The plasticity of WDR5 peptide-binding cleft enables
the binding of the SET1 family of histone
Pamela Zhang1, Hwabin Lee1, Joseph S. Brunzelle2and Jean-Francois Couture1,*
1Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of
Ottawa, Ottawa, ON, Canada K1H 8M5 and2Feinberg School of Medicine, Department of Molecular
Pharmacology and Biological Chemistry, Northwestern University, Chicago, IL 60611, USA
Received September 23, 2011; Revised November 10, 2011; Accepted November 28, 2011
In mammals, the SET1 family of lysine methyl-
SET1A and SET1B, catalyzes the methylation of
lysine-4 (Lys-4) on histone H3. Recent reports
have demonstrated that a three-subunit complex
stimulates the methyltransferase activity of MLL1.
On the basis of studies showing that this stimulation
is in part controlled by an interaction between WDR5
and a small region located in close proximity of the
MLL1 catalytic domain [referred to as the WDR5-
interacting motif (Win)], it has been suggested that
WDR5 might play an analogous role in scaffolding
the other SET1 complexes. We herein provide bio-
chemical and structural evidence showing that
WDR5 binds the Win motifs of MLL2-4, SET1A
and SET1B. Comparative analysis of WDR5-Win
complexes reveals that binding of the Win motifs
is achieved by the plasticity of WDR5 peptidyl-
arginine-binding cleft allowing the C-terminal ends
of the Win motifs to be maintained in structurally
divergent conformations. Consistently, enzymatic
assays reveal that WDR5 plays an important role
and SET1B methyltransferase
RbBP5-ASH2L heterodimer. Overall, our findings il-
lustrate the function of WDR5 in scaffolding the
SET1 family of KMTs and further emphasize on the
important role of WDR5 in regulating global histone
H3 Lys-4 methylation.
The landscape of each chromosome emanates from an
intricate nucleoprotein complex
nucleosome. Composed of two copies each of histone
H3, H4, H2A and H2B and wrapped by a DNA
fragment ?146bp, this protein–DNA complex constitutes
the first step of DNA compaction and plays an important
role in controlling the access of different DNA-binding
machineries to various regions of chromosomes. This
process is dynamically regulated by ATP-dependent chro-
matin remodelers along with histone-modifying enzymes
and regulates a myriad of biological processes including,
but not limited to, chromosome silencing, transcription,
DNA replication and DNA damage repair (1).
Several post-translational modifications such as acetyl-
ation, phosphorylation, ubiquitylation/sumoylation, ADP
ribosylation, PARylation and methylation, have been
mapped onto histone proteins and linked to various
cellular functions (2). Of interest, methylation of lysine
Variegation 3–9, Enhancer of Zeste and Trithorax) that
frequently exhibits preference towards a precise residue
and has the ability to transfer up to three methyl groups
to a lysine e-amine, has been linked to important biologic-
al processes. Notably, systematic mapping of methyl–
lysine marks on histone H3 demonstrated that histone
H3 Lys-4 mono-, di- and tri-methylation (H3K4me1/2/3)
decorate the promoter and strong enhancer regions of
actively transcribed genes. The same study also showed
that H3K4me1 localizes to poised enhancers and chroma-
tin regions undergoing transcriptional transition (3,4).
These results further support initial findings linking
H3K4 methylation with promoter regions of actively
transcribed genes (5–7).
In the budding yeast Saccharomyces cerevisiae, the
COMPlex ASsociated with SET1 (COMPASS) (8,9) is
referred to as the
*To whom correspondence should be addressed. Tel: +613 562 5800 8854; Fax: +613 562 5655; Email: firstname.lastname@example.org
Published online 20 January 2012 Nucleic Acids Research, 2012, Vol. 40, No. 94237–4246
? The Author(s) 2012. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
the sole protein catalyzing H3K4 methylation whereas
in mammals, this reaction is catalyzed by at least six
members: MLL1-4, SET1A and SET1B. Each mammalian
methyltransferase assembles in a COMPASS-like complex
and despite sharing similarities in substrate specificity, the
SET1 lysine methyltransferases (KMTs) are not function-
ally redundant (10–16), and, correlatively, binding studies
have demonstrated that members of the SET1 family
interact with unique subsets of proteins. The KMTs
MLL2 and MLL3 have been shown to interact with
activating signal cointegrator 2 (17) while WDR82
and menin associate with SET1A/SET1B (18–20) and
MLL1/MLL2 (11,15), respectively. Despite these differ-
ences, members of the SET1 family share several
features including the complexity of their domain organ-
ization and the presence of a catalytic SET domain.
