEBS Journal (2014) ª 2014 FEBS
Dynamics and architecture of the NuRD complex S. L. Kloet et al.
–15 –10 –5 0 5 10 15
01
23
45
67
Log2 (GFP/Control)
– Lo
g [F
DR
(t-te
st)]
GATAD2ACHD3
CDK2AP1
MTA2
MBD3
KPNA2
RBBP4
MTA1
HDAC1
CHD4
ZNF687
CHD5
GATAD2B
ZNF592
HDAC2
ZNF512BMTA3SALL4
FC > 3.96 FDR > 1.301
300 mM NaCl 0.1% NP-40
RBBP7
ZMYND8
–15 –10 –5 0 5 10 15
02
46
Log2 (GFP/Control)
– Lo
g [F
DR
(t-te
st)]
GATAD2A
CHD3
CDK2AP1 MTA2MBD3
KPNA2
RBBP4
MTA1
HDAC1
CHD4
ZNF687
CHD5
GATAD2B
ZNF592 HDAC2
ZNF512B
MTA3
SALL4
FC > 4.65 FDR > 1.301
300 mM NaCl0.5% NP-40
RBBP7
ZMYND8
–15 –10 –5 0 5 10 15
01
23
45
67
Log2 (GFP/Control)
– Lo
g [F
DR (t
-test
)]
GATAD2A
CHD3
CDK2AP1
MTA2
MBD3KPNA2
RBBP4
MTA1
HDAC1
CHD4
ZNF687
CHD5 GATAD2B
ZNF592HDAC2
ZNF512B
MTA3
SALL4
FC > 4.04 FDR > 1.301
1 M NaCl0.5% NP-40
ZMYND8
RBBP7
13
57
– Dox + DoxGFP-MBD3*
*
*MBD3
A
B
C
D
E
α-MBD3
F
Stoichiometry
Stoichiometry
3FEBS Journal (2014) ª 2014 FEBS
S. L. Kloet et al. Dynamics and architecture of the NuRD complex
plot. Proteins that bind nonspecifically to the GFP
beads will appear in the background cloud.
To determine the stability of the NuRD complex,
several NP-40 and NaCl concentrations were used dur-
ing the APs (Fig. 1B–D). In each of these pulldowns,
known NuRD core subunits were identified. In addi-
tion, several previously described NuRD interactors
were detected, including ZMYND8, ZNF592, and
SALL4 [8,14,17]. Interestingly, we did not pull down
LSD1 in any of our GFP–MBD3 purifications. LSD1
has previously been reported as a putative subunit of
the NuRD complex [18], but our data on the MBD3–NuRD complex do not agree with this observation.
Next, the intensity-based absolute quantification (iBAQ)
values were compared between all NuRD subunits and
interactors. The iBAQ algorithm normalizes the total
MS intensity for each protein according to the number
of theoretically observable peptides [19]. This allows
estimation of the relative abundance of large and small
proteins detected in APs. As the NuRD complex con-
tains many paralogs that overlap at the peptide level,
iBAQ values were summed for all paralogs (Table S1).
Comparison of the iBAQ values relative to the GFP–MBD3 bait revealed that most core subunits of
NuRD remained tightly bound to each other and to
MBD3 despite the presence of high salt (1 M NaCl)
and NP-40 (0.5%) concentrations in the wash steps
(Fig. 1E). Surprisingly, the core RBBP4 and RBBP7
subunits were very sensitive to NP-40. Increasing the
NP-40 concentration from 0.15% to 0.5% led to
decreases in the amounts of RBBP4 and RBBP7 asso-
ciated with the complex (stoichiometry of 5.5 reduced
to 4).
In addition, the association of several zinc finger
domain-containing proteins with NuRD was very sen-
sitive to high salt concentrations. The association of
ZNF512B decreased nearly eight-fold when the salt
concentration in the wash was increased from 300 mM
to 1 M NaCl (Fig. 1F). A reduction in SALL4 binding
was also observed with increased salt concentration. In
contrast, other substoichiometric interactors, such as
ZMYND8 and KPNA2, remained tightly associated
with the core complex under increased salt and NP-40
concentrations. Together, these results suggest that the
core NuRD complex is very stable when challenged
with high salt concentrations, indicating that interac-
tions within the NuRD complex are at least partially
driven by hydrophobic interactions. The partial disso-
ciation of RBBP4 and RBBP7 observed at high NP-40
concentrations may suggest that these proteins are less
tightly associated with the rest of the core complex.
Finally, although other detected interactors had a low
iBAQ value relative to the core complex (1–2% rela-
tive to the core subunits), some of these interactions
were very stable at high salt and NP-40 concentra-
tions, suggesting a functional role for these proteins in
relation to the NuRD complex.
RBBP4 and RBBP7 partially dissociate from the
NuRD complex in a subunit exchange assay
To further study the potential dynamics of NuRD sub-
unit interactions, a SILAC-based subunit exchange
assay was used (Fig. 2A) [15]. HeLa cells expressing
GFP–CDK2AP1 were labeled in culture with heavy
amino acids (forward) or light amino acids (reverse).
