BBA - Proteins and Proteomics 1865 (2017) 1613–1622

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BBA - Proteins and Proteomics

journal homepage: www.elsevier.com/locate/bbapap

Characterizing the molecular architectures of chromatin-modifying MARK

complexes☆
Dheva T. Setiaputra, Calvin K. Yip⁎
Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

A R T I C L E I N F O A B S T R A C T

Keywords: Eukaryotic cells package their genome in the form of a DNA-protein complex known as chromatin. This orga-
Chromatin-modifying complex nization not only condenses the genome to fit within the confines of the nucleus, but also provides a platform for
Nucleosome a cell to regulate accessibility to different gene sequences. The basic packaging element of chromatin is the
Post-translational modifications nucleosome, which consists of 146 base pairs of DNA wrapped around histone proteins. One major means that a
Electron microscopy
cell regulates chromatin structure is by depositing post-translational modifications on nucleosomal histone
X-ray crystallography
Nuclear magnetic resonance spectroscopy
proteins, and thereby altering internucleosomal interactions and/or binding to different chromatin associated
factors. These chromatin modifications are often catalyzed by multi-subunit enzyme complexes, whose large
size, sophisticated composition, and inherent conformational flexibility pose significant technical challenges to
their biochemical and structural characterization. Multiple structural approaches including nuclear magnetic
resonance spectroscopy, X-ray crystallography, single-particle electron microscopy, and crosslinking coupled to
mass spectrometry are often used synergistically to probe the overall architecture, subunit organization, and
catalytic mechanisms of these macromolecular assemblies. In this review, we highlight several recent chromatin-
modifying complexes studies that embodies this multipronged structural approach, and explore common themes
amongst them. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John
Baenziger, Albert Berghuis and Peter Tieleman.

1. Nucleosomes and chromatin [8,94].

In every cell of the human body, over one metre of genomic DNA
needs to be compacted into the nucleus, an organelle of approximately
six microns in diameter [125]. This remarkable degree of compaction,
which is observed in all eukaryotic cells, is mediated by a cornucopia of
proteins associated with the genome, forming a nucleoprotein structure
known as chromatin. The basic repeating unit of chromatin is the nu-
cleosome, which consists of approximately 146 base pairs of DNA
wrapped around an octameric disk-shaped protein complex made up of
four core histones: H2A, H2B, H3, and H4 [65]. Histones are highly
basic proteins, with their positively charged residues interacting with
the negatively charged DNA phosphate backbone [65]. In addition to
functioning as a major packaging element, the nucleosome serves as a
major regulatory platform for cellular processes that requires access to
genomic DNA. For example, post translational modifications at the level
of the nucleosome allow a cell to dynamically alter its chromatin
structure and gene expression pattern in response to changing en-
vironmental conditions such as genotoxic insults or nutrient scarcity
The crystal structure of the nucleosome core particle provided key
insights into its regulatory mechanisms. First, the N-terminal tails of
histones are highly unstructured, with the H3 and H4 tails projecting
towards the DNA entrance and exit sites, while the H2A and H2B tails
extending to the opposite side (Fig. 1B). These histone tails have been
shown to undergo different types of post-translational modifications,
ranging from small chemical groups such as methyl and acetyl groups,
all the way to a full protein such as ubiquitin (reviewed in [6]). These
modifications, together with the solvent accessible “faces” of the nu-
cleosome core particle, serve as platforms for recruiting different
chromatin-binding proteins. Another distinct feature of the nucleosome
core particle is an acidic patch formed by the H2A/H2B dimer [65].
Interaction between this acidic patch and a corresponding basic patch
found on the histone H4 tail has been shown to contribute to inter-
nucleosomal packing [90]. Subsequent finding that neutralizing the
positive charge of the H4 basic patch by acetylation of histone H4 lysine
16 (H4K16), a post-translational modification associated with tran-
scription activation, abrogates this compaction [34,90] suggested that

☆
This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman.
⁎
Corresponding author.
E-mail address: calvin.yip@ubc.ca (C.K. Yip).

http://dx.doi.org/10.1016/j.bbapap.2017.06.018
Received 22 March 2017; Received in revised form 9 June 2017; Accepted 21 June 2017
Available online 24 June 2017
1570-9639/ © 2017 Elsevier B.V. All rights reserved.
D.T. Setiaputra, C.K. Yip
histone post translational modifications could regulate transcription
activation by altering the high-order structure of chromatin. Interest-
ingly, a series of recent high-resolution structural studies of different
nucleosome-protein complexes found that this acidic patch also med-
iates electrostatic interaction between the nucleosome and its cognate
binding partner [3,7,66,69,70,111]. In this review, we will use selected
chromatin-modifying proteins and complexes to illustrate how recent
structural based studies advance our understanding on the properties
and mechanisms of action of these highly sophisticated and versatile
molecular machineries.