In addition, all but MLL5 associate with a three-subunit
complex composed of WD-repeat protein-5 (WDR5),
retinoblastoma-binding protein-5 (RbBP5) and absent,
referred to as the MLL core complex (21,22), which
is required forthe stimulation
methyltransferase activity both in vitro (23) and in vivo
assigned discrete functions to RbBP5, ASH2L and
WDR5 in the assembly of the core complex and stimula-
tion of MLL1 KMT activity. Apart from its DNA binding
domain (25,26), ASH2L binds RbBP5 via its SPIa and
ryanodine receptor domain and the ASH2L/RbBP5
heterodimer stimulates MLL1 methyltransferase activ-
ity (27). In addition to its ASH2L-interacting region,
RbBP5 comprises residues that directly bind WDR5, an
interaction important for maintaining the integrity of the
core complex and indirectly the stimulation of MLL1
KMT activity (28,29). On the opposite side of the
WDR5 b-propeller domain, WDR5 binds a small motif
preceding the SET domain of MLL1 (23,30). Also
referred to as the WDR5-interacting motif (Win), the inter-
action between the MLL1 Win motif (MLL1Win) and
WDR5 is important in the scaffolding of the MLL1–
WDR5–RbBP5–ASH2L complex and stimulation of
MLL1 KMT activity in vitro (23). Based on these
findings and sequence homology between the Win motifs,
the same authors surmised that WDR5 would play an
analogous role in binding the other members of the
SET1 family, namely MLL2-4, SET1A and SET1B (31).
We herein provide biochemical evidence demonstrating
that WDR5 binds, with different equilibrium dissociation
constants, peptides corresponding to the Win motifs of the
other SET1 family members. Correspondingly, the crystal
structures of WDR5 in complex with MLL2-4, SET1A
and SET1B Win motifs reveal that WDR5 accommodates
the different Win motifs using divergent networks of
hydrogen bonds and hydrophobic contacts. Finally, we
show thatWDR5, when
the ASH2L-RbBP5 heterodimer, is important for the
methyltransferase activity. Overall, these findings further
support the fundamental role of WDR5 in scaffolding the
of MLL1 di-/tri-
studies have recently
SET1A and SET1B
SET1 complexes and indirect stimulation of H3K4
MATERIALS AND METHODS
Constructs corresponding to the Win and SET domains of
MLL1 (3745–3969), MLL2 (5344–5536) and MLL4
(2512–2715) were cloned into a pGST-based vector while
those for MLL3 (4714–4911), SET1A (1487–1707) and
SET1B (1701–1923) were cloned in a home-made vector
(pSMT3). MLL1 and MLL3 were overexpressed as GST
and His-SUMO fusion proteins, respectively, in Rosetta
cells (Novagen) with 0.1mM IPTG for 16h at 18?C. Due
to their poor solubility, MLL2, MLL4, SET1A and
SET1B were overexpressed in Arctic cells (Stratagene)
with 1mM IPTG for 48h at 10?C. Cells were harvested
in either 50mM sodium phosphate pH 7.0, 500mM
sodium chloride and 5mM b-mercaptoethanol (pSMT3)
or phosphate buffered saline (pGST), lysed by sonication,
chromatography and ULP1- (pSMT3) or TEV- (pGST2)
cleaved on beads for 16h at 4?C. Following cleavage, the
enzymes were dialyzed into a final buffer of 20mM
Tris–HCl pH 7.5, 300mM sodium chloride and 5mM
b-mercaptoethanol. The members of the core complex,
including ASH2L, RbBP5 and WDR5 (S22–334) were
overexpressed and purified as previously described (33).
Following gel filtration, the core complex subunits were
dialyzed in 20mM Tris–HCl pH 7.5, 300mM sodium
of SET1 proteins were quantified either by UV or
coomassie-stained sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS–PAGE) gel.