Similarly, wild-type HeLa cells were labeled with light
or heavy amino acids. GFP pulldowns were performed
immediately after mixing of the nuclear extracts (T0)
or after overnight incubation (TON). Proteins that are
more dynamically associated with NuRD will dissoci-
ate from the complex during the overnight incubation
step and may be replaced by proteins from the other,
differentially labeled extract. This eventually results in
a decrease in detected SILAC ratios, whereby dynamic
core subunits and/or interactors will move towards the
background cloud in the scatter plot.
At T0, all NuRD core subunits were significantly
enriched according to boxplot statistics (Fig. 2B).
However, after overnight incubation, RBBP4 and
RBBP7 clearly separated from the other NuRD subun-
its, and migrated towards the background cloud
(Fig. 2C). To more directly compare the two scatter
plots, the difference in forward and reverse ratios
between the plots was visualized in a graph (Fig. 2D).
A protein with no change in ratios between experi-
ments would have a value of 0. This graph clearly
shows that RBBP4 and RBBP7 are the most dynamic
NuRD core subunits. These observations are in agree-
ment with recent structural studies suggesting that
MTA and histone H4 compete for RBBP binding [20].
Furthermore, RBBP4 and RBBP7 are present in many
protein complexes other than the NuRD complex
(Sin3 complex, PRC2), which also could explain their
observed dynamic behavior [21,22].
Cross-linking MS reveals novel architectural
features of the human NuRD complex
To further characterize the structural interactions
between NuRD complex core subunits, we conducted
cross-linking MS to identify interprotein interaction
surfaces (Fig. 3A). The NuRD complex was isolated
from HeLa nuclear extracts by the use of tandem AP
with a GFP–His-MBD3 bait, and subsequently cross-
linked with BS3 for LC-MS/MS analysis. Cross-linking
4 FEBS Journal (2014) ª 2014 FEBS
Dynamics and architecture of the NuRD complex S. L. Kloet et al.
was monitored by silver staining to ensure complete
cross-linking of all NuRD subunits (Fig. 3B). High-
confidence cross-linked peptides [false discovery rate
(FDR) of < 0.05] were then identified with PLINK by
using default settings and searching against a database
of NuRD core components (Fig. 3C,D) [23]. In total,
336 spectra matching with high confidence to 16 non-
ambiguous interprotein cross-links, 87 nonambiguous
0 2 4 6
0–2
–4–6
–8–1
0
Forward H/L (log2)
Rev
erse
H/L
(log
2)
HDAC1
MBD2
HDAC2
MTA1
CDK2AP1
MTA3
MTA2
CHD3
CHD4
GATAD2A
MBD3
GATAD2B
RBBP4
RBBP7
T0
core
transient
core
transient
Forward
Reverse
Light WT
Heavy GFPMix
Incubation for 0 h (T0) or
Overnight (TON)
10-min GFP pulldown
Tryptic digest,LC-MS/MS
Heavy WT
Light GFPMix
T0
TON
ReverseForwardA
B
D
1 3 5 0 1 2 3 4 5 6
0–2
–4–6
–8
Forward H/L (log2)
Rev
erse
H/L
(log
2)
HDAC2HDAC1
MTA1
MTA3
MTA2
MBD3
CHD3
CHD4
MBD2GATAD2A
GATAD2B
RBBP4RBBP7
TON
CDK2AP1
C
Fig. 2. RBBP4 and RBBP7 are the most dynamic NuRD core subunits. (A) Schematic representation of the SILAC subunit exchange assay.
(B, C) Scatter plots of GFP–CDK2AP1 forward and reverse pulldowns immediately after extract mixing [(B), T0] and after overnight
incubation [(C), TON]. The SILAC ratio for each protein in the forward pulldown is plotted against the ratio in the reverse pulldown. Boxplot
statistics were used to determine statistically significant GFP–CDK2AP1 interactors. The arrow and circle in (C) illustrate the movement of
RBBP4 and RBBP7 towards the background cloud, indicating that these proteins exchange between the heavy (H) and light (L) extracts. (D)
Bar graph showing the difference in forward and reverse SILAC ratios between T0 and TON. WT, wild type.