2. Chromatin-modifying complexes
The relatively large overall size of the nucleosome necessitates nu-
cleosome-binding proteins to make multiple contact points to bind ef-
fectively and with a high degree of specificity. Consistent with this
view, many enzymes that mediate post translational modifications of
chromatin were found to function in the context of large multisubunit
complexes. These complexes are often modular, with groups of subunits
forming subassemblies that confer a particular activity or biochemical
function. The catalytic functions of chromatin-modifying complexes can
be highly versatile, with their chromatin-modifying activities yielding
different outcomes depending on the context of their sites of action. For
example, the mammalian polycomb-repressive complex 2 (PRC2)-cat-
alyzed H3K27 trimethylation defines regions of highly compact, tran-
scriptionally silent heterochromatin in most cell types (reviewed in
[91]). However, this silencing modification has been found alongside
the activating H3K4 trimethylation in active embryonic stem cell pro-
moters to fine tune expression of the associated genes [11]. Another
intriguing example can be found in the yeast nucleosomal acetyl-
transferase of histone H4 (NuA4) complex, which is most commonly
associated with activated transcription of housekeeping genes through
promoter histone H4 acetylation [81]. However, NuA4 also responds to
DNA damage, where it acetylates nucleosomes flanking the site of da-
mage. Acetylation of nucleosomal histones by NuA4 decondenses the
surrounding chromatin structure, increasing accessibility to DNA repair
machinery [13]. Finally, virtually all chromatin-modifying complexes
catalyze post translational modification of non-histone targets (PRC2
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BBA - Proteins and Proteomics 1865 (2017) 1613–1622
Fig. 1. Structure of the nucleosome core particle. High
resolution crystal structure of Xenopus laevis nucleo-
some (PDB ID: 1AOI). A. Basis of histone H3/H4 tet-
ramer and H2A/H2B dimer assembly to form the his-
tone octamer. B. The histone octamer associates with
146 bp of DNA to form the intact nucleosome core
particle. The N-terminal tails of the histones are
shown.
and NuA4 examples: [64,86]), further expanding their roles into other
nuclear and non-nuclear processes.
The recruitment of chromatin-modifying complexes plays a critical
role in regulating their activities. Many of these complexes are recruited
to target sites within the genome by DNA-binding proteins that re-
cognize specific DNA sequences through specialized domains such as
the zinc finger, leucine zipper, and helix-turn-helix motifs [96]. Ad-
ditionally, chromatin-modifying complexes often contain “chromatin
reader domains”, which recognize specific types of histone modification
and allow their cognate complex to form a positive feedback loop.
Many chromatin-modifying complexes contain multiple subunits with
chromatin reader domains as well as DNA-binding subunits. Although
this property allows chromatin-modifying complexes to potentially
engage in a multitude of interactions with the nucleosome, it remains
poorly understood how these complexes coordinate its multiple re-
cruitment modules to exert their diverse functions. A comprehensive
understanding of the molecular structures of chromatin-modifying
complexes in their apo and nucleosome-bound states could yield an
answer to this fundamental question.
3. Structural studies of chromatin-modifying complexes
The sheer size and sophisticated composition of chromatin-mod-
ifying complexes pose formidable challenges to their structural char-
acterization. The biggest impediment is arguably the isolation of suf-
ficiently large quantities (micrograms to milligram level) of pure and
homogeneous samples. The large number of subunits, some of them
large proteins, often preclude the reconstitution of these complexes in
vitro using isolated recombinant components or in vivo by co-over-
expression in the same host. Purifying the intact native complex from
endogenous sources is an alternative strategy. However, sufficiently
large yields of homogeneous and intact complexes are often difficult to
achieve. Finally, most chromatin-modifying complexes are inherently
dynamic and conformationally flexible, a property that potentially fa-
cilitates multivalent interactions with the nucleosome. This property
often prevents these complexes from forming stable lattice interactions
required for crystallization. To overcome these challenges, a common
strategy is to apply a “divide-and-conquer” approach that combines the
Fig. 2. Techniques for structural characterization
of chromatin-modifying complexes. Approximate
molecular weight ranges for nuclear magnetic
resonance (NMR), X-ray crystallography, and
single-particle electron microscopy (EM) struc-
tural analyses. Example structures obtained using
these techniques are depicted (PDB ID: 1KDX,
5I9E, 1AOI, 5CH1, 4R8P, 5J9Q, 2RNZ, 3M99;
EMDB ID: 9536, 6300).
D.T. Setiaputra, C.K. Yip
strengths of different structural biology techniques to devise a unified
model (Fig. 2).
For conventional structural approaches, nuclear magnetic resonance
(NMR) spectroscopy has been extensively used to explore the high-re-
solution structures and dynamics of domains and motifs of chromatin
readers and regulatory protein binding sites of subunits of chromatin-
binding complexes. On the other hand, X-ray crystallography is used to
delineate the structures of subunits and multi-protein subassemblies of
chromatin-modifying complexes at atomic resolution, allowing us to
gain extensive insights into the catalytic mechanisms, subunit organi-
zation, and binding interfaces of different chromatin-modifying com-
plexes. In recent years, single-particle electron microscopy (EM) has
allowed the visualization of chromatin-modifying complexes in their
fully assembled states. This emerging molecular imaging technology
involves using tens to hundreds of thousands of individual images of the
complex acquired by a transmission electron microscope and re-
constructing the three-dimensional overall shape of the complex of
interest. In addition to its lower sample demand, single-particle EM can
also capture different conformational states of a protein complex.
Single-particle EM, until recently, was limited to intermediate resolu-
tions that nevertheless provided highly useful architectural informa-
tion. Cross-linking coupled to mass spectrometry (CXMS) is a structural
proteomics approach that elucidates the intersubunit connectivity
within chromatin-modifying complexes. It has recently gained popu-
larity by providing distance restraints for mixed molecular models
generated by in silico approaches [110].
In the following sections, we highlight several examples of chro-
matin-modifying complexes to illustrate this multipronged approach to
structural analysis and comment on how recent technological advances
will affect future structural studies of these sophisticated cellular ma-
chineries.
