Structural Basis for the Requirement
of Additional Factors for MLL1 SET Domain
Activity and Recognition of Epigenetic Marks
Stacey M. Southall,1,2Poon-Sheng Wong,1,2Zain Odho,1S. Mark Roe,1and Jon R. Wilson1,*
1Section of Structural Biology, The Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, Chelsea,
London SW3 6JB, UK
2These authors contributed equally to this work
The mixed-lineage leukemia protein MLL1 is a tran-
scriptional regulator with an essential role in early
development and hematopoiesis. The biological
function of MLL1 is mediated by the histone H3K4
methyltransferase activity of the carboxyl-terminal
SET domain. We have determined the crystal
structure of the MLL1 SET domain in complex with
cofactor product AdoHcy and a histone H3 peptide.
This structure indicates that, in order to form a well-
ordered active site, a highly variable but essential
component of the SET domain must be repositioned.
To test this idea, we compared the effect of the
addition of MLL complex members on methyltrans-
ferase activity and show that both RbBP5 and
Ash2L but not Wdr5 stimulate activity. Additionally,
we have determined the effect of posttranslational
modifications on histone H3 residues downstream
and upstream from the target lysine and provide
a structural explanation for why H3T3 phosphoryla-
tion and H3K9 acetylation regulate activity.
As organisms have become more complex, they have evolved
more elaborate mechanisms to make use of chromatin to
increased complexity has been the evolution of large numbers
of enzymes responsible for the highly coordinated addition or
removal of posttranslational marks on histone proteins. The
methylation of specific histone lysine and arginine residues in
the transcription regulation of genes has a very prominent role
in this ‘‘epigenetic’’ regulation (for reviews see Berger, 2007;
Kouzarides, 2007; Shilatifard, 2008). The biological function of
the MLL(KMT2) protein family arises from the histone methyl-
transferase activity of their carboxyl-terminal SET domains
(Milne et al., 2002). They specifically modify the side chain of
histone H3 lysine 4 (H3K4), by addition of mono-, di-, or trimethyl
groups in a highly regulated manner. H3K4 methylation is asso-
ciated with the regulation of the promoter regions of actively
transcribed genes, with the level of methylation state (mono-,
di-, or trimethyl) varying across the coding region (Bernstein
et al., 2005; Pokholok et al., 2005; Schneider et al., 2004; Shila-
tifard, 2008). With advances in the analysis of global and specific
patterns of histone modifications, the emerging physiological
role of this H3K4 methylation is becoming much more complex
than simply marking areas of active transcription (Berger,
2007; Guenther et al., 2007). MLL1 is an essential gene required
for positive gene regulation in early development, it is expressed
in most adult cell types, and is known to be required for normal
hematopoiesis and the regulation of the cell cycle (for reviews
see Hess, 2004; Liu et al., 2008). Many of the homeotic transfor-
mations associated with MLL1 knockdown can be correlated to
loss of SET domain activity (Terranova et al., 2006). MLL1 has
for two reasons. First, it has a defined role in mammalian
development, where it has been shown to be required for the
regulation of important homeobox (Hox) genes and therefore is
essential for early patterning in the embryo (Guenther et al.,
2005; Yu et al., 1995). Second, in acute myeloid and lymphoid
leukemia the MLL1 gene is frequently targeted in oncogenic
gene translocations, leading to the expression of chimeric
fusions between the amino-terminal 1400 residues of MLL1
and one of over 50 partner genes (Meyer et al., 2006). MLL1
translocations are more prevalent in childhood leukemias and
in therapy-related cases, where they are associated with
a poor prognosis.
In common with the vast majority of lysine methyltransferase
proteins, the MLL family have an evolutionary conserved SET
domain (Milne et al., 2002; Xiao et al., 2003b). The SET domains
of a small number of proteins have been structurally character-
ized (Manzur et al., 2003; Min et al., 2002; Trievel et al., 2003;
Wilson et al., 2002; Xiao et al., 2005; Zhang et al., 2003). From
this structural work, a molecular mechanism has been estab-
lished with two important features. First, the S-C bond of the
AdoMet methyl-leaving group and the 3-amine-receiving group
adopt positions on the enzyme that optimize the distances and
geometry for methyl transfer. Second, the active site provides
a chemical environment for the amine lysine and the AdoMet
methyl that favors methyl transfer via a SN2 nucleophilic substi-
tution mechanism, by surrounding the reactants with a carbonyl
cage (Dillon et al., 2005; Xiao et al., 2006). To achieve this, a key
structural feature of the SET domain is the hydrophobic channel
Molecular Cell 33, 181–191, January 30, 2009 ª2009 Elsevier Inc. 181
or pore that connects the substrate- and cofactor-binding faces
and accommodates the alkyl chain of the target lysine. A partic-
ular feature of lysine methylation as a functional group in
signaling is that each lysine residue can be decorated with
one, two, or three methyls (Schotta et al., 2004). A defining
characteristic of individual SET domain proteins is therefore
whether they can catalyze mono-, di-, or trimethylation and
whether they favor prior methylation of their target substrates.
MLL1 has been shown to be capable of catalyzing multiple
methylations; however, sequence analysis indicates that key
tyrosine residues that have previously been attributed to
constrain Set7/9 and PR-Set7 enzymes to be monomethylases
are conserved (Couture et al., 2005; Xiao et al., 2005, 2003a).