5FEBS Journal (2014) ª 2014 FEBS
S. L. Kloet et al. Dynamics and architecture of the NuRD complex
MTA2
MBD3
RBBP4
CHD3 HDAC1
CHD4
RBBP7GATAD2A
GATAD2B
HDAC2
MTA1 MTA3
F
A
Hela cells
GFP-His MBD3
Collect cells
Extract nuclear proteins
GFPGFP
beadsMBD3
On bead cross-linking
MBD3
Enzymatic digestion
Cross-linksLoop-links
Mono-links
LC-MS/MS analysis
b3
b4
b5
b6
y2y3
y4
y5
validation of cross-linked peptides
P HVK WT R
Q VFKL Kb3 b5b4
y2
y3 y4 y5
b6
b7
b7y1
y6
y6
y1
B
TEV protease cleavage
MBD3
His
HisNi2+–NTA
beads
Ni2+–NTAbeads
C
200
150
100
50
0151050 2520 30
FDR (%)
Num
ber o
f spe
ctra
Estimated FDR curve
Charge 3+Charge 4+
His
58
46
80
175
RBBP4/7
CHD3/4
D
E
180°
y1+
y2+
y3+
y4+
y2+
y3+
y4+
y5+
y5+
y6+
y8+
y7+
+2b
+4b
+3b
00
60
50
40
30
20
10
100
90
80
70Re
lativ
e in
tens
ity (%
)
200 300 400 500 600 700 800 900
m/z1000
N SPM TK E
L VDAK Fy5
y8 y7 y6
b2 b3
y4
y5
L VI Ry4 y3 y2 y1
y3 y2
b4
K
HDAC1/2
MBD3
MTA1/2/3
TEVelute
Ni2+ - NTA
purificationsupernatant
Cross-linkingFraction
GFP bead purification
and Ni2+–nitrilotriacetic acidpurification
6 FEBS Journal (2014) ª 2014 FEBS
Dynamics and architecture of the NuRD complex S. L. Kloet et al.
intraprotein cross-links and 46 ambiguous cross-links
encompassing a variety of homomers or heteromers or
intraprotein cross-links were identified (Table S2).
Structural validation was confounded by a lack of
overlap between the few existing NuRD crystal struc-
tures and our identified cross-links. For example,
although five HDAC1 and 11 HDAC2 intraprotein
cross-links and four ambiguous HDAC1/HDAC2
cross-links were identified, all of them were located in
the undetermined region of the known HDAC1 crystal
structure [Protein Data Bank (PDB) 4BKX]. However,
seven cross-links mapping to the CHD4 chromodo-
main-ATPase region (residues 500–1298), which has a
yeast CHD1 ortholog with known structure (PDB
3MWY), were identified. To our knowledge, this
represents the highest number of mappable cross-links
from our dataset to any single structure. Structural
validation of our CHD4 chromodomain-ATPase cross-
links was performed with a homology modeling
approach. The human CHD4 sequence was aligned to
the yeast CHD1 template with HHPRED, and homology
models were constructed with MODELLER (Human
CHD4 Uniprot: Q14839) [24–27]. In 10 overlapping
homology models, well-ordered regions from the crys-
tal structure yielded consistently low divergence mod-
els, whereas short alignment gaps or structurally
undetermined loops were clearly disordered (Fig. 3E).
All homology models were highly similar, except for
very small, flexible regions, so one model was chosen
as representative, and cross-links were validated by
calculating the solvent-accessible surface (SAS) dis-
tance with XWALK [28]. All seven cross-links showed an
SAS distance of < 30 �A, as expected from the maxi-
mum length of the BS3 cross-linker. Three cross-links
mapped to a well-ordered region, and four mapped to
a disordered region that probably possesses a highly
flexible structural conformation. Therefore, all seven
CHD4 chromodomain-ATPase cross-links seem to be
structurally plausible, and we conclude that our cross-
linking data are of good quality.
Few NuRD subcomplexes have resolved structures,
most notably the MBD2–GATAD2A coiled-coil sub-
complex, the RBBP4–MTA1 subcomplex, and the
HDAC1–MTA1 subcomplex [20,29,30]. None of these
studies described comprehensive protein structures for
both binding partners, so the complete interaction
interfaces remain unknown. Therefore, our interpro-
tein cross-linking data reveal novel features of the
NuRD complex architecture and extend our under-
standing of NuRD structure–function relationships
(Fig. 3F). For example, robust contacts were observed
between MTA1–MTA3 and RBBP4 and RBBP7
subunits. Our data do not show whether these RBBP4
and RBBP7 cross-link sites indicate binding of a single
MTA paralog or a multimer, nor do they enable us to
distinguish between potentially dynamic or paralog-
specific interactions at each site. However, mapping
cross-linked residues onto the known RBBP4 structure
shows that Lys25 and Lys307 fall on either side of the
known MTA1 interaction interface (PDB 4PC0 [20])
(Fig. 4A). As RBBP4 Lys25 maps relatively close to
the known MTA1-binding pocket, whereas Lys307 sits
across a large, adjacent hydrophobic region, these
cross-links could outline the opposite ends of the
MTA1–MTA3 binding platform (Fig. 4B). Intrigu-
ingly, this would explain our observation that MTA1–MTA3 binding to RBBP4 and RBBP7 is stabilized by
hydrophobic interactions under high-salt conditions.
Similarly, although the MBD3 structure is not known,
we identified a short MBD3 region (residues 92–124)that contained five cross-links with GATAD2A/GA-
TAD2B, two of which represented unique cross-links
for GATAD2A, one that was unique for GATAD2B,
and two that were ambiguous for the GATAD2A/GA-
Fig. 3. Cross-linking MS offers insight into the NuRD molecular architecture. (A) MBD3–NuRD was obtained by tandem AP (GFP affinity
enrichment, TEV cleavage, and Ni2+–nitrilotriacetic acid (NTA) enrichment) and cross-linked on-bead with BS3. Proteolytic digestion and
analysis by AP-MS/MS was followed by identification and validation of high-confidence cross-linked peptides (FDR < 0.05) with PLINK. (B)
NuRD complex purification and cross-linking was monitored by silver staining to ensure complete cross-linking of all complex components.