Isothermal titration calorimetry
Isothermal titration calorimetry (ITC) experiments were
performed using a VP-ITC calorimeter (MicroCal) as pre-
viously described (29,33). Briefly, a solution of peptide
(0.5–1mM) derived from the Win motif of MLL1,
MLL2, MLL3, MLL4, SET1A or SET1B was injected
into a solution of WDR5 (0.06mM). Titrations were
performed at 19?C in 20mM sodium phosphate pH 7.0,
100mM NaCl and 5mM b-mercaptoethanol. To decrease
the rate of saturation of WDR5 peptidyl-arginine-binding
cleft, the volume of injection of the MLL4Winpeptide was
decreased by half (from 10 to 5ml) for injections 9–19.
Titration curves were analyzed using the Origin software.
Protein crystallization, data collection and structure
Equimolar amounts of purified WDR5 and the Win
peptides were incubated on ice for 1h and crystallized
using the sitting- or hanging-drop vapor diffusion
methods. Crystals of WDR5–Win complexes were ob-
tained in conditions shown in Supplementary Table S1.
Briefly, crystallization conditions for each complex vary
but the predominant components in the mother liquors
are ammonium sulfate and polyethylene glycol 3350.
Crystals were sequentially soaked in the mother liquor
4238Nucleic Acids Research, 2012,Vol.40, No. 9
supplemented with 5, 10, 15 and 20% of glycerol, har-
vested and flash frozen in liquid nitrogen. Data sets were
collected at the Life Science Collaborative Access Team
(LS-CAT) beamline at the Advance Photon Source of the
HKL2000 (34). The structure of each WDR5–Win
complex was solved by molecular replacement using
Phaser and apo-WDR5 as a search model (RCSB
2H13.pdb) (35). The structure of each WDR5–Win
complex was further improved using iterative cycles of
refinement, using either Refmac (36) or Buster (37) and
model building with COOT (38). The quality of the struc-
tures was validated using Molprobity (39).
In vitro methyltransferase assay
Methyltransferase assays were performed as previously
described (29,33). Briefly, each recombinantly purified
SET1 KMT was incubated with equimolar amounts of
WDR5, RbBP5 and ASH2L in 50mM Tris pH 8.0,
200mM NaCl,3mM Dithiothreitol
MgCl2, 5% glycerol and 1mM histone H3 or peptide
corresponding to the first N-terminal 15 residues or
10mM of recombinantly purified H3. Reactions were
initiated by adding 1mCi of radiolabeled S-adenosyl-L-me-
thionine (AdoMet) (0.07mM of tritiated AdoMet) and
stopped by spotting the reactions onto Whatman P-81
filter papers. Free AdoMet was removed by washing the
filter papers four times in 250ml of 50mM sodium bicar-
bonate pH 9.0 and methyltransferase activity was
quantified by liquid scintillation counting.
Equilibrium dissociation constants of the Win motifs
A recent biochemical analysis of WDR5 peptidyl-
arginine-binding cleft revealed that this protein binds a
10-residue peptide corresponding to MLL1Win with an
equilibrium dissociation constant (KD) of 1.7mM. The
crystal structure of WDR5 in complex with MLL1Win
identified that R3765 of MLL1 was critical in conferring
binding to WDR5, scaffolding the MLL1–WDR5–
RbBP5–ASH2L complex and, correlatively, stimulation
of MLL1 methyltransferase activity (23). Accordingly,
the same authors posited that a similar region found on
other members of the SET1 family would play an analo-
gous role (31) while another study proposed that only
MLL1 and MLL4 could bind WDR5 (30). In order to
investigate these hypotheses, we first sought to measure
the KDvalues of WDR5 for MLL1-4 and SET1A/BWin
motifs. Using ITC, we determined that WDR5 binds
MLL1Winand MLL4Winpeptides with a KDof 1.13mM
and 0.03mM, respectively. Similar titration experiments of
WDR5 with MLL2Winand MLL3Winpeptides resulted in
KDof 0.16mM and 2.03mM, respectively, while SET1AWin
and SET1BWinpeptides bound to WDR5 with a KDof
0.26mM and 0.10mM, respectively. Overall, these results
demonstrate that WDR5 binds the Win region of all the
members of the SET1 family of KMTs. However, given
the divergence in the amino acid sequence succeeding the
conserved arginine residue and the ?37-fold range of
binding affinities of WDR5 for the Win motifs (Table 1
and Figure 1), our binding analysis suggests that WDR5
interacts with SET1 KMTs using divergent modes.