4. PRC1 complex
Polycomb group (PcG) proteins are key regulators of gene silencing
in higher eukaryotes, facilitating the formation of highly compacted
and silenced heterochromatin (reviewed in [91]). These proteins play
crucial roles during embryonic development, and their misregulation
has been associated with different types of human cancer (reviewed in
[9]). PcG proteins form several chromatin-modifying complexes with
distinct activities. One of these complexes is the Polycomb repressive
complex 1 (PRC1) that catalyzes the ubiquitylation of histone H2AK119
[20]. PRC1 can also compact chromatin independently of its catalytic
activity [40]. The catalytic PRC1 core is a heterodimer of two RING-
domain proteins: one of either RING1A or RING1B ubiquitin E3 ligase,
and the PCGF-family protein BMI1 [20,73,109]. This catalytic hetero-
dimer associates with several other subunits important for its recruit-
ment and regulation (Fig. 3A). Intriguingly, there is a plethora of PRC1
variants with different subunit compositions, complicating its char-
acterization. However, several recent structural studies have shed light
on aspects of PRC1's mechanism of action.
A series of high-resolution crystal structures of the RING1B-BMI1
core have generated insights into the catalytic activity of PRC1
[10,17,60,100]. These structures revealed that the C-terminus of BMI1
interacts with the N-terminus of RING1B to form a short four-helix
bundle [17,60]. BMI1 stimulates RING1B catalytic activity [17,60], and
mutations within their interface has been shown to abrogate PRC1
activity in vivo and in vitro [17,42,60,109]. The RING1B-BMI1 hetero-
dimer requires the association of a ubiquitin E2 transferase for its
ubiquitylation activity [17]. Bentley and colleagues crystallized the
trimeric complex of RING1B-BMI1 bound to the E2 conjugating enzyme
UbcH5c, revealing that the E2 enzyme contacts only RING1B [10].
Intriguingly, this study also showed that RING1B-BMI1 and UbcH5c
complex is active only on nucleosomal substrates but not individual
histones or the octameric histone complex [10]. This observation hints
at a possible role for DNA binding as part of PRC1's substrate
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BBA - Proteins and Proteomics 1865 (2017) 1613–1622
recognition motif. More recently, the groundbreaking crystal structure
of RING1B-BMI1 and UbcH5c in complex with the nucleosome core
particle revealed how PRC1 engages its substrate (Fig. 3B) [69]. More
specifically, the trimeric PRC1 module was found to bind the nucleo-
some in a rigid manner without inducing large structural changes.
RING1B-BMI1 make extensive contacts with the face of the nucleosome
and interact with all four core histones. Interestingly, the RING1B-BMI1
heterodimer was observed to use an ‘arginine anchor’ to interact with
the H2A/H2B acidic patch [69], a common feature captured by several
published crystal structures of nucleosome-protein complexes
[3,7,66,69,70,111]. Consistent with the previously described nucleo-
somal specificity of PRC1, this structure further revealed that UbcH5c
makes several interactions with nucleosomal DNA.
In addition to the catalytic RING1B-BMI1 core, PRC1 complexes
contain several other subunits. Many variants of the PRC1 complex
contain one copy of the chromodomain-containing chromobox (Cbx)
protein (Cbx2, -4, -6, -7, -8) [102]. Chromodomains specifically bind
methylated histones, and the PRC1 Cbx subunits in Drosophila recruit
the complex to trimethylated H3K27 through their chromodomains
[39]. However, mammalian Cbx homologues do not require their
chromodomains for chromatin association [104]. NMR and crystal-
lographic analysis of human Cbx2, -6, -7, and -8 by Kaustov and col-
leagues revealed that these chromodomains have reduced specificity
and affinity for H3K27me3 due to increased hydrophobicity in the
binding site (Fig. 3C) [53]. This study also found that PRC1-associated
Cbx chromodomains are able to bind non-histone methyllysines [53].
Another NMR study by Yap and colleagues found that Cbx7 binds both
RNA and H3K27me3, with both interactions being important for si-
lencing of the INK4b-ARF-INK4a tumor suppressor locus [120]. These
findings suggest that the chromodomains of PRC1-associated Cbx ex-
panded their specificity and facilitates recruitment of the complex
through the canonical methyllysine binding as well as through other
interactions.
The structural studies highlighted above, as well as other bio-
chemical analysis provide deeper insights into the molecular function of
the PRC1 complex. The catalytic module structures revealed how the
enzymatic subunit recognizes its nucleosomal substrate, while the Cbx
studies hinted at a possible adaptation of chromodomains towards
binding non-histone substrates. However, the lack of structural in-
formation on the full PRC1 complex has prevented further under-
standing of how the different functionalities of this complex coordinate
and cooperate with one another and how PRC1 can compact chromatin
via nonenzymatic mechanisms. The major challenge in delineating the
structure of intact PRC1 is the presence of multiple variants. Even
“canonical” PRC1 carry different versions of every subunit, including its
catalytic core: RING1A can substitute RING1B, and BMI1 can be re-
placed by a number of PCGF-family proteins [43]. Furthermore, non-
canonical PRC1 complexes also exist, such as the PRC1.1 complex that
lacks a Cbx subunit and binds specific DNA sequences directly [114].
The presence of many PRC1 variants may hint at a compositionally
dynamic complex, a feature that can play an important role in its cel-
lular function.
5. PRC2 complex
Polycomb repressive complex 2 (PRC2) is another PcG complex that
participates in transcription silencing. It catalyzes the trimethylation of
H3K27 through its SET domain-containing catalytic subunit EZH2. In
addition to EZH2, the PRC2 core complex consists of EED, SUZ12, and
RbAp46/48 (Fig. 3D) [19,57]. Recent structural studies of the PRC2
core complex as well as its binding partners have generated critical
insights into its function.