Note that, after the cross-linking reaction, NuRD subunits migrate at an apparent higher molecular mass in the SDS/PAGE gel. (C) High-
confidence spectra were matched to putative cross-links by use of a target-decoy approach in PLINK. Representative FDR curves are shown
for cross-links of charge states +3 and +4. (D) An annotated high-scoring spectrum from a CHD4 intraprotein cross-link, Lys959–Lys969. (E)
Structural validation was performed with a human CHD4 chromodomain-ATPase homology model aligned to a yeast CHD1 ortholog
template structure. Yeast CHD1 is colored red, with 10 overlapping CHD4 homology models in blue. Validated cross-links are colored
purple. Below the homology models are close-up views of seven CHD4 cross-links in the chromodomain-ATPase region obtained with a
representative CHD4 homology model; the lower left image shows Lys959–Lys969, Lys959–Lys1189 and Lys969–Lys1189 cross-links in a
well-ordered region, and the lower right image shows Lys690–Lys693 and Lys693–Lys696 cross-links in a gapped region, and Lys833–
Lys838 and Lys833–Lys839 cross-links in a structurally undetermined region. (F) Cross-link map for human NuRD subunits. Subunits
connected by at least one unambiguous cross-link are indicated by solid lines, and subunits connected only by ambiguous cross-links are
indicated by dashed lines. In our data, all ambiguous cross-links are ambiguous for peptides from a paralogous subunit. The relative
numbers of interprotein and intraprotein cross-links are designated by the area of gray shading surrounding that link.
7FEBS Journal (2014) ª 2014 FEBS
S. L. Kloet et al. Dynamics and architecture of the NuRD complex
TAD2B peptide (Fig. 4C). The MBD2 and MBD3
sequences were aligned in EBI’s CLUSTAL OMEGA in
order to map any MBD3–GATAD2A cross-links with
the solved MBD2–GATAD2A coiled-coil structure
(PDB 2L2L [30,31]). Only two cross-linked lysines
could be mapped to a representative conformer GA-
TAD2A structure (Lys163 and Lys178), and no cross-
links could be mapped to the MBD2 structure
(Fig. 4D). Furthermore, one GATAD2A cross-link
site, Lys178, was located at the tail end of the con-
former, which showed a highly flexible conformation
and thus offers little structural information. It is possi-
ble that we observed low cross-link coverage within
the MBD2–GATAD2A coiled-coil domain because, as
the authors of that study noted, this interaction hides
at least 1337 �A2 of the solvent-accessible area, poten-
tially hindering BS3 accessibility. Thus, our cross-links
may outline an interaction interface rather than reveal
a binding footprint. However, in agreement with previ-
ous work, our data show a single binding interface
between MBD3 and GATAD2A/GATAD2B paralogs.
Overall, these cross-linking MS data greatly improve
MBD3BD331 200 291
GATAD2A
1 200 400 633
GATAD2B
1 200 400 593
Coiled-coildomain
Coiled-coildomain
MTA2A21 200 400 600 668
MTA3A31 200 400 594
MTA1A11 200 400 600 715
RBBP4P41 200 425
RBBP7P71 200 425
RBBP4 interaction domain
RBBP4 crystalstructure
90°
GATAD2A-K163
RBBP4-K307
RBBP4-K25
GATAD2A-K178
MTA1 Fragment/H4 Binding pocket (MTA1 Fragment Removed)
Uncharacterized RBBP4 hydrophobic pocket
C D
A
B
Fig. 4. Structural analysis of cross-linked residues reveals novel features of NuRD subcomplex interactions. (A) Twelve identified cross-links
between RBBP4 and RBBP7 and MTA1–MTA3 map to two residues on RBBP4, i.e. K25 and K307 (K24 and K306 on RBBP7). (B) Residues
from (A) are mapped onto the known crystal structure for RBBP4. These residues surround the binding site of the MTA1 peptide. RBBP4-
K25 sits near the MTA1-binding site, whereas RBBP4-K307 lies across a large hydrophobic region representing a potential MTA1-binding
platform. Hydrophobic regions are colored red, and hydrophilic regions are colored blue. (C) Five cross-links identified between MBD3 and
GATAD2A/GATAD2B map to MBD3 residues K92, K109, and K124. (D) Coiled-coil conformer model of GATAD2A and MBD2. The
GATAD2A coil is colored light red, and the MBD2 coil is colored blue. All lysines are colored dark red, and the cross-linked residues are
colored yellow.
8 FEBS Journal (2014) ª 2014 FEBS
Dynamics and architecture of the NuRD complex S. L. Kloet et al.
our understanding of the NuRD complex subunit
architecture, and will help to direct future structural
studies.