Crystal structures of the WDR5–MLLWincomplexes
To gain insights into the mechanisms underlying WDR5
ability to bind the structurally divergent C-terminal ends
of the Win peptides (Table 1), we determined the crystal
structures of WDR5 in complex with peptides correspond-
ing to the Win motifs of MLL2 (1.4A˚resolution), MLL3
(2.2A˚ resolution), MLL4 (1.57A˚
(Supplementary Table S1). The WDR5–Win complexes
superimpose with overall r.m.s.d. of<0.3A˚
aligned atoms, showing that binding of the Win pep-
tides does not result in noticeable changes in the overall
structure of WDR5. Simulated annealing omit maps
revealed clear electron density for several residues
flanking the central arginine residue of each Win motif
(Supplementary Figure S1). However, the distal residues
in the peptides, including the M4715 side chain (single
letter code refer to residues of the Win motifs), S4716
and A4717 of MLL3Win, K2518 of MLL4Win, P1501 and
I1502 of SET1AWinand G1882 and I1892 as well as the
side chains of F1889 and Y1890 of SET1BWinlack inter-
pretable electronic density and were not modeled. Close
inspection of the WDR5-Win structures reveals that all
the peptides bind WDR5 peptidyl-arginine-binding cleft,
a crevice located on one side of the WDR5 b-propeller
structure (Figure 1), in which each peptide engages in
several hydrogen bonds, van der Waals contacts and
hydrophobic interactions with WDR5. To facilitate
the discussion of the interactions between WDR5 and
the Win motifs, we have used a modified version of the
Schechter–Berger notation for assigning residues of
the Win motifs in which the central arginine residue is
denoted as P0 (Table 1). In addition, interactions
between WDR5 and the Win motifs have been separated
into two categories based on interactions with residues
preceding and succeeding the P+2glutamate residue of
the Win motifs (Table 1).
Consistent with the sequence homology of residues
flanking the central arginine (P0), including those at P?3,
P?2, P?1, P+1and P+2positions (Table 1), this region of
the peptide adopts similar orientations and engages in sev-
eral shared direct and water-mediated hydrogen bonds
and hydrophobic interactions with WDR5. Similar to
MLL1Win, the P?2to P+2region folds as a 310helix in
which the side chains of P?3, P?2and P?1residues pack
tightly within WDR5 peptidyl-arginine-binding cleft and
make hydrophobic contacts with Ala-65, Gly-89, Ile-90
and Asp-107 side chains. The same residues in WDR5
form a wall that maintains the side chain of the P?2
residue in an orientation permissive to make a hydrogen
bond with the backbone amide of the P+1residue. The
alanine residue in the P?1position binds analogously in
all WDR5–Win complexes. Indeed, the P?1amide group
engages in a hydrogen bond with the Asp-107 carboxylate
group and its side chain makes hydrophobic contacts with
Nucleic Acids Research,2012, Vol.40, No. 94239
Figure 1. WDR5 binds MLL1-4, SET1A and SET1B Win motifs. ITC titration experiments with MLL1Win (A), MLL2Win (B), MLL3Win
(C), MLL4Win(D), SET1AWin(E) and SET1BWin(F) peptides and WDR5 (upper trace) and the fitted binding curve (lower trace). The sequence
of the Win motifs and the KDare indicated as insets. Titration of WDR5 with Win peptides displays binding stoichiometries (N values) between
0.9 and 1.0.
Table 1. Alignment of SET1 Win motifs
Amino acid sequence
Underlined residues have not been modeled in the crystal structure. !, C4708S substituted peptide.
aSide chain is missing.