The histone methyltransferase activity of PRC2 requires the asso-
ciation of EZH2 with EED and SUZ12 [18]. Crystal structures of the
WD40 domain-containing EED provided the first glimpses into PRC2
structure [46,67,116]. These structures determined the region of the
D.T. Setiaputra, C.K. Yip
EED WD40 domain that binds EZH2 and trimethylated lysines
[46,67,116]. Upon EED binding to H3K27me3, PRC2's own catalytic
product, the catalytic activity of EZH2 is allosterically stimulated [67].
Residues of EED trimethyllysine binding identified from these studies
are indispensable for PRC2 activity in vivo [67]. A subsequent crystal
structure of the EZH2 catalytic SET domain revealed a puzzling con-
formation of the active site: the lack of a binding site for the constitutive
SET domain cofactor S-adenosylmethionine (SAM) [2]. Furthermore,
the C-terminus of the SET domain seems to be occluding the active site,
possibly acting as an autoinhibitory module [2]. Soon after, Jiao and
Liu determined the crystal structures of the PRC2 catalytic module
consisting of EED, EZH2, and SUZ12 from the thermophilic fungus C.
thermophilum in complex with a stimulating H3K27me3 peptide bound
to EED and an H3K27M mutant peptide bound to its active site (Fig. 3E)
[51]. These two striking structures revealed a large conformational
change of EZH2, with substrate binding inducing rotation of the puta-
tive autoinhibitory domain away from the active site and completing
the SAM-binding site [51]. Additionally, trimethylated histone binding
to EED stabilizes the structure of EZH2's stimulation-response motif
(SRM), which makes multiple contacts with the enzyme active site,
likely serving to activate its catalytic activity [51]. A more recent
crystal structure of the PRC2 trimer showed that Jarid2, another PRC2
target that also activates the enzyme allosterically, also acts through the
SRM of EZH2 [52]. These studies on the PRC2 core elucidated many
subtle features of how the catalytic site coordinates its substrate, but the
structure of the full PRC2 complex remains out of reach of X-ray crys-
tallography.
In order to analyze the architecture of the intact PRC2 complex,
Ciferri and colleagues turned to single-particle EM [23]. Initially they
expressed four PRC2 components, EZH2-EED-SUZ12-RbAp48, using the
Sf9 insect cell expression system. Initial negative stain EM based as-
sessment, however, revealed that this tetrameric assembly is con-
formationally heterogeneous and highly flexible. Intriguingly, the ad-
dition of AEBP2, another PRC2-associated protein that stimulates its
catalytic activity, was found to greatly stabilize the complex and reduce
the conformational heterogeneity. Ciferri and colleagues subsequently
determined a 21 Ångstrom 3D reconstruction of this reconstituted
pentameric complex. This structure revealed a four-lobed organization
consistent with what has been observed from crystallographic analysis
of the catalytic trimer [51]. These researchers also complemented the
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BBA - Proteins and Proteomics 1865 (2017) 1613–1622
Fig. 3. Structural investigation of polycomb re-
pressive complexes (PRC) 1 and 2. A. Schematic
of the subunit composition of canonical PRC1.
PRC1 consists of a catalytic RING-family subunit,
and accessory PCGF-, HPH-, and CBX-family
subunits. B. Crystal structure of RING1B and
BMI1 with the E2 ubiquitin conjugase UbcH5c
bound to the nucleosome core particle (PDB ID:
4R8P). C. NMR structures of different CBX-family
subunits of PRC1 bound to methylated lysine
peptides (PDB ID: 2L1B, 3H91, 3I90, 3I91). D.
Schematic of the canonical PRC2 subunit com-
position. EZH2 is the catalytic methyltransferase
subunit of PRC2. E. Crystal structure of the PRC2
catalytic core consisting of EZH2, SUZ12, and
EED (PDB ID: 5CH1). F. Molecular architecture of
intact PRC2 generated using a single-particle
electron microscopy map and X-ray crystal-
lography structures modeled in (EMDB ID: 2236;
PDB: 5CH1, 2YB8).
limited resolution of their EM studies by performing crosslinking cou-
pled to mass spectrometry (CXMS) experiments, which utilizes a bi-
functional amine crosslinker of a specific length to determine the dis-
tances between lysine pairs. The amalgamation of high resolution
crystal structures, low resolution EM reconstruction, and CXMS dis-
tance restraints enabled the generation of a PRC2 pseudoatomic model
(Fig. 3F). Using this structure, the authors predicted many architectural
features that were later confirmed through the crystal structure of the
PRC2 trimer [51]. Interestingly, PRC2 has increased activity on dinu-
cleosomal templates [68]. Ciferri and colleagues used the positions of
EED and EZH2 in the PRC2 model to propose that EED binds the sti-
mulatory H3K27me3 of a nucleosome adjacent to the substrate nu-
cleosome. Due to extensive studies of both isolated portions of PRC2 as
well as the intact complex through a variety of techniques, many as-
pects of the molecular function of PRC2 have now been elucidated.
Future high-resolution studies of the full PRC2 complex is poised to
further advance our understanding of its structural dynamics and reg-
ulatory mechanisms.