Discussion
Here, we have used a variety of MS-based methods to
increase our understanding of the dynamics, stability
and architecture of the MBD3–NuRD complex. GFP-
based APs combined with a label-free method to esti-
mate protein complex stoichiometry revealed that the
RBBP4 and RBBP7 core subunits of the NuRD com-
plex are sensitive to high detergent concentrations.
Increasing the NP-40 concentration from 0.1% to 0.5%
in the wash buffer resulted in a reduced amount of
RBBP4 and RBBP7 copurified with MBD3. This find-
ing illustrates an important limitation of AP-MS experi-
ments, namely that some of the interactions that occur
in vivo may be lost during AP. Conversely, interactions
can be formed in the extract that may not occur in vivo.
To overcome these limitations, cross-linking prior to
cell lysis can be used as an alternative approach. How-
ever, this approach is technically very challenging,
owing to experimental and computational issues.
Except for MBD3 and HDAC1/HDAC2, the stoichi-
ometry values that we obtained in our GFP–MBD3
purifications are generally in good agreement with our
previously published NuRD stoichiometry results
[14,32]. Previously, we made use of a BAC GFP–MBD3 HeLa cell line that expresses MBD3 with a
GFP tag at near-endogenous levels. In the current
study, we used a doxycycline-inducible GFP–MBD3
cell line. This cell line may express MBD3 at slightly
higher levels, which may result in different MBD3 and
HDAC1/HDAC2 stoichiometry values. It is important
to note that the final stoichiometry values that we
obtained represent an average value based on a large
number of potentially heterogeneous complexes. On the
basis of our data, we cannot be sure whether paralo-
gous proteins such as MTA1–MTA3 or GATAD2A/
GATAD2B are present within the same complex, or
whether these proteins are, in fact, mutually exclusive,
as we have previously shown for MBD2 and MBD3
[33]. It is highly likely that a large number of distinct
NuRD complexes exist. However, our cross-linking
data cannot distinguish unambiguously between homo-
meric and heteromeric interactions for HDAC1/
HDAC2, MTA1–MTA3 and GATAD2A/GATAD2B.
Interestingly, we were able to detect unambiguous inter-
protein cross-links for CHD3 and CHD4, indicating
that these proteins may form a heterodimer. Neverthe-
less, NuRD complex heterogeneity most likely creates a
significant challenge for high-resolution determination
of the NuRD molecular architecture.
In addition to the core NuRD subunits, a number
of substoichiometric interactors were identified in the
GFP–MBD3 purifications. Interestingly, these interac-
tors showed differential sensitivity to salt and deter-
gent. ZMYND8 and ZNF532/592/687 copurified with
GFP–MBD3 even when challenged with 1 M NaCl
and 0.5% NP-40. The stability of these interactions
indicates that they may be functionally relevant. Inter-
estingly, these proteins have previously been reported
to interact with each other as a central hub in a large
transcriptional network [34]. Given the presence of a
large number of putative DNA-binding zinc fingers in
the ZMYND8/ZNF module, we hypothesize that this
module may recruit the NuRD complex to a subset of
its target genes. The same holds true for other substoi-
chiometric interactors. Further work is needed to
determine the functional significance of the ZMYND8/
ZNF–NuRD interaction.
Experimental procedures
Cloning
A tandem AP (TAP) tag was cloned into pcDNA5 FRT/
TO. This tag consists of enhanced GFP followed by two
tobacco etch virus (TEV) protease cleavage sites and a His6tag. The TEV sites and His tag were added to enhanced
GFP via PCR. MBD3 was amplified with BamHI and
XhoI, and cloned into the multiple cloning site of
pcDNA5 FRT/TO.
Cell culture and SILAC
HeLa FRT cells were transfected with pcDNA5 FRT/TO
TAP–MBD3 by the use of Lipofectamine LTX Plus (Invi-
trogen, Glasgow, Scotland). Cells underwent selection for
10 days with hygromycin, and single colonies were picked,
expanded, and screened for expression of TAP–MBD3.
TAP–MBD3 expression was induced by treatment with
1 lg�mL�1 doxycycline for 16 h. SILAC treatment of HeLa
cells was performed as described previously [35].
Nuclear extracts
Nuclear extracts were prepared essentially according to Dig-
nam et al. [36]. Cells were harvested with trypsin, washed
twice with PBS, and centrifuged at 400 g for 5 min at 4 °C.Cells were allowed to swell for 10 min at 4 °C in five volumes
of Buffer A (10 mM Hepes/KOH, pH 7.9, 1.5 mM MgCl2,
10 mM KCl), and then pelleted at 400 g for 5 min at 4 °C.Cells were resuspended in two volumes of Buffer A plus pro-
9FEBS Journal (2014) ª 2014 FEBS
S. L. Kloet et al. Dynamics and architecture of the NuRD complex
tease inhibitors and 0.15% NP-40, and transferred to a Do-
unce homogenizer. After 30–40 strokes with a Type B pestle,
the lysates were spun at 3200 g for 15 min at 4 °C. The
nuclear pellet was washed once with PBS, and spun at 3200 g
for 5 min at 4 °C. The nuclear pellet was resuspended in
Buffer C (420 mM NaCl, 20 mM Hepes/KOH, pH 7.9,
20% v/v glycerol, 2 mM MgCl2, 0.2 mM EDTA) with 0.1%
NP-40, protease inhibitors, and 0.5 mM dithiothreitol. The
suspension was incubated with rotation for 1 h at 4 °C, andthen spun at 18 000 g for 15 min at 4 °C. The supernatant
was saved, aliquoted, and stored at �80 °C until further use.