This suggests that there must be differences in the configuration
of the active site of MLL to allow multiple methylation.
Alongside issues surrounding product specificity, the target-
ing of chromatin modification enzymes is also critical to their
function. Although the SET domain architecture includes
elements, such as the variable SET-I region, that contribute to
the binding of peptide substrates, a feature of the structurally
interactions have been dominated by main-chain interactions.
As a result there are few clear determinants of specificity.
Although both Set7/9 and MLL1 target H3K4, there are no
obvious sequence features that explain their common speci-
ficity. An additional aspect of the targeting of MLL1 is that
physiologically it is known to associate with a multiprotein
complex comprising Wdr5, RbBP5, Ash2L, and Dpy30 (Popovic
and Zeleznik-Le, 2005; Yokoyama etal., 2004). This organization
is evolutionarily conserved, and the homologous yeast Set1
complex COMPASS has provided many insights into the regula-
tion of the methyltransferase activity by complex members
(Dehe et al., 2006; Yokoyama et al., 2004). However, some key
domains of yeast Set1, which have been shown to be involved
in the regulation of the methyltransferase activity, are not
conserved in MLL1, and therefore the mechanism of regulation
may differ (Schlichter and Cairns, 2005; Tresaugues et al.,
2006). In the absence of any of the core complex members,
Ash2L, RbBP5, or Wdr5, the methyltransferase activity of MLL
is severely compromised (Dou et al., 2006; Steward et al.,
2006). We therefore wished to determine if any of the complex
members contributed directly to the activity of a minimal SET
We have solved the structure of the SET domain from MLL1 in
complex with its cofactor product S-adenosylhomocysteine
(AdoHcy or SAH), with and without a short peptide based on
the amino terminus of histone H3 with a dimethylated lysine 4
residue (H3K4me2). Analysis of this structure indicates that,
unlike previous SET domain proteins, MLL1 requires the involve-
ment of another factor to order the active site to obtain optimal
methyltransferase activity. In a defined in vitro methyltransferase
assay, with minimal components, we compare the activity of the
MLL complex, both individually and in combination. We find that
inclusion of either Ash2L or RbBP5 significantly promotes meth-
yltransferase activity. Finally, we show how physiologically
relevant histone modifications affect the activity of the enzyme
and account structurally for their role in the regulation of MLL1.
Overall Structure of the MLL1 SET Domain
Sequence analysisof the MLL1 SET domain, which occupies the
extreme carboxyl terminus of the protein, suggests that it has all
the conserved sequence elements required for methyltransfer-
ase activity (Figures 1A and 1B). Yet, both in vivo and in vitro
studies have indicated that other proteins from the MLL complex
are required for efficient methylation (Dehe et al., 2006; Krogan
et al., 2002; Schneider et al., 2005; Steward et al., 2006;
complex of a MLL1 SET domain construct consisting of residues
3785–3969 with the cofactor product AdoHcy and a ternary
complex containing AdoHcy and a short substrate peptide
(Figure 1C). The binary complex structure was refined to a reso-
lution of 2.0 A˚, and the ternary complex to 2.2 A˚(Table 1). There
are no significant changes in the structure of the SET domain of
the binary and ternary complexes, and the cofactor binds in
a well-defined surface pocket. In the ternary complex the
peptide binds in a deep open channel. Overall the MLL1 SET
domain topology is the same as that of other SET domains,
and the relationship between the N-flanking, SET-N, SET-I,
SET-C including the pseudo-knot, and postSET subdomains is
well conserved (Figure 1A). However, our data suggest that the
SET-I sub domain is not in the optimal position to form the
binding site of the target lysine, and this provides the structural
explanation for the requirement of other factors to promote
The MLL1 Active Site and Activity
An archetypal feature of SET domain structures to date has been
the channel through the protein linking the cofactor-binding
surface on one side with the substrate-binding surface on the
other (Xiao et al., 2003b). This channel, made up of residues
fromthe SET-I, SET-C, and postSET regions, encloses the lysine
position for methyl transfer to take place. However, in MLL1 the
SET-I region and postSET domains form two separate lobes
(Figure 1C). The substrate binds in an open channel at the
bottomof the cleftbetween theselobes, with the target lysine re-
maining relatively exposed and therefore flexible. Despite
a different arrangement of the elements of the lysine channel to
other SET domains, the important active site residues are
conserved compared to the other SET domain proteins
(Figure 1B). The essential active site residues (Phe3884,
Tyr3942, Tyr3944, and Phe3946 and the main chain of the
tetrapeptide, residues Cys3882 to Phe3885) have a similar
arrangement to other SET domains (Figure 2A). In the absence
of peptide substrate, the electron density for the side chain of
Tyr3942 suggests two orientations, but upon substrate binding
structures (see Figure S1 available online).