6. CBP/p300
CBP and p300 are two highly similar chromatin-modifying proteins
that stimulate transcription of target genes through several mechan-
isms, most notably their catalytic histone acetyltransferase activities
(reviewed in [108]). Histone acetyltransferases transfers an acetyl
moiety from acetyl-CoA to the unstructured histone tails, and this his-
tone modification correlates with an open chromatin configuration and
increased transcription (reviewed in [95]). CBP/p300 are transcrip-
tional coactivators, a class of proteins and protein complexes that ac-
tivates the transcription of target genes after recruitment by DNA-
binding transcription factors [5,74]. CBP/p300 mutations are asso-
ciated with Rubinstein-Taybi syndrome [76], while chromosomal
translocations centred about the CBP/p300 gene has been observed in
acute myeloid leukemia [14,50,93]. At a molecular level, CBP/p300 is a
~300 kDa protein consisting of its catalytic histone acetyltransferase
domain and four transactivation domains (TADs) that interact with
DNA sequence specific transcription factors: a cysteine-histidine-rich
region 1 (CH1) containing the transcriptional adaptor zinc domain 1
(TAZ1) [25], a kinase-inducible domain interacting (KIX) domain [79],
a CH3 region containing another TAZ domain (TAZ2) and a ZZ-type
D.T. Setiaputra, C.K. Yip
zinc finger domain [27], and finally a nuclear receptor co-activator
binding (NCBD) domain [54]. Although the binding of an individual
TAD is relatively weak, multivalent interactions involving multiple
TADs may give rise to binding cooperativity. CBP/p300 also possesses
two chromatin reader domains: the methylated histone-binding plant
homeodomain (PHD) within CH2 and an acetylated histone-binding
bromodomain (Fig. 4) [30,71]. Although CBP/p300 is not a protein
complex, it is a massive protein containing many functional domains
that interact with a multitude of binding partners. The arrangement of
these functional domains and the sheer size of the protein mean that
structural determination of CBP/p300 utilizes many of the approaches
used for analyzing multi-protein complexes.
The TADs of CBP/p300 are peculiar in that they are able to bind a
large variety of transcription factors, including oncogenic factors such
as c-Myc as well as tumor suppressing factors such as p53 [4,61,103].
The malleability of these domains brings to light to power of NMR in
elucidating their structures. The TAZ domains consist of four amphi-
pathic helices with three zinc binding sites [25,27], and many solution
NMR structures of these domains bound to transcription factors have
been determined [25,28,38,41,72,112]. These studies determined that
the two CBP/p300 TAZ domains are structured in the apo state, while
their ligands are often unstructured in solution. Though TAZ1 and TAZ2
bind using similar surfaces, their specificity is mediated in large part by
the different orientations of their fourth alpha helices and surface
charges [112]. The crystal structure of a single TAZ2 domain simulta-
neously binding three MEF2-DNA complexes on three different surfaces
further illustrate the versatility of this transactivation domain [47].
Meanwhile, the other zinc-containing motif of CBP/p300, the ZZ-type
zinc finger, has been shown to bind a unique region of the small ubi-
quitin-like modifier (SUMO protein) distinct from the canonical SUMO
interacting motif target [33].
Similar to the TAZ domains, the KIX transactivation domain of CBP/
p300 was characterized through NMR [16,29,58,79,107,124]. The KIX
domain consists of a three-helix bundle and two additional 310 helices,
and is also a versatile mediator of protein-protein interactions [79]. The
kinase inducible domain (KID) of CREB becomes structured upon
binding to the CBP KIX domain, and this interaction is sensitive to the
phosphorylation state of KID [79]. This same binding interface is used
by the transcription factor c-Myb, while an alternate face binds MLL,
another transcription factor [29]. Strikingly, the NMR structure of the
ternary KIX-MLL-c-Myb complex reveals a cooperativity where MLL
stabilizes and extends a KIX alpha helix, which in turn forms additional
interactions with c-Myb [29]. The KIX domain also binds two p53 ac-
tivation domains upon the two faces of KIX to cooperatively strengthen
their interaction [58].
In contrast to the TAZ and KIX domains, the NCBD domain of CBP/
p300 is largely unstructured in the apo state that is stabilized upon
transcription factor binding [54]. NMR and X-ray crystallography stu-
dies of the NCBD domain found that it adopts different structures de-
pending on its binding partners [31,54,78], adding conformational se-
lection to the already extensive malleability of the CBP/p300 TADs.
Although NMR studies have been invaluable in characterizing the
CBP/p300 TADs, the breakthrough in structural understanding of the
CBP/p300 catalytic core came from X-ray crystallography. Crystal
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BBA - Proteins and Proteomics 1865 (2017) 1613–1622
Fig. 4. Structural determination of the CBP/p300
functional domains. The individual domains of
CBP/p300 and their corresponding re-
presentative structures are shown (PDB ID: 2KA4,
1KDX, 4BHW, 1TOT, 2KA6, 2KKJ).
structures detailing the CBP/p300 HAT and bromodomain were pre-
viously determined independently [63,71], but the striking structure of
the p300 region encompassing its bromodomain, CH2, and HAT do-
mains determined by the Panne group elucidated the interplay between
these domains (Fig. 4) [30]. This structure revealed a previously uni-
dentified RING domain interrupting the PHD domain within CH2. RING
domains often act as ubiquitin ligase E3 enzymes [32], though its
geometry within the p300 catalytic core renders this unlikely. Instead,
this RING domain is tightly associated with the HAT domain, and
mutations of this interface observed in B-cell lymphomas and Rubin-
stein-Taybi syndrome markedly increase p300's HAT activity [30]. This
RING-HAT interaction suggests that the HAT activity of CBP/p300 is
also regulated in cis, though additional investigation of its functional
implications is necessary. The bromodomain-RING-PHD (BRP) module
can recognize acetylated nucleosomes and may also serve to recruit
p300 to modified nucleosomes [30,80]. Together, the catalytic core of
CBP/p300 itself is potentially subject to multiple levels of regulation
both in cis through the RING domain and in trans through interactions
with modified nucleosomes.