Label-free pulldowns and cross-linking MS
Label-free GFP pulldowns, LC-MS/MS and data analysis
were performed as described previously [14]. For cross-link-
ing MS, 8 mL of HeLa nuclear extract (5 mg�mL�1 plus
50 lm�mL�1 ethidium bromide) containing doxycycline-
induced TAP–MBD3 was incubated with 400 lL of GFP-
trap beads (Chromotek) for 2 h at 4 °C in a rotation
wheel. Beads were then washed three times with 10 mL of
wash buffer (300 mM NaCl, 0.5% NP-40, 20 mM Hepes/
KOH, pH 7.9, 0.5 mM dithiothreitol, 2 mM MgCl2, 0.2 mM
EDTA and complete protease inhibitors) and twice with
5 mL of TEV cleavage buffer (150 mM NaCl, 10 mM Tris/
HCl, pH 8.0, 0.1% NP-40, and 1 mM dithiothreitol). Beads
were then resuspended in 0.5 mL of TEV cleavage buffer
containing 100 units of GST–TEV protease (US Biological)
and incubated overnight at 4 °C in a rotation wheel. The
supernatant was collected, and GST–TEV protease was
removed from the eluate by adding 10 lL of GST beads
and incubating for 15 min at 4 °C in a rotation wheel. The
eluate was then incubated with 20 lL of Ni2+–nitrilotriace-
tic acid beads (Qiagen) for 45 min at 4 °C in a rotation
wheel. Beads were washed once with 1 mL of TEV cleav-
age buffer and three times with 1 mL of cross-linking buf-
fer (25 mM Hepes/KOH, pH 7.9, 300 mM KCl, 5%
glycerol, 0.05% Tween-20). Beads were then resuspended in
50 lL of cross-linking buffer containing 250 lM BS3 cross-
linker (Sigma), and incubated for 30 min at 37 °C in a
thermoshaker (1100 r.p.m.). The cross-linking reaction was
quenched by adding 7 lL of 0.5 M ammonium bicarbonate
and incubating for 5 min at room temperature. Finally,
beads were washed twice with 0.5 mL of PBS, after which
bound cross-linked proteins were digested overnight with
on-bead LysC/trypsin digestion. Tryptic peptides were de-
salted and purified with stagetips prior to LC-MS/MS
analysis.
Analysis of cross-linking MS data
Thermo RAW MS files were converted to mgf format with
the MSCONVERT tool from PROTEOWIZARD, and analyzed with
PLINK, with default settings for HCD with BS3 as a cross-
linking agent [23,37]. Cross-linked peptides matched to
spectra with an FDR of < 0.05 were considered to be high
confidence, and were used for further analysis. Spectra were
visually analyzed and annotated with PLABEL [38]. Template
structures for homology modeling were searched with
HHPRED, and homology models were built with the MODEL-
LER interface in UCSF CHIMERA [24,26,39]. The yeast CHD1
chromodomain and ATPase residues 38–800 (PDB 3MWY)
were used as a template for residues 500–1298 from human
CHD4 (Uniprot Q14839) for homology modeling [27].
Calculations of rmsd values were performed in UCSF CHI-
MERA. SAS distances were calculated for a representative
CHD4 model with XWALK. XINET was used to visualize
high-confidence intraprotein and interprotein cross-links in
network form (Rappsilber Laboratory, University of Edin-
burgh, Scotland; crosslinkviewer.org). All protein structure
visualizations were produced with UCSF CHIMERA.
Acknowledgements
This work was supported by the EU FP7 framework
program 4DCellFate. We thank I. Poser and A.
Hyman for providing the BAC GFP–CDK2AP1 cell
line. We thank N. Vermeulen-Hubner and H. Stunnen-
berg for providing access to the Q Exactive mass
spectrometer.
Author contributions
SLK, HIB, VG, PWTCJ, MB, HN, and MV per-
formed experiments. SLK, HIB, MM, and VG ana-
lyzed data. GJK contributed reagents. SLK, MM and
MV wrote the paper with input from all authors.
References
1 Zhang Y, LeRoy G, Seelig HP, Lane WS & Reinberg
D (1998) The dermatomyositis-specific autoantigen Mi2
is a component of a complex containing histone
deacetylase and nucleosome remodeling activities. Cell
95, 279–289.
2 Wade PA, Jones PL, Vermaak D & Wolffe AP (1998)
A multiple subunit Mi-2 histone deacetylase from
Xenopus laevis cofractionates with an associated Snf2
superfamily ATPase. Curr Biol 8, 843–846.
3 Tong JK, Hassig CA, Schnitzler GR, Kingston RE &
Schreiber SL (1998) Chromatin deacetylation by an
ATP-dependent nucleosome remodelling complex.