The yeast COMPASS and mammalian MLL1 complexes are
associated with all three levels of methylation in vivo, but an
intact complex has been shown to be a prerequisite for full
activity (Dehe et al., 2006; Steward et al., 2006; Wysocka et al.,
2005). Nevertheless, for the isolated MLL1 SET domain we are
able to measure activity in the absence of the other complex
Structure of the MLL1 SET Domain
182 Molecular Cell 33, 181–191, January 30, 2009 ª2009 Elsevier Inc.
members, using a sensitive3H-methyl incorporation assay with
synthetic peptide substrates (Figure 2B). The activity observed
is only around one-thousandth of that observed for Set7/9,
with the same substrate in equivalent assay conditions (data
not shown). This illustrates that the isolated SET domain has
the elements required to catalyze methylation, but that without
additional factors MLL1 catalytic efficiency is compromised.
MLL1 SET domain can incorporate methyl groups into unmodi-
lation activity. In the assay conditions tested the trimethylation
activity of the isolated MLL1 SET domain was at the limits of
The importance of the key tyrosines (Tyr3858 and Tyr3942)
to methyl transfer was investigated with site-directed mutants
(Figure 2B). Activity for MLL1 constructs with Tyr3858 and
Tyr3942 to Ala mutations was significantly reduced for
Figure 1. Overall Structure of the MLL1 SET Domain
(A) Schematic representation of the full-length MLL1 protein and the construct containing the SET domain used in structural studies (residues 3785–3969),
indicating the subdomains referred to in the text. The N-flanking region is in pale blue, the SET-N in pale green, the SET-I in blue, the SET-C in bright green,
and the postSET domain in orange.
(B) Sequence of the MLL1 SET domain aligned with MLL1, Dim-5, Suv39h2, Set7/9, and PRSet7. The SET domain region secondary structure elements derived
from the structure are indicated above the sequence. Identical residues are highlighted in red, conserved cysteines involved in Zn binding in orange, and active
site residues in blue.
Structure of the MLL1 SET Domain
Molecular Cell 33, 181–191, January 30, 2009 ª2009 Elsevier Inc. 183
unmodified and monomethylated substrates as was a Tyr3858
to Phe mutant. The effect of the Tyr3942 to Phe mutation was
less significant, and it is noted that equivalent residue in both
Dim5 and Suv39h2 is a phenylalanine. Neither individual
phenylalanine mutation promoted trimethylation, but a double
mutation Tyr3858 and 3942 to Phe, while inactivating mono-
reminiscent of the effect of similar site directed mutations in
Set7/9, where it was found that a Tyr to Ala mutant was no
promoted di- and trimethylation (Xiao et al., 2003a; Zhang
et al., 2003).
The MLL1 SET Domain Requires Ordering
to Promote Activity
Given that the MLL1 domain has the same overall topology and
the essential residues for methyl transfer activity are conserved,
we were interested to understand why the isolated SET domain
exhibits such low activity. Analysis of the structure reveals that
the open nature of the peptide-binding groove, and the more
spacious active site, can be attributed to a shift in the orientation
of the SET-I region of the enzyme. With the MLL1, Set7/9,
cules, most elements of the SET domains align very well, but the
SET-I region of MLL1 is moved relative to the C-flanking region
(Figure S2). In Figure 3A, focusing on part of the C-flanking
domain, the cofactor, and SET-I region, it is clear that the helix
of the SET-I region in MLL1 is displaced away from the C-flank-
ing region compared to Dim-5. Consequently the loop following
the SET-I helix is also shifted. This is significant because it is this
tide), that forms one face of the substrate lysine alkyl binding
Table 1. Crystallographic Data and Refinement Statistics for the
MLL1 SET Domain Binary Complex with AdoHcy and the MLL1
SET Domain Ternary Complex with AdoHcy and Substrate
Bank Code 2W5Y (Binary)2W5Z (Ternary)
a, b, c (A˚)48.8, 56.2, 78.248.9, 56.5, 78.4
a, b, g (deg)
90, 90, 9090, 90, 90
Wilson B factor27.828.3
No. of reflections
No. of atoms
1 (Zn2+), 26 (AdoHcy),
1 (Zn2+), 26 (AdoHcy),
Bond lengths (A˚)0.0080.005
Bond angles1.266 0.911
aRwork= Sj jFoj ? jFcj j/SjFoj.
bRfree= STj jFoj ? jFcj j/STjFoj, where T is a test data set of 5% of the total
reflections randomly chosen and set aside before refinement.
cTheaverage valueacross theresolution range,while thatinparentheses
is the value for the highest resolution bin (2.1–2.0 A˚and 2.3–2.2 A˚,
Figure 2. The MLL1 Active Site and Activity
(A) Residues which form the active site of MLL1 and help form the lysine
for these residues.
(B) Activity assay of the MLL1 SET domain carried out in vitro with substrate
peptides based on histone H3 either unmodified, or carrying K3K4me1 or
H3K4me2 modifications for wild-type and site-directed mutants. Activity is
represented as the mean of triplicate measurements, with error bars indicating
one standard deviation.
Structure of the MLL1 SET Domain
184 Molecular Cell 33, 181–191, January 30, 2009 ª2009 Elsevier Inc.
channel. Whereas the AdoHcy and the conserved tyrosine
Tyr3944 align very well, the Ca of MLL1 Cys3882 is about 8 A˚
from the Ca of the equivalent residue in Dim-5 (Figure 3B). The
net result of the change in orientation of the SET-I helix and the
channel tetrapeptide is that the target lysine side chain in
MLL1 is not constrained. In the electron density maps the posi-
tions of the Caand Cb atoms are clearly defined, but the location
of density into which we are able to build two potential
orientations of the N(3)me2 group (Figure S3A). This freedom of
movement is not permitted in other, more active SET domain
methyltransferases, and in these the lysine side chain is in
a single clearly defined position (Xiao et al., 2003a; Zhang
et al., 2003).