The structural investigations of CBP/p300 summarized above, as
well as many others, paint the picture of a versatile and highly regu-
lated histone acetyltransferase. The power of NMR techniques is high-
lighted in the analysis of the malleable structures of CBP/p300 TADs
bound to the activation domains of transcription factors, while X-ray
crystallography provides high resolution information of the archi-
tecture of the 4-domain catalytic core. The logical next step is a more
holistic analysis of CBP/p300 structure, elucidating the structure of the
intact protein and how it interacts with chromatin. However, large
regions of CBP/p300 connecting its functional domains are predicted to
be disordered [108], presenting a formidable challenge for structure
determination. Regardless, a supercomplex consisting of CBP/p300
bound to transcription factors and a nucleosome array will provide an
unprecedented understanding of the enzyme in its cellular context.
7. SAGA complex
The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is a histone
acetyltransferase complex from budding yeast Saccharomyces cerevisiae,
with several orthologous complexes present in higher eukaryotes [95].
SAGA is a multifunctional coactivator complex, activating transcription
of stress-response genes through histone acetylation, histone deubi-
quitination, and TATA-binding protein (TBP) recruitment (reviewed in
[56]). With 19 unique subunits and a total mass of approximately
2 MDa, SAGA is the largest HAT complex in yeast. Its subunits are or-
ganized into four distinct modules: the HAT module containing the
Gcn5 acetyltransferase subunit, the DUB module containing the Ubp8
deubiquitinase subunit, the TAF module containing subunits shared
with transcription factor II D (TFIID), and the SPT module containing
subunits that associate with TBP and transcription factors that recruit
SAGA (Fig. 5A) [59]. The subunit composition of SAGA is highly con-
served in the mammalian STAGA complex. The PCAF and ATAC com-
plexes are paralogues of STAGA, and contain subsets of its subunits
[56]. Of particular note is the direct clinical relevance of human STA-
GA, with a polyglutamine expansion within its SCA7 DUB module
D.T. Setiaputra, C.K. Yip
subunit giving rise to the neurodegenerative disease spinocerebellar
ataxia type 7 [26]. The modularity and multifunctionality of SAGA
makes it particularly important to determine its architecture to un-
derstand how its different modules cooperate to exert their biological
activities.
Similar to the complexes discussed above, much of our structural
understanding of SAGA was obtained through X-ray crystallography
and NMR spectroscopy of its subunits or fragments these proteins
(Fig. 5B). The structure of the Gcn5 catalytic domain, the prototype of
the large Gcn5-related N-acetyltransferase (GNAT) family of acetyl-
transferase enzymes, was determined using both X-ray crystallography
and NMR methods [24,62,82,101]. These structures were pivotal in
determining the catalytic mechanism of GNAT acetyltransferases, and
revealed a CoA-binding motif that is highly conserved in other acetyl-
transferase families [118]. Upon binding to acetyl-CoA, the peptide-
binding groove of Gcn5 widens to accommodate the incoming sub-
strate. However, Gcn5 can only acetylate nucleosomes in the context of
the HAT module [44], and the structural basis of this catalytic activa-
tion remains unknown.
In contrast to the HAT module, SAGA's deubiquitylation module has
been extensively characterized. The catalytic subunit of this module,
Ubp8, specifically cleaves monoubiquitylated substrates including
ubiquitinated H2BK123 [48]. Similar to Gcn5, Ubp8 is inactive outside
the context of the four-subunit DUB module. Crystal structures of the
intact module consisting of Ubp8, Sgf73, Sus1, and Sgf11 were initially
reported by the Zheng and Wolberger groups [55,85]. These striking
structures revealed the highly intertwined nature of the DUB module,
which adopts a two-lobed structure centred about Ubp8. The complex is
held together by the N-terminal zinc finger domain of Ubp8 that as-
sociates with the other three subunits. Sgf73 bridges the N-terminal
Ubp8 zinc finger and its C-terminal catalytic domain, while Sgf11 forms
an elongated structure that spans both lobes of Ubp8 and interacts
extensively with its catalytic domain. Mutations in the interface be-
tween the Sgf11 zinc finger and the Ubp8 catalytic lobe disrupt Ubp8
catalytic activity, suggesting that this interaction stimulates Ubp8 ac-
tivity [55,85]. Recently, the Wolberger group determined the crystal
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BBA - Proteins and Proteomics 1865 (2017) 1613–1622
Fig. 5. Determining the structures of the SAGA
and NuA4 histone acetyltransferase complexes.
A. Subunit composition and modular organiza-
tion of the yeast SAGA complex. B. Structures of
individual SAGA subunit domains (PDB ID:
1QST, 3MP1, 1F68, 2CUJ). C. Crystal structure of
the SAGA deubiquitination module bound to the
nucleosome core particle (PDB ID: 4ZUX). D.
Molecular architecture of SAGA based on a
single-particle electron microscopy reconstruc-
tion (EMDB ID: 6300), crosslinking coupled to
mass spectrometry, and X-ray crystallography
(PDB ID: 3M99). E. Subunit composition and
modular organization of the yeast NuA4 histone
acetyltransferase complex. F. Pseudoatomic
model of the catalytic Piccolo module of NuA4
bound to a nucleosome core particle (EMDB ID:
9536; PDB: 5J9Q, 2RNZ). G. Structures of several
NuA4 chromatin reader domains (PDB ID: 5I9E,
3E9G, 3FK3, 2RNZ).
structure of the SAGA DUB module in complex with the nucleosome
core particle; a staggering structure encompassing almost 6000 residues
(Fig. 5C) [70]. The DUB module contacts the nucleosome exclusively
through histones H2A and H2B, with the conserved H2A acidic patch
being bound by the Sgf11 zinc finger through a series of arginine re-
sidues reminiscent of the arginine anchors observed in PRC1 and other
nucleosome-bound structures. An interesting observation made from
this structure is the relatively small interface between the nucleosome
and the DUB module [70], with histone H3, SAGA's main acetylation
target, possibly being accessible to the HAT module in the full complex
for simultaneous modification.