Nature 395, 917–921.
4 Spruijt CG, Bartels SJ, Brinkman AB, Tjeertes JV,
Poser I, Stunnenberg HG & Vermeulen M (2010)
CDK2AP1/DOC-1 is a bona fide subunit of the Mi-2/
NuRD complex. Mol BioSyst 6, 1700–1706.
5 Allen HF, Wade PA & Kutateladze TG (2013) The
NuRD architecture. Cell Mol Life Sci 70, 3513–3524.
10 FEBS Journal (2014) ª 2014 FEBS
Dynamics and architecture of the NuRD complex S. L. Kloet et al.
6 Miccio A, Wang Y, Hong W, Gregory GD, Wang H,
Yu X, Choi JK, Shelat S, Tong W, Poncz M, et al.
(2010) NuRD mediates activating and repressive
functions of GATA-1 and FOG-1 during blood
development. EMBO J 29, 442–456.
7 Lauberth SM & Rauchman M (2006) A conserved 12-
amino acid motif in Sall1 recruits the nucleosome
remodeling and deacetylase corepressor complex. J Biol
Chem 281, 23922–23931.
8 Eberl HC, Spruijt CG, Kelstrup CD, Vermeulen M &
Mann M (2013) A map of general and specialized
chromatin readers in mouse tissues generated by label-
free interaction proteomics. Mol Cell 49, 368–378.
9 Baubec T, Ivanek R, Lienert F & Schubeler D (2013)
Methylation-dependent and -independent genomic
targeting principles of the MBD protein family. Cell
153, 480–492.
10 Whyte WA, Bilodeau S, Orlando DA, Hoke HA,
Frampton GM, Foster CT, Cowley SM & Young RA
(2012) Enhancer decommissioning by LSD1 during
embryonic stem cell differentiation. Nature 482, 221–
225.
11 Reynolds N, Latos P, Hynes-Allen A, Loos R, Leaford
D, O’Shaughnessy A, Mosaku O, Signolet J, Brennecke
P, Kalkan T, et al. (2012) NuRD suppresses
pluripotency gene expression to promote transcriptional
heterogeneity and lineage commitment. Cell Stem Cell
10, 583–594.
12 Rais Y, Zviran A, Geula S, Gafni O, Chomsky E,
Viukov S, Mansour AA, Caspi I, Krupalnik V, Zerbib
M, et al. (2013) Deterministic direct reprogramming of
somatic cells to pluripotency. Nature 502, 65–70.
13 Dos Santos RL, Tosti L, Radzisheuskaya A, Caballero
IM, Kaji K, Hendrich B & Silva JC (2014) MBD3/
NuRD facilitates induction of pluripotency in a
context-dependent manner. Cell Stem Cell 15, 102–
110.
14 Smits AH, Jansen PW, Poser I, Hyman AA &
Vermeulen M (2013) Stoichiometry of chromatin-
associated protein complexes revealed by label-free
quantitative mass spectrometry-based proteomics.
Nucleic Acids Res 41, e28.
15 Mousson F, Kolkman A, Pijnappel WW, Timmers HT
& Heck AJ (2008) Quantitative proteomics reveals
regulation of dynamic components within TATA-
binding protein (TBP) transcription complexes. Mol
Cell Proteomics 7, 845–852.
16 Walzthoeni T, Leitner A, Stengel F & Aebersold R
(2013) Mass spectrometry supported determination of
protein complex structure. Curr Opin Struct Biol 23,
252–260.
17 van den Berg DL, Snoek T, Mullin NP, Yates A,
Bezstarosti K, Demmers J, Chambers I & Poot RA
(2010) An Oct4-centered protein interaction network in
embryonic stem cells. Cell Stem Cell 6, 369–381.
18 Wang Y, Zhang H, Chen Y, Sun Y, Yang F, Yu W,
Liang J, Sun L, Yang X, Shi L, et al. (2009) LSD1 is a
subunit of the NuRD complex and targets the
metastasis programs in breast cancer. Cell 138, 660–
672.
19 Schwanhausser B, Busse D, Li N, Dittmar G,
Schuchhardt J, Wolf J, Chen W & Selbach M (2011)
Global quantification of mammalian gene expression
control. Nature 473, 337–342.
20 Alqarni SS, Murthy A, Zhang W, Przewloka MR, Silva
AP, Watson AA, Lejon S, Pei XY, Smits AH, Kloet
SL, et al. (2014) Insight into the architecture of the
NuRD complex: structure of the RbAp48–MTA1 sub-
complex. J Biol Chem 289, 21844–21855.
21 Kuzmichev A, Nishioka K, Erdjument-Bromage H,
Tempst P & Reinberg D (2002) Histone
methyltransferase activity associated with a human
multiprotein complex containing the Enhancer of Zeste
protein. Genes Dev 16, 2893–2905.
22 Zhang Y, Iratni R, Erdjument-Bromage H, Tempst P &
Reinberg D (1997) Histone deacetylases and SAP18, a
novel polypeptide, are components of a human Sin3
complex. Cell 89, 357–364.
