Strategy for "Detoxification" of a cancer-derived histone mutant based on mapping its interaction with the methyltransferase PRC2

Article abstract of DOI:10.1021/ja5060934

The histone methyltransferase PRC2 plays a central role in genomic stability and cellular development. Consequently, its misregulation has been implicated in several cancers. Recent work has shown that a histone H3 mutant, where the PRC2 substrate residue Lys27 is replaced by methionine, is also associated with cancer phenotypes and functions as an inhibitor of PRC2. Here we investigate the mechanism of this PRC2 inhibition through kinetic studies and photo-cross-linking. Efficient inhibition is dependent on (1) hydrophobic lysine isosteres blocking the active site, (2) proximal residues, and (3) the H3 tail forming extensive contacts with the EZH2 subunit of PRC2. We further show that naturally occurring post-translational modifications of the same H3 tail, both proximal and distal to K27M, can greatly diminish the inhibition of PRC2. These results suggest that this potent gain of function mutation may be "detoxified by modulating alternate chromatin modification pathways.

Full text of DOI:10.1021/ja5060934

Communication
pubs.acs.org/JACS
Strategy for “Detoxification” of a Cancer-Derived Histone Mutant
Based on Mapping Its Interaction with the Methyltransferase PRC2
Zachary Z. Brown,†,∥ Manuel M. Muller,†,∥ Siddhant U. Jain,‡ C. David Allis,§ Peter W. Lewis,‡
̈
and Tom W. Muir*,†
†Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States
‡Epigenetics Theme, Wisconsin Institute for Discovery, University of Wisconsin, Madison, Wisconsin 53715, United States
§Laboratory of Chromatin Biology & Epigenetics, The Rockefeller University, New York, New York 10065, United States
S
* Supporting Information
currently unknown. Finding a way to restore K27me3 to the rest
ABSTRACT: The histone methyltransferase PRC2 plays
a central role in genomic stability and cellular develop-
ment. Consequently, its misregulation has been implicated
in several cancers. Recent work has shown that a histone
H3 mutant, where the PRC2 substrate residue Lys27 is
replaced by methionine, is also associated with cancer
phenotypes and functions as an inhibitor of PRC2. Here
we investigate the mechanism of this PRC2 inhibition
through kinetic studies and photo-cross-linking. Efficient
inhibition is dependent on (1) hydrophobic lysine
isosteres blocking the active site, (2) proximal residues,
and (3) the H3 tail forming extensive contacts with the
EZH2 subunit of PRC2. We further show that naturally
occurring post-translational modifications of the same H3
tail, both proximal and distal to K27M, can greatly
diminish the inhibition of PRC2. These results suggest that
this potent gain of function mutation may be “detoxified”
by modulating alternate chromatin modification pathways.
of the genome would be a strategy to mitigate the consequences
of this deleterious mutation. The intricacies of PRC2 regulation,
along with the frequency by which its activity is perturbed in
human pathologies, create urgency in understanding how
substrates and inhibitors interact with this multisubunit enzyme
complex. Here we present a detailed biochemical investigation
into how PRC2 recognizes the mutated H3 tail. A comprehensive
structure−activity relationship (SAR) analysis of the H3 tail
revealed key orthosteric and allosteric contributions to binding
and recognition by PRC2. Photo-cross-linking studies served to
identify the subunits of PRC2 responsible for H3 tail recognition.
Finally, we show that inhibition of PRC2 can be significantly
diminished by post-translational modifications (PTMs) on the
same H3 peptide, providing a potential mechanism for how these
cancer-derived H3 mutations might be overcome.
We began by characterizing the inhibition of PRC2 activity by
H3K27M mutant mononucleosomes. Wild-type and H3K27M
nucleosomes were assembled using purified recombinant histone
proteins and a strong nucleosome positioning DNA sequence
(Widom-601).10 Methyltransferase activity was measured by
scintillation counting upon incubation of wild-type nucleosome
substrates with 3H-containing S-adenosylmethionine (3H-SAM)
in the presence of PRC2, purified from HeLa cells (Figure 1A, see
Supporting Information for details). In agreement with previous
measurements,11 an apparent Km value (Km,app) of 67 26 nM
was obtained for the wild-type nucleosome substrate (Figure S1).