Many SAGA subunits within the SPT and HAT modules also contain
chromatin reader domains. The structures of several SAGA reader do-
mains have been determined either in isolation of in complex with its
binding target through X-ray crystallography or NMR spectroscopy
(Fig. 5B) [12,49,75,77,121]. Although these isolated structures are
critical to understanding the molecular basis of chromatin recognition,
they provide limited understanding of how the full complex engages its
substrates.
SAGA's subunit composition is highly conserved from yeast to hu-
mans. Its complexity, and by extension its architecture, must therefore
be an integral part of its cellular role. Several studies, including one by
our research group, have attempted to characterize the intact SAGA
complex using negative stain single-particle EM approaches
[36,88,113]. These efforts encountered several significant challenges.
First and foremost is the difficulty in obtaining sufficient quantity of
purified complexes. Recombinant overexpression is virtually impossible
due to the number of subunits and their sizes, with SAGA's largest
subunit Tra1 being over 400 kDa. These studies therefore used native
complexes purified endogenously from yeast. The purified sample,
however, is heterogeneous in composition and conformation. Since
excessive heterogeneity is not amenable for EM analysis, the Schultz
group and our group utilized limited crosslinking to stabilize the
complex [36,88]. Although this treatment limited the flexibility of the
complex, we observed three major conformations that SAGA adopts
[88]. Due to the challenges outlined above, however, these structures
D.T. Setiaputra, C.K. Yip
are limited to low resolution and do not provide information on the
architecture at the level of individual subunits. To address this, the
Hahn group and our group used crosslinking coupled to mass spectro-
metry (CXMS) to obtain information on the subunit proximity and the
spatial arrangement of the different subunits within the full complex
[45,88]. By combining our EM and CXMS data, we generated a model
for the subunit arrangement of the intact SAGA complex (Fig. 5D) [88].
SAGA consists of a highly interconnected structural core with periph-
erally associated catalytic modules. Based on our model, we found that
the HAT and DUB module arrangement points to the possibility of si-
multaneous engagement of nucleosomes. Furthermore, the chromatin
reader-containing subunits are arrayed along one flexible surface of
SAGA, hinting at a nucleosome-binding groove amenable to multivalent
interactions.
Relative to the number of SAGA subunits, there is a general lack of
available high resolution structural information. The HAT module has
eluded crystallography analysis, possibly due to the high degree of
flexibility that we observed [88]. Furthermore, the arrangement of
SAGA-transcription factor complex remains a mystery. The foremost
missing piece of the puzzle, however, is precisely how SAGA utilizes its
many chromatin readers to bind its nucleosomal substrates. How does
its chromatin-reading binding site accommodate non-histone substrates
of SAGA? High-resolution information of the full SAGA complex bound
to different substrates will be invaluable in elucidating how SAGA
utilizes its complex architecture to facilitate its catalytic activity.
8. NuA4 complex
The nucleosomal acetyltransferase of histone H4 (NuA4) is another
histone acetyltransferase complex found in S. cerevisiae. Its catalytic
subunit, Esa1, is the only essential HAT in yeast [92]. NuA4 stimulates
the transcription of numerous housekeeping genes, including those
encoding ribosomal subunits [81]. Similar to SAGA, NuA4 is a large
1 MDa complex consisting of 13 subunits that organize into four func-
tional modules: the Piccolo HAT module, the Eaf5/7/3 module, the
recruitment module, and the shared SWR1C module (Fig. 5E) (reviewed
in [35]). Piccolo is NuA4's catalytic module, Eaf5/7/3 associates with
the RNA polymerase II holoenzyme for cotranscriptional acetylation,
the recruitment module associates with transcription factors to target
the complex, while the shared SWR1C module consists of subunits
shared with the SWR1 complex that is responsible for incorporating the
histone variant H2A.Z in place of H2A within nucleosomes [35]. In-
triguingly, NuA4 and SWR1 seem to have fused in higher eukaryotes,
forming the TIP60 complex containing both the histone acetyl-
transferase activity of NuA4 and the remodeling activity of SWR1 [35].
How the different functional modules of NuA4 cooperate is an intri-
guing and unanswered question.
Similar to SAGA, fragments of NuA4 have been characterized to
high resolution through X-ray crystallography. The catalytic domain of
Esa1, which belongs to the MYST (MOZ-Ybf2/Sas3-Sas2-Tip60) family
of acetyltransferases, has been extensively characterized by the
Marmorstein group [118,119,122]. Despite low sequence similarity, the
CoA-binding and active site of the Esa1 catalytic domain is highly si-
milar to GNAT family HATs, diverging in the flanking regions that
determines substrate specificity [118]. Interestingly, Esa1 contains an
autoacetylated lysine essential for its activity [122]. Similar to Gcn5,
however, Esa1 requires integration into the Piccolo HAT module to
acetylate nucleosomes [15]. The mechanism behind this dependence
was elucidated through a recently reported crystal structure of the in-
tact four-subunit Piccolo module by Xu and colleagues (Fig. 5F) [117].
This remarkable crystal structure, together with a cryoEM reconstruc-
tion of Piccolo bound to a nucleosome, revealed a series of multivalent
interactions between the module and its substrate. Interestingly, the
catalytic site of Esa1 seems to be specific only to a limited number of
residues flanking its lysine substrate, and Piccolo's selectivity is medi-
ated instead through a series of interaction with the nucleosome face.