4240 Nucleic Acids Research, 2012,Vol.40, No. 9
the side chains of Phe-133 and Phe-149. Similar to histone
H3 R2 (35,40–42) and MLL1 R3765 (30,31), the central
arginine (P0) of the Win motifs extends inside the central
toroid cavity of WDR5 in which its guanidinium group
interacts with the side chains of Phe-133 and Phe-263. The
P0residue also makes several direct hydrogen bonds with
the main chains of Ser-91, Phe-133 and Cys-261 and a
water-mediated hydrogen bond with the Ser-175 back-
bone carbonyl group. Following the central arginine
residue, the P+1position is occupied by a small residue
such as serine (MLL2, SET1A and SET1B) or alanine
(MLL3 and MLL4) while the P+2position is occupied by
between the P+1and P+2residues, this portion of the Win
motifs shares several interactions with WDR5. The P+1
residue makes van der Waals contacts with the side chains
of Ala-47 and Tyr-260 while in MLL4 the P+2carboxylate
moiety makes two additional water-mediated hydrogen
bonds with the backbone amides of the P+3 and P?3
residues. In addition, the hydroxyl group of the MLL2Win
and SET1BWinP+1serine residue makes a hydrogen bond
with the sulfhydryl group of the P?2cysteine residue.
binding modes of the P?3, P?2, P?1and P0residues, struc-
tural alignment of the WDR5–Win complexes reveals
MLL4WinP?3residue is rotate 180?, which results in add-
itional water-mediated and direct hydrogen bonds with
WDR5 (Figure 3). This rotation leads to a small structural
displacement of the P?2backbone with negligible conse-
quences on the positioning of C5063 (MLL2), S4708
(MLL3), A2509 (MLL4), S1493 (SET1A) and C1883
(SET1B) side chains and their interactions with WDR5.
However, in clear contrast to the P?2, P?1, P0, P+1and P+2
residues, the P+3to P+5residues adopt divergent orienta-
tions. MLL2Win and MLL3Win peptides, which bind
analogously to WDR5, interact in such a way that their
respective C-termini point towards blade 5 of WDR5
while MLL4Win and MLL1Win adopt a conformation
placing their C-terminal ends towards blade 4 of the
b-propeller. SET1AWinand SET1BWin peptides bind in
an intermediate orientation such that their C-termini are
maintained on the surface of blades 4 and 5 of WDR5
highsequence homology andanalogous
Figure 2. Crystal structure of WDR5 in complex with MLL2-4, SET1A and SET1B Win motifs. (A) Overall structure of WDR5 (dark gray) showing
the seven blades and the relative orientations of MLL2 (yellow), MLL3 (turquoise), MLL4 (green), SET1A (firebrick) and SET1B (cyan) Win motifs.
(B) Crystal structure of WDR5–MLL4Wincomplex. Zoomed view of WDR5 peptidyl-arginine-binding cleft in which WDR5 and MLL4 carbon
atoms are rendered in gray and green, respectively. (C) Crystal structures of WDR5–MLL2Winand WDR5–MLL3Wincomplexes. MLL2 and MLL3
carbon atoms are highlighted as in (A). (D) Crystal structures of WDR5–SET1AWinand WDR5–SET1BWincomplexes. SET1A and SET1B carbon
atoms are colored as in (A). Water molecules and key hydrogen bonds are depicted as red spheres and orange dash lines, respectively.
Nucleic Acids Research,2012, Vol.40, No. 9 4241
(Figure 2A). Given the structural differences adopted by
residues succeeding the P+2residue (Figure 3), we pur-
ported that each peptide would engage in distinct inter-
actions with WDR5. The backbone of P+3 Y2514 of
MLL4Win, which binds similarly to H3769 of MLL1Win,
forms direct and water-mediated hydrogen bonds with
Asp-172 and Lys-259 carboxylate and carbonyl groups,
respectively. In addition, its aromatic ring makes hydro-
phobic contacts with the side chains of Tyr-191, Pro-173
and Phe-149. In contrast to the solvent exposed P+4
L2516, the P+5R2517 side chain is nestled in a small
cleft formed by the surface of blade 4 and the loop
connecting blades 4 and 5 of WDR5 (Figure 3B). This
crevice, which is composed of Tyr-191, Asp-192, Gly-193,
chain in a permissive orientation to engage in direct
hydrogen bonds with the backbone carbonyl groups of
Tyr-191 and Asn-214 and van der Waals contacts with
?37-fold more tightly to WDR5 than the highly homolo-
gous MLL1Winpeptide (Figure 1 and Table 1). Owing to
the high sequence homology between these two motifs and
structural homology between the WDR5-bound MLL1Win
and MLL4Win complexes, the differences in binding
affinities are seemingly at odds. However, while a com-
parative analysis of WDR5–MLL1Win and WDR5–
MLL4Win complexes reveals that both MLL1Win and
MLL4WinP+4residues share extensive interactions with
WDR5, it also shows that a water-mediated interaction
of the MLL1WinP+4H3769 side chain with the WDR5
Asp-172 carboxylate group is replaced by a direct
hydrogen bond between the same residue in WDR5 and
the side chain hydroxyl group of MLL4WinP+4Y2514; a
difference that presumably explain the dissimilarity
between MLL1Win and MLL4Win binding affinities for
Owing to the presence of a proline residue in the P+3
position of MLL2Winand MLL3Win, we postulated that
these peptides would bind WDR5 differently than
MLL1Winand MLL4Win. Consistent with this hypothesis,
structural alignment of WDR5–MLL2Win and WDR5–
MLL1Wincomplexes reveals that the C-terminal end of
MLL2Winshifts towards the loop preceding b18, resulting
in several novel interactions between MLL2Win and
WDR5 (Figure 3B). In MLL2Win, the P+3P5068 residue
makes close van der Waals contacts with the side chains of
Lys-259 and Tyr-260. The WDR5 peptidyl-arginine-
binding cleft also undergoes notable structural changes.