H3K27M-containing nucleosomes inhibited PRC2 with KI = 2.1
0.9 nM. Under these conditions, the mechanism of inhibition
appears competitive (vs mononucleosome substrates), indicated
by a linear dependence of the K27M IC50 values on the
concentration of substrates (Figure 1B).12,13 In contrast, the IC50
value of K27M-nucleosomes was independent of the concen-
tration of SAM, consistent with noncompetitive inhibition (vs
SAM, Figure S1C). These values are in agreement with the strong
inhibition of PRC2 activity observed in tumor tissues, despite the
overwhelming excess of wild-type histones over mutant
congeners. While H3K27M mononucleosomes are remarkably
potent inhibitors, with a molecular mass of almost 200 kDa, they
have many sites of potential interaction with PRC2. Therefore,
ovalent modifications of chromatin facilitate the dynamic
Corganization of eukaryotic genomes and fine-tuning of
transcriptional outputs.1 Proteins that install or remove func-
tional groups, or specifically recognize modified chromatin,
mediate downstream biochemical processes and are essential for
cell growth, homeostasis, and lineage commitment. Conse-
quently, misregulation of chromatin-associated proteins is
frequently correlated with disease states. In particular, mutations
altering EZH2, the catalytic subunit of the polycomb repressor
complex 2 (PRC2), are often found in cancers where they
interfere with PRC2’s role in gene silencing.3 At the molecular
level, PRC2 functions by methylating Lys27 within the N-
terminal tail region of histone H3.4 Intriguingly, Lys27 is
frequently mutated to methionine (H3K27M) in a subpopula-
tion of histone H3 in pediatric glioblastomas.5,6 Despite
representing only a few percent of the total H3 pool in glioma
cells, H3K27M is able to strongly diminish global levels of H3K27
methylation by directly binding to PRC2.7−9 Paradoxically
though, an analysis of the chromatin landscape in K27M-carrying
tumor tissues revealed that small regions of the genome escaped
inhibition.8,9 These islands of K27me3 probably contribute to the
mechanism of K27M-mediated pathogenesis, but how certain
regions overcome the inhibitory effects of the K27M mutation is
Received: June 17, 2014
Published: September 2, 2014
© 2014 American Chemical Society
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inhibition of methyltransferase activity using a scintillation assay
3
containing PRC2, H-SAM, and a substrate peptide (20 μM).
Under these conditions, the K27M peptide (1) inhibited
methyltransferase activity by 53% 7%. Norleucine (2) as well
as (S)-aminoheptanoic acid (3) exhibited complete inhibition. In
a full IC50 determination, peptides 1 and 2 had respective IC50
values of 56 7 and 3.3 0.5 μM (Figure S2A). Shortening the
carbon chain by one methylene group to norvaline (peptide 5)
provided a modest loss in activity. Some branching aliphatic side
chains (peptides 6 and 9) may be tolerated, although provide no
improvement over the more potent straight chain aliphatics (5
and 2, respectively). Other branching geometries (7 and 8), as
well as the peptide which included (R)-aminoheptanoic acid (4),
did not provide measurable inhibition.
We further sought to assess the effect of heteroatom
substitution within the side-chain. To probe whether cation−π
interactions could be harnessed with various lysine analogs, we
synthesized several diamine building blocks (Table 1). However,
the corresponding series of peptides showed little to no inhibition
(peptides 10−15). We therefore resorted to less polar
heteroatom containing side-chains. Conversion of the thioether
moiety of K27M to an ether (peptide 16) resulted in modest loss
of inhibition. Oxidation of methionine to the sulfoxide (peptide
17) abolished inhibition. Trifluoro-methionine (peptide 18)
inhibited PRC2 substantially better than methionine (IC50 of 11
2 μM for 18 vs 56 7 μM for 1, Figure S2A). Methionine
analogs with diverse steric properties (peptides 20−25) were
generated through chemoselective alkylation of thiols at the
peptide level (Scheme S2). Both cysteine and homocysteine were
derivatized with various π-electron-containing electrophiles.
Although several of the peptides (21−24) provided measurable
inhibition, none were a significant improvement over the
aliphatic entries 2 or 3. Thus, the EZH2 active site binds strongly
to linear, hydrophobic side chains with little tolerance to extra
steric bulk and polar groups.
Figure 1. Model for PRC2 activity and inhibition. (A) Schematic
representation of PRC2 activity. (B) Cheng−Prusoff analysis of the
mechanism of PRC2 inhibition by K27M. IC50 values were determined
at varying concentrations of unmodified mononucleosome substrates.