The arrangement of the active site in MLL1 therefore, in
contrast to the sites of other characterized SET domains, and
despite high sequence conservation, is not in an optimal confor-
mation for methyl transfer. This accounts for the low activity that
Figure 3. Ordering of the MLL1 Active Site
(A) Overlay of the SET-I region and C-flanking
region of MLL1 (gray), and Dim-5 (yellow, PDB;
1PEG). Structures are superposed on their cofac-
tors and overlap on their C-flanking domains.
(B) Superposition of active site residues in the SET
domains of MLL1 and Dim-5 showing the
displacement of the channel tetrapeptide in MLL1.
(C) Reorientation of SET-I; surface representation
of the MLL1 ternary complex on the left showing
open configuration of the active site. On the right,
the MLL1 Set-I domain (green) has been modeled
to align with the position observed in Dim5.
(E) Methyltransferase activity of MLL1 in the pres-
ence of Ash2L-Dpy30 heterodimer Wdr5-RbBP5
heterodimer, Wdr5, RbBP5, and Ash2L-DPY30 +
Wdr5-RbBP5. Activity is represented as the
mean of triplicate measurements, with error bars
indicating one standard deviation.
of the MLL1
we observe in in vitro assays with the
isolated SET domain. We propose there-
fore that another protein binds to the SET
domain, promoting a shift in the position
of the SET-I region, and moves the
channel tetrapeptide into the equivalent
position to other SET domains. The effect
of this proposed reorientation of SET-I is
modeled in Figure 3C, in which a rigid
body movement of the SET-I aligns it to
the equivalent position of Dim-5. This
movement would complete the lysine-
binding channel, constrains the position
of the target side chain, and generates
The obvious candidate proteins that
might produce such a shift in SET-I
orientation are members of the MLL complex (Figure 3D). We
therefore determined the effect of introducing equimolar equiva-
lents of complex members into our minimal SET-domain-only
methyltransferase assay system (Figure 3E). Initially, we found
that both recombinant Ash2L-Dpy30 and Wdr5-RbBP5 hetero-
dimers, isolated from insect cells, promoted methyltransferase
activity. This suggests that Ash2L is able to interact directly
with the SET domain independently of Wdr5-RBP5. However,
the Wdr5-RbBP5 heterodimer promoted a greater enhancement
of activity. To separate the contribution of these two b propeller
proteins, we added the individual proteins to the assay. Surpris-
ingly, we found no enhancement of activity with Wdr5 but found
that RbBP5 gave the same enhancement of activity, approxi-
mately 6-fold, as the heterodimer. Previously it has been
suggested that Wdr5 might bridge the interaction with Ash2L
and was essential for methyltransferase activity (Dou et al.,
2006; Steward et al., 2006), and our data certainly do not
preclude a more extensive MLL1-Wdr5-Ash2L interface in the
Structure of the MLL1 SET Domain
Molecular Cell 33, 181–191, January 30, 2009 ª2009 Elsevier Inc. 185
context of full-length MLL1. Furthermore, the addition of
equimolar concentrations of Ash2L-Dpy30 and Wdr5-RbBP5
together leads to a nearly 20-fold enhancementof activity, which
is greater than the additive contribution of the two heterodimers
(Figure 3E). This indicates an important role for the intact
complex in the stabilization of the active MLL1 SET domain
with any of these proteins in our assay conditions (data not
The molecular mechanism by which the MLL complex
proteins contribute directly to the activity of the SET domain
will require further structural investigation. However, given the
conservation of essential catalytic features in MLL1, our anal-
ysis favors a mechanism that leads to reorientation of the
SET-I helix to properly form the lysine channel. We wished to
test if residues on the SET-I helix, not directly involved in the
formation of the catalytic site, could mediate changes in the
activity of the SET domain in the presence and absence of
complex and identified three candidate pairs of surface resi-
dues (Figure 4A). These residues were mutated to alanine,
and the methyltransferase activity against unmodified histone
peptide was determined with the SET domain construct alone
(Figure 4B). The intrinsic SET-domain-only methyltransferase
activity was reduced by mutations of residues on the outward
face of the SET-I helix, only slightly for pair A (Gln3867 and
Arg3871) near the open end of the substrate groove, and
more markedly for pair B (Tyr3874 and Lys3878). However,
even though the B mutants were apparently intrinsically
compromised, the methyltransferase activity was equivalent to
the wild-type in the presence of complex. For the A mutants,
however, the addition of complex only increased activity to
approximately 50% that of wild-type. The observation that
binding of complex is able to compensate for the effects of
the B mutations demonstrates that these do not affect the
optimal catalytic conformation, but instead push the isolated
domain toward a less active conformation. By contrast, the
less intrinsically disrupted A mutant cannot be fully activated
by the complex, and more likely this can be attributed to
reduced complex binding. The methyltransferase activity of C
mutations (Asp3869 and Glu3872) on the inside of the helix
Figure 4. Mutagenesis of the SET-I Helix
(A) Representation of the SET-I helix showing the
electrostatic surface potential, negative in red
and positive in blue. Three pairs of residues are
(B) Methyltransferase assay with unmodified
histone peptide for wild-type and SET-I mutant
proteins. Activity is represented as the mean of
triplicate measurements, with error bars indicating
one standard deviation.
to form surface
did not produce significant changes in
activity in assays with both the SET
domain only and with added complex.