23 Yang B, Wu YJ, Zhu M, Fan SB, Lin J, Zhang K, Li
S, Chi H, Li YX, Chen HF, et al. (2012) Identification
of cross-linked peptides from complex samples. Nat
Methods 9, 904–906.
24 Soding J, Biegert A & Lupas AN (2005) The HHpred
interactive server for protein homology detection and
structure prediction. Nucleic Acids Res 33, W244–W248.
25 Sali A & Blundell TL (1993) Comparative protein
modelling by satisfaction of spatial restraints. J Mol
Biol 234, 779–815.
26 Eswar N, Webb B, Marti-Renom MA, Madhusudhan
MS, Eramian D, Shen MY, Pieper U & Sali A (2007)
Comparative protein structure modeling using
MODELLER. Curr Protoc Protein Sci, Chapter 2,
Unit 2.9.
27 Hauk G, McKnight JN, Nodelman IM & Bowman GD
(2010) The chromodomains of the Chd1 chromatin
remodeler regulate DNA access to the ATPase motor.
Mol Cell 39, 711–723.
28 Kahraman A, Malmstrom L & Aebersold R (2011)
Xwalk: computing and visualizing distances in
cross-linking experiments. Bioinformatics 27,
2163–2164.
29 Millard CJ, Watson PJ, Celardo I, Gordiyenko Y,
Cowley SM, Robinson CV, Fairall L & Schwabe JW
(2013) Class I HDACs share a common mechanism
of regulation by inositol phosphates. Mol Cell 51,
57–67.
30 Gnanapragasam MN, Scarsdale JN, Amaya ML, Webb
HD, Desai MA, Walavalkar NM, Wang SZ, Zu Zhu S,
Ginder GD & Williams DC Jr (2011) p66Alpha-MBD2
coiled-coil interaction and recruitment of Mi-2 are
11FEBS Journal (2014) ª 2014 FEBS
S. L. Kloet et al. Dynamics and architecture of the NuRD complex
critical for globin gene silencing by the MBD2–NuRD
complex. Proc Natl Acad Sci USA 108, 7487–7492.
31 Goujon M, McWilliam H, Li W, Valentin F, Squizzato
S, Paern J & Lopez R (2010) A new bioinformatics
analysis tools framework at EMBL-EBI. Nucleic Acids
Res 38, W695–W699.
32 Smits AH & Vermeulen M (2014) Exploring chromatin
readers using high-accuracy quantitative mass
spectrometry-based proteomics. In Systems Analysis of
Chromatin-related Protein Complexes in Cancer (Emili
A, Greenblatt J & Wodak S, eds), pp. 133–148.
Springer, New York.
33 Le Guezennec X, Vermeulen M, Brinkman AB,
Hoeijmakers WA, Cohen A, Lasonder E &
Stunnenberg HG (2006) MBD2/NuRD and MBD3/
NuRD, two distinct complexes with different
biochemical and functional properties. Mol Cell Biol 26,
843–851.
34 Malovannaya A, Lanz RB, Jung SY, Bulynko Y,
Le NT, Chan DW, Ding C, Shi Y, Yucer N,
Krenciute G, et al. (2011) Analysis of the human
endogenous coregulator complexome. Cell 145, 787–799.
35 Baymaz HI, Fournier A, Laget S, Ji Z, Jansen PW,
Smits AH, Ferry L, Mensinga A, Poser I, Sharrocks A,
et al. (2014) MBD5 and MBD6 interact with the
human PR–DUB complex through their methyl-CpG-
binding domain. Proteomics doi: 10.1002/
pmic.201400013. [Advance online publication].
36 Dignam JD, Lebovitz RM & Roeder RG (1983)
Accurate transcription initiation by RNA polymerase II
in a soluble extract from isolated mammalian nuclei.
Nucleic Acids Res 11, 1475–1489.
37 Kessner D, Chambers M, Burke R, Agus D & Mallick
P (2008) ProteoWizard: open source software for rapid
proteomics tools development. Bioinformatics 24, 2534–
2536.
38 Wang LH, Li DQ, Fu Y, Wang HP, Zhang JF, Yuan
ZF, Sun RX, Zeng R, He SM & Gao W (2007)
pFind 2.0: a software package for peptide and protein
identification via tandem mass spectrometry. Rapid
Commun Mass Spectrom 21, 2985–2991.
39 Pettersen EF, Goddard TD, Huang CC, Couch GS,
Greenblatt DM, Meng EC & Ferrin TE (2004) UCSF
Chimera – a visualization system for exploratory
research and analysis. J Comput Chem 25, 1605–1612.
Supporting information
Additional supporting information may be found in
the online version of this article at the publisher’s web
site:Table S1. iBAQ values used to calculate NuRD sub-
unit stoichiometry.
Table S2. Overview of all assigned cross-links with
FDR < 0.05 as calculated in PLINK.
12 FEBS Journal (2014) ª 2014 FEBS
Dynamics and architecture of the NuRD complex S. L. Kloet et al.