The phenolic side chain of Tyr-191 rotates of 90?position-
ing its aromatic ring in a near parallel orientation with
addition, the same lysine residue in MLL2Win/MLL3Win
forms a novel hydrogen bond with the Lys-259 carbonyl
group and hydrophobic contacts with Leu-234. Finally, in
contrast to all other Win motifs, the MLL2WinP+5L5071
residue lies within a hydrophobic cleft composed of
Pro-216 and Leu-234. Overall, the crystal structure of
MLL2Winbound to WDR5 suggests that the P+3residue
acts as a structural determinant underlying the binding
mode of a peptide within WDR5 peptidyl-arginine-
After observing that the P+3 position in MLL2Win/
MLL3Winpeptides is a key point of structural divergence
between the Win peptides, we postulated that the P+3
glycine residue of SET1AWin/ SET1BWin motifs would
Figure 3. Comparative
Superimposition of WDR5-bound MLL1 (beige) and MLL2 (yellow)
peptides. (B) Overlay of MLL1 (beige) and MLL4 (green) peptides in
complex with WDR5 (grey). (C) Structural alignment of WDR5–
MLL1Win and WDR5–SET1AWin complex in which carbons of the
peptides are highlighted as in Figure 2. Residues of WDR5 peptidyl-
complexes are rendered as pink carbon atoms. Hydrogen bonds and
water molecules are rendered as in Figure 2.
analysisWin motif bindingmodes.(A)
4242 Nucleic Acids Research, 2012,Vol.40, No. 9
also exhibit structural differences compared to other Win
motifs. Accordingly, in contrast to MLL1-4 Win peptides,
the backbone carbonyl of the SET1AWinP+2glutamate
residue undergoes a 180?rotation that results in novel
hydrogen bonds between the amide groups of the P+3
and P+4residues and the backbone carbonyls of the P?1
residue and Lys-259 of WDR5, respectively. Notably, in
comparison with the WDR5–MLL1Wincomplex, the side
chains of Lys-259 and Tyr-191 shift toward the Win
peptides, which results in the formation of additional
hydrophobic contacts with the SET1AWin P+3 glycine
and P+5tyrosine residues (Figure 3C). In addition, the
same tyrosine residue in SET1A engages in several hydro-
phobic contacts with the lateral chain of Leu-234 and
Overall, the crystal structures of the WDR5–Win
complexes highlight WDR5’s ability in binding the Win
motifs using alternative structural determinants. More im-
portantly, our structural studies suggest that the plasticity
of WDR5 peptidyl-arginine-binding cleft stems from its
ability to operate subtle but key structural changes that
residues in position P+3. Finally, crystal structures of the
WDR5–Win complexes suggest that the type of residue at
the P+3position will likely play an important role in the
binding mode adopted by the peptide within the WDR5
Role of WDR5 in stimulating the KMT activity of the
SET1 family of methyltransferases
The Win motif of MLL1 is important for its interaction
with the WDR5–RbBP5–ASH2L complex and thereby
stimulation of its methyltransferase activity (23). After
showing that WDR5 binds the Win motifs of MLL1-4
and SET1A/B, we examined the role of the WDR5–
members of the SET1 family of methyltransferases.