These results are consistent with a view
that a fully active SET domain conforma-
tion is only achieved upon complex binding and that the interac-
tion of the complex with the SET-I region is important for this
Substrate Specificity and the Epigenetic Code
The substrate peptide binds in a defined cleft in MLL1; however,
it makes few specific side-chain contacts with MLL1 that could
account for substrate specificity (Figure 5A). This feature of there
being few determinants of substrate specificity is found not only
in other SET domain histone methyltransferases, but also in
recent structures of JmjC domain demethylase enzymes, which
recognize the same histone tails (Couture et al., 2007). In MLL1
the amino-terminal end of the peptide chain packs alongside
the edge of the b sheet made by the SET-I domain, which
extends from the Cys3882 to Phe3885 tetrapeptide discussed
above. Several main-chain-to-main-chain hydrogen bonds
anchor the peptide to the SET domain, including the carbonyl
and amine groups of the target lysine, a feature conserved in
other SET domains. The C-terminal end of the short peptide
used in crystallization adopts a helical conformation stabilized
by several intrapeptide hydrogen bonds. The main-chain
carbonyl of Arg8 makes a water-mediated hydrogen bond to
the main-chain amide of Ser3915. The cocrystallization peptide
had a C-terminal tyrosine (added to facilitate quantification),
which replaces the lysine 9 of histone 3. This tyrosine binds in
a well-defined pocket in MLL1 formed from Phe3885 and the
hydrophobic side chains Ile3887, Val3917, and Ile3926. We think
that this pocket accommodates the alkyl component of the
lysine in the native sequence and probably contributes to spec-
ificity. The side chains of two substrate residues, Gln5 and Arg8,
tron density for these side chains is relatively poorly defined
compared to the main chain of the peptide, and it would appear
they are able to adopt two conformations each in this complex,
both conformations having their own set of interactions
Physiologically, many of the residues close to lysine 4 are
themselves targets for posttranslational modification. These
include Thr3, which is phosphorylated by Haspin; Arg8, which
is methylated by PRMT5; and Lys9, which is methylated by
a range of enzymes, notably the Suv39 enzymes, and is
Structure of the MLL1 SET Domain
186 Molecular Cell 33, 181–191, January 30, 2009 ª2009 Elsevier Inc.
acetylated by, among others, GCN5 (Dai et al., 2005; Marmor-
stein, 2001; Pal et al., 2004; Rea et al., 2000). One component
of the epigenetic code is that modification enzymes should be
sensitive to the posttranslational marks left by other modifiers.
We therefore carried out a series of methyltransferase assays
with MLL1 using peptide substrates carrying modified residues
(Figure 5B). Activity was dramatically reduced for a peptide
substrate phosphorylated at position 3. This residue fits into
a defined pocket in the structure (Figure 5A), and although the
threonine hydroxyl does not hydrogen bond to MLL1, it is clear
that the residue would have to move in order to accommodate
the bulky negatively charged phosphate group. Repositioning
of Thr3 would affect the positioning of the adjacent target lysine
4 residue and accounts for the loss of activity. Symmetric
dimethylation of Arg8 reduced the observed methylation activity
by around 30%. As discussed above, Arg8 can adopt two
conformations in the ternary complex, and it is possible that
the methylation of this arginine could potentially favor one of
these. Interestingly, flexibility of the interaction with Arg8 was
also observed in the recent structure of the Ing4 PHD finger
bound to H3 peptide, but in this case Arg8 monomethylation
was shown not to significantly affect binding (Palacios et al.,
2008). Lysine 9 methylation also reduced activity by about
30% with respect to an unmodified peptide, whereas acetylation
of lysine 9 doubled the level of observed methylation. The
enhanced methylation of a H3K9 acetylated substrate may arise
from increased affinity for its binding pocket, as the charge
neutralization would favor a hydrophobic interaction.
The N-Flanking and PostSET Regions of MLL1
A striking feature of the overall structure is the N-terminal region,
which forms an extended tail stretching away from the globular
catalytic domain (Figure 1B). In fact, this tail is ordered by crystal
lattice contacts with the surface of the SET domain of the
adjacent molecule and even contacts the next but one molecule
(Figure S4). In the context of the full MLL1 protein, this region
would link the SET and adjacent FYRC domain, and it is likely
that it forms a flexible linker between them. Within the tail is
a short acidic helix (Figure 6B). We were unable to build residues
prior to Pro3793, and this region (including a short sequence
introduced by the vector) is presumed to be flexible within the
crystal lattice. The extended tail of MLL1 leads into a short
N-flanking domain comprising two small helices that pack
against a section of b sheet formed from two strands of SET-C
and one from SET-N. The two helices are linked by a sharp,
proline-mediated turn, with the shorter helix packing against
a loop from the SET-N region. All SET domain structures contain
an N-terminal flanking region, which is required for the structural
integrity of the SET domain. However, apart from a subset that
has a classical cysteine-rich preSET domain, there is significant
sequence and structural divergence within this region.