1619
BBA - Proteins and Proteomics 1865 (2017) 1613–1622
Although the crystallized complex lacks the region of Piccolo that in-
teracts with the acidic patch of histones H2A and H2B, these acidic
residues are important for NuA4 activity and likely form another im-
portant binding interface.
Several other NuA4 subunits and functional domains have been
characterized by X-ray crystallography (Fig. 5G). Two subunits within
the shared SWR1C module, Arp4 and Act1 (actin) have been crystal-
lized both individually and in complex [21,37]. The dimer between
Act1 and Arp4 are found in several other chromatin-associated com-
plexes including SWR1 and Ino80, where it is bound by an HSA domain
[99]. In contrast to cytoplasmic actin that polymerizes to form elements
of the cytoskeleton [1], nuclear actin such as the one found in NuA4 is
monomeric. The crystal structure revealed that Arp4 and the HSA do-
main, which is contributed by the subunit Eaf1 in NuA4, serve to cap
the region of Act1 required for polymerization [21]. However, the
functional role of actin in chromatin-associated complex remains un-
known. The structures of three NuA4 chromatin reader domains have
been solved either by X-ray crystallography or NMR spectroscopy: the
methyllysine-binding Tudor and chromodomains of Esa1 and Eaf3, and
the crotonylated-lysine-binding YEATS domain of Yaf9
[89,98,106,115,123]. As with SAGA, however, the arrangement of
these domains in the context of intact NuA4 is required to fully un-
derstand their contribution to chromatin association.
The structure of budding yeast NuA4 has been investigated through
electron microscopy by the Asturias group [22]. Their low resolution
negative stain EM structure shows NuA4 as a two-lobed complex, with
the nucleosome binding site in the junction of the two lobes [22].
However, the location of the other NuA4 modules was not determined.
One notable observation is the similarity between NuA4 and the ‘head’
region of SAGA, despite the two complexes only having one common
subunit, Tra1 [22,88]. The authors hypothesized that the Tra1 occupies
one of the two NuA4 lobes, but this is inconsistent with our observation
that the corresponding SAGA region is too small to accommodate the
400 kDa subunit [88]. A more detailed structural dissection of NuA4
will be vital for our understanding of its molecular architecture.
As with many of the complexes discussed above, structural data
shed key insights into the catalytic core of NuA4. The crystal structure
of the Piccolo module shows that NuA4 engages its substrates on the
disk face of the nucleosome, with its specificity largely mediated
through multivalent interactions. The vital next step builds upon the
catalytic module, solving the structures of the outlying modules and
observing how they engage with NuA4's substrates. Similar to SAGA,
structural information of the intact complex will elucidate how NuA4's
substrate binding site can accommodate non-histone substrates and
how its different modules cooperate to stimulate transcription.
9. Perspectives
In this review, we highlighted five chromatin-modifiers that are
uniquely challenging for structural studies. Usage of multiple structural
techniques such as X-ray crystallography, NMR spectrometry, and
single-particle electron microscopy has eked out vital knowledge of
how these complexes act within the cell. Time and again we see that
these chromatin-modifying complexes engage their nucleosome sub-
strate through multiple interactions, taking advantage of the same
acidic patch on the nucleosome surface [69,70,117]. This multivalent
mode of interaction provides a rationale for the complexity and size of
these complexes, with a large surface area required for multiple con-
tacts with the nucleosome. Chromatin-modifying complexes are also
generalists able to function in many chromatin sites. Much of their
specificity is determined by DNA-binding transcription factors and
other regulatory partners, also requiring a binding interface. The next
threshold to break through, however, is the high-resolution structure of
intact complexes to study the cooperativity within these multi-
functional complexes.
A few short years ago, solving the atomic structure of protein
D.T. Setiaputra, C.K. Yip
complexes hundreds to thousands of kiloDaltons in size was a virtual
impossibility. Recent technological advances, however, have thrust
structural biology into the spotlight. Foremost in this structural re-
volution is the advent of new detector technology in single-particle
cryoelectron microscopy, which enabled the determination of multiple
atomic and near-atomic resolution structures of giant protein com-
plexes at an unprecedented pace [105]. The relatively small amount of
purified protein required and the lack of an upper size limit makes
cryoEM an extremely powerful technique for studying chromatin-
modifying complexes. Single-particle EM, derided in its infancy as
‘blob-ology’ due to its limited resolution, has now taken the field by
storm. On the other hand, the development of X-ray free electron lasers
and serial femtosecond crystallography that utilize microscopic crystals
circumvents the primary bottleneck of X-ray crystallography: obtaining
large protein crystals [87]. This technique has been used to solve the
20-subunit photosystem II complex at room temperature [97]. Even
NMR spectroscopy is innovating past its size limitations, with methyl-
TROSY (transverse relaxation optimized spectroscopy) experiments
providing structural insights into the 1.1 MDa proteasome core particle
[83,84]. The development of these and other techniques will usher in a
golden age of structural understanding of chromatin-modifying com-
plexes and other challenging targets. Each of these powerful new
technologies come with their own sets of limitations. Just like the dif-
ferent modules of chromatin-modifying complexes, cooperativity be-
tween multiple next-generation structural approaches will be key for a
comprehensive understanding of these enigmatic molecular machines.
Transparency document
The http://dx.doi.org/10.1016/j.bbapap.2017.06.018 associated
with this article can be found, in online version.
Acknowledgments
This work was supported by a Natural Sciences and Engineering
Research Council of Canada (NSERC) Discovery Grant (418157-2012),
a Michael Smith Foundation for Health Research Career Investigator
Award, a Canadian Institutes of Health Research (CIHR) New
Investigator Award, and an infrastructure grant from the Canadian
Foundation for Innovation (CFI) to CY and a NSERC CGS fellowship to
DS.
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