A comparison of SET1 KMT activity in the absence
or presence of the WDR5–RbBP5–ASH2L complex
methyltransferase activity of all members, with the excep-
tion of MLL5, but with differences in the fold of stimula-
tion. Specifically, the WDR5–RbBP5–ASH2L complex
stimulated MLL2/MLL3 and MLL4 activity by ?52/
256- and ?15-fold, respectively, while co-incubation of
SET1A and SET1B with the core complex subunits
increased methylation of histone H3 by ?40- and
?1290-fold (Figure 4). The stimulatory roles of the core
complex on SET1B can be rationalized by the nearly un-
detectable activity of these KMTases on histone H3 in the
absence of the WDR5–RbBP5–ASH2L complex. Overall,
these observations support initial in vivo data showing that
the members of the core complex are critical for global
levels of histone H3K4 methylation.
After showing that the core complex subunits stimulate
the methyltransferase activity of MLL2-4, SET1A and
SET1B, we sought to measure the contribution of
(Figure 4). Interestingly, incubation of the ASH2L/
RbBP5 heterodimer with each KMT failed to fully
activity of thecomplex
stimulate, to various extent, the enzymatic activity of
each member of the SET1 family. Specifically, the
absence of WDR5 resulted in a 2-fold decrease of
ASH2L-RbBP5 heterodimer decreased the methylation
of histone H3 by 5-fold and 1.5-fold, respectively
(Figure 4). Overall, these results clearly demonstrate the
importance of WDR5 in stimulating the methyltransferase
activity of MLL2-4, SET1A and SET1B and support
recent studies suggesting that WDR5 is key for the
activity for all members of the SET1 family of KMTs.
the andMLL2 with
Figure 4. Stimulation of the SET1 family of KMTs activity on histone
H3 by the core complex. Radiometric methyltransferase assays per-
formed with MLL2 (A), MLL3 (B), MLL4 (C), SET1A (D) and
SET1B (E) either in the absence or presence of WDR5 and the
RbBP5–ASH2L complex. Methyltransferase assays were performed at
a concentration of enzyme in the linear range of activity of MLL2
(1mM), MLL3 (0.5mM), MLL4 (1mM), SET1A (1mM) and SET1B
(0.5mM) complexes. Activity is represented as the average of three in-
dependent experiments performed in triplicate. Error bars indicate the
standard deviation between the assays.
Nucleic Acids Research,2012, Vol.40, No. 94243
Following the identification that the WDR5–RbBP5–
ASH2L complex stimulates all members of the SET1
family, we sought to evaluate the methylation of histone
H3 by MLL1-4 in the absence of the core complex
subunits. To examine the catalytic activity of each MLL,
we incubated each KMT with a histone H3 peptide and
a tritiated cofactor. As shown in Figure 5, we failed to
and MLL4 while a similar assay performed with MLL3
resulted in a notable accumulation of the methylated
peptide. These observations suggest that MLL3 enzymatic
activity may play a role independent of the WDR5–
The results presented herein provides biochemical and
arginine-binding cleft is important in the binding of all
the members of the SET1 family of methyltransferases
stimulated by the core complex.
Of interest, we observed that MLL2 and MLL3 Win
motifs bind similarly to WDR5. Owing to the presence of a
proline residue in P+3, Song et al. (30) had previously
proposed that MLL2/MLL3 might interact with WDR5
using a different mode of binding. Comparative analyses
of WDR5–MLL2Win and WDR5–MLL1Win complexes
support this hypothesis as the C-terminal ends of each
peptide are maintained by specific sets of hydrogen
bonds, hydrophobic contacts and van der Waals inter-
actions. Similarly, we also observed noticeable differ-
ences between WDR5–SET1AWinand WDR5–MLL1Win
complexes that include rotation of the SET1AWin P+2
backbone, a change that could be attributed to the
presence of a glycine residue in P+3, which results in
reorientation of theP+4
structural differences are accommodated by the reorgan-
izationof residues forming
arginine-binding cleft including Lys-259 and Tyr-191.
Although, it was evidenced by several mutational studies
(23,28,30,33,35,41) that the N-terminal end of the Win
motifs is important for binding to WDR5, our compara-
tive analysis suggest that the propensity of WDR5 in
binding the members of the SET1 family of KMTs is
achieved by the plasticity of its peptidyl-arginine-binding
cleft and more specifically its ability to accommodate the
divergent C-terminal region of the Win motifs.