The C-flanking region of different SET domain families are
often divergent, but MLL1 fits within the largest subset, which
have what may be termed a classical Su(var)-like postSET
domain (Dillon et al., 2005). The prominent feature of this domain
is a zinc-binding cage formed from three cysteine residues from
the C-terminal region with the fourth tetrahedral cysteine ligand,
Cys3909, provided by the loop linking the SET-I and SET-C
regions of the SET domain (Figure 6A). This ZnCys4 cage pins
the C-flanking domain to the main body of the SET domain,
and disruption of the cage by mutation of this cysteine to alanine
resulted in an insoluble protein (data not shown). Given the
conservation of the position of the postSET region with those
of Dim-5 and Suv39h2, it seems unlikely that this region is
involved in the active site-ordering mechanism (Figure S2). In
the MLL1 ternary complex we were able to build a complete
canonical postSET region. However, despite the ZnCys4 cluster
atoms themselves being very prominent, the electron density for
residues in this area is poorly defined compared to the rest of the
Figure 5. Substrate Binding
(A) Details of the residues involved in binding of the substrate peptide. Water
molecules are shown as red spheres, and hydrogen bonds are shown as
(B) Methyltransferase activity of the isolated MLL1 SET domain with substrate
peptides carrying physiologically relevant posttranslational modifications.
Activity is represented as the mean of triplicate measurements, with error
bars indicating one standard deviation.
Structure of the MLL1 SET Domain
Molecular Cell 33, 181–191, January 30, 2009 ª2009 Elsevier Inc. 187
protein. This suggests that, although fixed at the ZnCys4 cluster,
the restof the postSET loop isrelatively flexible. Indeed, wewere
unable to build the region between Phe3946 through Asn3953 in
the binarycomplex.This isreminiscent of thepostSET domainof
Dim-5 (Zhang et al., 2003) and the related mammalian protein
Suv39h2 (unpublished data; PDB entry 2R3A). This flexibility
may aid substrate and cofactor docking and undocking. The
surface of the MLL1 SET domain has several basic and acidic
patches that could potentially be binding sites for interaction
partners (Figure 6B); in particular there are two acidic lobes,
which form the entrance to the groove in which the substrate
peptide binds. These arise from the edge of the postSET domain
and the SET-I helix on the opposite sides of the cleft. It is
tempting to speculate that these could potentially form an inter-
action surface with either substrate nucleosomes or members of
the MLL complex.
The AdoHcy moleculein
conformation that is a characteristic of SET domain proteins
and differs from the linear conformation associated with other
methyltransferases (Xiao et al., 2006). The AdoHcy binding
site is a surface pocket (Figure 1B), and the cofactor makes
an extensive set of interactions with the protein (Figure 6C).
Many of these interactions are highly conserved, for example,
hydrogen bonds from the side chain of Asn3906 and main chain
of His3907 of the ‘‘NHS’’ motif in the SET-C region to the
adenine ring and Tyr3883 in the SET-N, which links cofactor
binding to the lysine binding region. The AdoHcy adenine ring
MLL1 adoptsa U-shaped
(A) Representationofthe Cys(4)Zn cage formed by
three residues from the postSET domain and one
from the SET-C region. Bond distances between
the cysteine sulfur and Zn ion are indicated in
(B) Surface view of the MLL1 SET domain showing
the surface potential and illustrating the peptide-
binding groove and cofactor-binding pocket.
Areas of negative potential are in red, and areas
of positive potential are in blue.
(C) Details of cofactor binding. Residues involved
in AdoHcy binding are shown in stick representa-
tion colored according to Figure 1A, and key
hydrogen bonds are indicated by a red dotted line.
6. The C-FlankingDomainand
does not stack against an aromatic
residue, as in many other SET domains,
but instead sits between two hydro-
phobic side chains, Ile3838 on one side
and Leu3698. There is also a hydrogen
bond to the amine of Asn3858, which
links the cofactor to the ZnCys4 cluster.
Effectively, the cofactor interacts with
all regions of the SET domain, including
those involved in substrate binding. The
dynamics involved in the breaking of
these interactions in order to release
AdoHcy to allow binding of a new
AdoMet molecule remain to be resolved for MLL1 and other
SET domain methyltransferases.
Notwithstanding both sequence and structural conservation of
MLL1 with respect to other SET domain proteins, the current
structure points toward a mechanism of allosteric control that
regulates MLL1 activity. The requirement for additional factors
from the MLL complex to correctly order the active site means
that the full methyltransferase activity will only be achieved
when correctly targeted to an appropriate chromatin region by
the complex. Deletion of individual yeast COMPASS members
or downregulation of mammalian MLL complex members has
been shown to reduce the observed H3K4 trimethylation of
histones isolated from those cells (Dehe et al., 2006; Krogan
et al., 2002; Schneider et al., 2005; Steward et al., 2006;