In metazoans, WDR5 plays a critical role in regulating/
maintaining proper levels of histone H3K4 methylation
(21,22,24) while in Drosophila, deletion of the will die
slowly protein (WDR5 homolog) leads to global loss of
methylation and ultimately to lethality (43). Similarly,
morpholino knockdown of WDR5 in Xenopus leads to
severe developmental deficiencies including gut, hemato-
poietic and somatic defects (21). We propose that the plas-
ticity of the WDR5 peptidyl-arginine-binding cleft plays
an important role in scaffolding all of the SET1 complexes
and underlies the fundamental roles of WDR5 in
regulating various developmental programs (21,44).
MLL3, a unique member of the SET1 family of HKMTs
The characterization of SET1 enzymatic activity on
histone H3 presented herein provides the first comparative
analysis for the SET1 family of methyltransferases. While
the SET domains of MLL1, MLL2, and MLL4 displayed
negligible enzymatic activity in the absence of the WDR5–
RbBP5–ASH2L complex, we observed detectable histone
H3 methyltransferase activity for MLL3 independent of
the core complex. This observation that MLL3 efficiently
methylates histone H3 in the absence of the core complex
while MLL2 (MLL4 in rodents) strictly depends on the
presence of the core complex subunits is interesting.
Indeed, while initial studies had suggested that MLL3
and MLL4 are functionally redundant in retinoic acid
receptor transactivation in mouse embryonic fibroblasts
(45), targeted inactivation of MLL3 resulted in several
defects including lower body mass, hypofertility and
increased formation of ureter epithelial tumors (45,46).
Based on our findings, we propose that the ability of
MLL3 to methylate histone H3 in the absence of the
core complex subunits may underlie some of the
non-overlapping functions between MLL3 and MLL4.
The expanding role of WDR5
The recent findings that WDR5 binds to proteins unre-
lated to the trithorax group proteins shed new light on
the ever-expanding role of WDR5. It has been shown
recently that Oct4 forms a complex with WDR5 independ-
ent of the presence of RbBP5, ASH2L or the SET1 family
members (44). By identifying an interaction between
WDR5 and the embryonic stem cell core transcriptional
network, the authors of this study established that WDR5
expression is necessary for the efficient formation of
induced pluripotent stem cells (44). In addition, Gan
et al. (47), recently demonstrated that WDR5 regulates
smooth muscle cell-selective gene activation by interacting
Similarly, WDR5 has been shown to interact with the
infection-triggered IRF3 and NF-kB activation (48) and
with the NRC/NCoA6 interacting factor 1 (49). Finally,
recent MudPit analysis identified WDR5 as a subunit of
the non-specific lethal complex (50). These examples
further emphasize the important role of WDR5 in scaf-
folding these complexes and we propose that the plasticity
Figure 5. MLL3 methylates histone H3 in the absence of the core
complex subunits. Comparative analysis of MLL’s enzymatic activity
in the absence of the core complex subunits. Assays were performed in
the presence of 5.0mM of enzymes. Activity is represented as in Figure 4.
4244Nucleic Acids Research, 2012,Vol.40, No. 9
of WDR5 peptidyl-arginine-binding cleft, or its recently
characterized V-shape cleft (29), plays an important role
in these biological processes.
Coordinates and structure factors for the WDR5-
WDR5-SET1AWin, WDR5-SET1BWin complexes have
been deposited in the protein data bank (rcsb.org) with
3UVM.pdb, 3UVN.pdb and 3UVO.pdb respectively.
Supplementary Data are available at NAR Online:
Supplementary Table 1 and Supplementary Figure 1.
We would like to thank Dr Alain Doucet, Sylvain
comments on the manuscript.
Canadian Institute of Health Research grant (to J.-F.C.);
an early research award from the Ministry of Research
and Innovation (province of Ontario). Dr Couture holds
a Canada Research Chair in Structural Biology and
Epigenetics. Natural Sciences and Engineering Research
Council of Canada (CREATE) (scholarship to P.Z.).
Funding for open access charge: Canadian Institute of
Health Research grant (to J.-F.C.); an early research
award from the Ministry of Research and Innovation
(province of Ontario).
Conflict of interest statement. None declared.
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