Wysocka et al., 2005). Ash2L or its yeast counterpart (Bre2/
Cps60) has in several reports been shown to be required for
the H3K4 trimethyl activity of MLL1/Set1, and loss of Wdr5 or
RbBP5 has been shown to reduce the overall activity of MLL1
(Dou et al., 2006; Steward et al., 2006). While Ash2L, RbBP5,
and Wdr5 have all been to shown to contribute to the overall
activity of MLL1, it is not always possible to separate the effects
on targeting or complex integrity from direct interaction with the
SET domain. In our assays, as only a minimal SET domain
construct is used, the enhanced activity must arise from direct
Structure of the MLL1 SET Domain
188 Molecular Cell 33, 181–191, January 30, 2009 ª2009 Elsevier Inc.
binding to the SET domain. Full-length MLL1 will make more
extensive contacts with the core complex through other
domains, and thus more effectively stabilize an active conforma-
tion. There is an apparent discrepancy between the role of Wdr5
in promoting methylation in our assay system and that reported
previously (Dou et al., 2006; Steward et al., 2006). This is
resolved by two recent reports showing the structure of Wdr5
in complex with a short MLL1-derived peptide (Patel et al.,
2008; Song and Kingston, 2008). In these the MLL1 peptide is
bound in what was previously shown to be the histone
peptide-binding site on Wdr5 (Ruthenburg et al., 2006; Schuetz
et al., 2006). The peptide corresponds to a region of MLL1 that
is amino terminal to the construct used in our studies and is
consistent with a view that Wdr5 does not directly affect the
catalytic mechanism of MLL1 but is required for complex
integrity. Further elucidation of the mechanisms underlying the
direct regulatory control of the MLL1 SET domain will become
clearer with the characterization of the sites of interaction of
Ash2L and RbBP5 with the SET domain. Trimethylation activity
was particularly weak in our assay system. We note that for
a recombinantly expressed intact complex from insect cells,
the trimethylation activity was also lower than for dimethylation
(Dou et al., 2006). We are currently unable to account for the
levels of trimethylation we have observed in assays.
In addition to regulatory control by the MLL complex, our
in vitro data show that MLL1 activity issensitive to the epigenetic
landscape on the histone tail to which it has been targeted. Prior
posttranslational modification of Thr3 and Lys9 had the most
significant effects on the activity of the isolated MLL1 SET
domain. In the present structure the Arg2 substrate side chain
and amino-terminal alanine were not visible, implying flexibility
in this region of peptide. An antagonistic relationship between
Arg2 methylation by PRMT6 and H3K4 methylation has been
shown previously (Guccione et al., 2007; Hyllus et al., 2007),
and we also saw no activity with an asymmetrically dimethylated
Arg2 peptide in our assay system (data not shown). A conse-
quenceof reorienting theSET-I domainintoa positionequivalent
to that seen in other SET domains would be to bring it closer to
the amino-terminal residues of the histone substrate. This could
increase the contribution of these residues in the enzyme/
substrate interaction. The residues from H3K4 onward were
not affected in our modeling. Synergy or antagonism between
H3T3 phosphorylation and H3K4 methylation is not currently
well documented. However, it was found that H3K4me3 binding
bythe tandem chromodomains of CHD1 was greatly reduced for
a peptide also carrying a H3T3ph modification (Flanagan et al.,
2005), and H3T3 phosphorylation by Mut9p kinase in Clamydo-
monas was shown to be antagonistic to H3K4me2 and
H3K4me3 modifications (Casas-Mollano et al., 2008). A binary
switch, involving phosphorylation and methylation of adjacent
residues, has been shown for H3K9 and H3S10, with phosphor-
ylation of the latter by Aurora B triggering the release of HP1
bound to H3K9me3 at the onset of mitosis (Dormann et al.,
2006). A similar binary switch may operate through H3T3 phos-
phorylation and H3K4 methylation.
Both H3K4 methylation and H3K9 acetylation are broadly
tion that H3K9 acetylation promotes H3K4 methylation by MLL1
is in keeping with the expectation that such marks should be
synergistic. In our assay system the stimulation of H3K4 methyl-
ation by prior H3K9 acetylation is greater than the inhibitory
effect of prior H3K9 methylation. It is tempting to postulate
that, in an epigenetic context, H3K9 acetylation might drive
H3K4 methylation and thus promote active transcription. In
a physiological context MLL1 presumably also makes further
interactions with chromatin substrate. The combinatorial effects
of targeting and direct regulatory control by the complex
combined with the sensitivity of the SET domain to other histone
modifications combine to make MLL1 a very specific enzyme.
The advantage of this to the cell is to ensure that H3K4 methyl-
ation and its downstream effects are under tight regulatory
Protein Expression and Purification
Human MLL1 constructs were subcloned into the vector pTHREE-E (in-house
GST-fusion vector modified to contain the multiple cloning site of pET-17b,
following an encoded rhinovirus 3C-protease site; AW. Oliver Institute of
Cancer Research). Expression was carried out at 30?C in LB media supple-
mented with 10 mM ZnSO4for 5 hr following induction with 0.5 mM IPTG. Cells
were lysed and purified on glutathione Sepharose affinity resin (Generon),
separated from the GST tag by cleavage with rhinovirus 3C protease, and
finally purified by gel filtration (GE Healthcare, Superdex 200). The purification
buffer was 50 mM Tris (pH 7.5), 500 mM NaCl, 2 mM 2-mercaptoethanol, 10%
v/v glycerol. Note that there is 100% sequence identity between mouse and
human MLL1 for the construct used.
Full-length (residues 1–538) mouse RbBP5 was expressed as 6xHis-fusion
proteins in S. frugiperda (Sf9) and purified on Ni-NTA agarose (QIAGEN),
followed by anion exchange (Mono-Q, GE Healthcare) and gel filtration
(GE Healthcare, Superdex 200). Mouse WDR5 was expressed as GST-fusion
proteins in E. coli BL21 (DE3) RIL cells and purified on glutathione resin
(Generon). The GST tag was subsequently removed using 3C protease, and
the protein was subjected to a final gel filtration step. 6xHis-RbBP5-WDR5
and 6xHis-ASH2L-DPY-30 complexes were expressed in Sf21 cells and puri-
fied on Ni-NTA agarose (QIAGEN), followed by gel filtration (GE Healthcare,
Superdex 200), anion exchange (Mono-Q, GE Healthcare), and a second gel
filtration step. Final purification buffer consisted of 40 mM HEPES (pH 7.5),
10 mM 2-mercaptoethanol, 150 mM NaCl, and 10% glycerol in the case of
6xHis-ASH2L-DPY-30 and 50 mM NaCl for RbBP5-Wdr5.
Initial crystallization trials where carried out at 4?C with a 600 mM protein
solution containing 1.2 mM AdoHcy. Trials were set up with 200 nl drops using
a Phoenix crystallization robot (Alpha Biotech, UK), with initial needles
detected in condition 85 of the JSCG+ suite (QIAGEN). Crystallization was
optimized manually, and the final reservoir solution used was 0.1 M sodium
cacodylate (pH 6.5), 2% w/v PEG 8000, 30% v/v 2-methyl-2,4-pentanediol
(MPD) for both MLL1 SET domain binary complex and ternary complex. The
ternary complex contained a 9-mer synthetic peptide based on the amino-
terminal tail of histone H3, ARTKQTARY carrying a dimethyl group on lysine
at position 4 (University of Bristol). Crystals were frozen in a cryoprotectant
consisting of 30 v/v MPD, 0.1 M sodium cacodylate (pH 6.5).
Data Collection and Processing
Data were collected at the Diamond Light Source (Diamond Light Source Ltd,
binary and ternary complexes crystallized inspace groupP212121withasingle
protein molecule in the asymmetric unit. Data were indexed using the program
Mosflm (Leslie, 1992) and reduced/scaled with programs from the CCP4i suite
(Bailey, 1994). The structure of the binary complex was solved by molecular
replacement using the program Phenix (Adams et al., 2002) using conserved
Structure of the MLL1 SET Domain
Molecular Cell 33, 181–191, January 30, 2009 ª2009 Elsevier Inc. 189
elements (SET-N, SET-C, and postSET) of the Dim-5 structure (PDB entry
1PEG) as the search model. Difference maps were used to rebuild and extend
the initial model using the program Coot (Emsley and Cowtan, 2004). Iterative
cycles of refinement were carried out using the phenix-refine module within
Phenix or Refmac within CCP4. Structural figures in this manuscript were
preparedusingthePyMOLmolecularvisualizationpackage (DeLano Scientific
Methyltransferase assays were performed using synthetic peptides based on
the sequence (ARTQTARYKSTGGKAPRY) with posttranslational modifica-
tions as indicated in the text (University of Bristol and Centre for Cancer Ther-
apeutics at ICR). Reactions followed the incorporation of tritiated S-adenosyl-
methionine (GE Healthcare) by separating peptide from the unincorporated
AdoMet using ‘‘Sep Pak’’ C18 cartridges (Waters) followed by scintillation
counting as described previously (Xiao et al., 2005). Final peptide concentra-
tion was 1 mM, AdoMet 0.5 mM (including 0.625 mM3H AdoMet), and the
assay buffer was 40 mM HEPES (pH 7.5) and 25 mM NaCl. Assays were
carried out at 22?C for 1 hr with a final enzyme concentration of 50 mM. Assays
with complex members were performed as above but with a final equimolar
concentration of both MLL1 and complex members of 25 mM. The final NaCl
concentration for assays with heterodimers was 78 mM and with individual
members was 148 mM. MLL1 SET domain did not show sensitivity to NaCl
concentration over a range of 0–300 mM NaCl (data not shown). All assays
were carried out in triplicate and expressed as mean ± SD.
The coordinates for the binary complex of MLL1 SET domain with AdoHcy and
ternary complex with AdoHcy and peptide have been deposited with the PDB
with accession numbers 2W5Y and 2W5Z, respectively.
The Supplemental Data include four figures and can be found with this
article onlineat http://www.cell.com/molecular-cell/supplemental/
The authors wish to thank LaVerne Rennalls for assistance with baculovirus
expression in insect cells, A.W. Oliver for assistance with data collection and
colleagues for critical reading of the manuscript. This work was supported
by the Career Development Faculty Programme of The Institute of Cancer
Research (to J.R.W), and benefits from infrastructural support for structural
biology at ICR by Cancer Research UK. Z.O. is supported by a studentship
from the Medical Research Council. We acknowledge NHS funding to the
NIHR Biomedical Research Centre.
Received: July 9, 2008
Revised: November 20, 2008
Accepted: December 30, 2008
Published: January 29, 2009
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