(C) Homology model of the EZH2 active site (cyan) bound by a
methionine residue (white). The cofactor SAM is depicted in black. (D)
Molecular architecture of PRC2; adapted from Ciferri et al. (EMD-
2236).2
we sought to dissect the various contributions of allosteric and
orthosteric inhibitor binding to PRC2.
SET domain methyltransferases, including EZH2, bind the
hydrophobic (alkyl) portion of the lysine side chain in an
aromatic cage (Figure 1C).14 Additionally, the active site utilizes
cation−π interactions to recognize and distinguish its substrates:
unmethylated and mono- and dimethylated lysine 27. A
hydrogen-bond donor deep in the pocket assists in aligning the
ε-amine for nucleophilic attack on the methyl donor SAM.
Methionine and to a lesser extent isoleucine are able to bind in
this pocket and inhibit PRC2 in vivo, suggesting that recognition
of a hydrophobic moiety is a primary factor for potent inhibition.7
Further in vitro work established norleucine (Nle) as a more
potent methionine isostere.7 To gain more insight into the steric
and electronic factors governing inhibitor binding to the EZH2
active site, we designed a series of short peptide constructs
(residues 23−34 of the H3 variant H3.3) that differed only in
residue 27 (Table 1). Each peptide (50 μM) was assayed for
To probe whether distal regions of the H3 tail are also
recognized by the PRC2 complex, we prepared a range of
truncated H3 peptides bearing norleucine at position 27 (Figure
2A). Peptide 26, encompassing residues 1−37 was left with a free
N-terminus to reflect the biologically prevalent form of the H3
tail, while truncated constructs were acetylated at their N-termini.
IC50 values were calculated from methyltransferase assays using
a
Table 1. SAR of PRC2 Inhibition by H3.3 Peptides (residues 23−34) Bearing Substitutions at the Orthosteric Residue K27
a
Peptide sequence: KAARXSAPSTGG; where X is varied as indicated. The cancer-derived methionine mutant is boxed. Blue indicates >50%
inhibition of PRC2 activity; black denotes an intermediate level of inhibition, red indicates no measurable inhibition. S.D. is one standard deviation.
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Figure 2. Molecular recognition of the H3 tail by PRC2. (A) Schematic view of truncated H3 tail segments used in (B), bitopic constructs used in (C),
and the cross-linking constructs used in (D). Solid lines indicate peptide segments; red dots indicate the orthosteric inhibitor residue, Nle; dotted lines
represent artificial linkers; blue squares represent a cysteine residue; yellow stars a Bpa residue; green triangles a biotin moiety. (B) Residues along the
entire H3 tail contribute to PRC2 binding. IC50 values were determined for peptides 26−34 and plotted as a function of peptide length; n = 2−3, error of
the fit is indicated. (C) PRC2 binds the H3 tail in a compact fashion. IC50 values were determined for indicated peptides; n = 2−3, error of the fit is
indicated. (D) EZH2 engages the entire H3 tail. PRC2 was photo-cross-linked with the indicated peptides. Cross-linked samples were enriched by
streptavidin pull-down and detected by Western blot.
H3(23−34) peptide substrates (Figure 2B). In agreement with
previous results, a peptide encompassing residues 18−37 (29)
displayed an IC50 = 1.6 μM.7 Truncations of this construct lead to
a small decrease (peptides 31, 33, 34) or even a modest increase
(peptide 30) in inhibitor potency. Excessive shortening of the
peptide (32) abolished measurable inhibition. In contrast, longer
peptide constructs bind more strongly to PRC2. Indeed, the full
length tail H3(1−37)K27Nle (peptide 26) potently inhibits
methylation of both peptide and mononucleosome substrates
(Figures 2B and S2B), the latter with an IC50 = 4.7 1.5 nM
which rivals the potency of H3K27M nucleosomes (IC50 = 2.6
0.5 nM). The steady increase in H3 tail binding as a function of
peptide length suggests that PRC2 engages residues along the
entire length of the histone tail (Figure 2B). In particular, residues
surrounding Lys27 are critical, and the N-terminal 10 residues
contribute substantially to inhibition. As peptides 28 (residues
11−37) and 30 (residues 21−37) display similar potency,
intervening residues appear to contribute little binding energy.
To investigate whether the binding of the H3 tail to PRC2 is
bivalent in nature, i.e., involving residues 1−10 and 21−37, we
designed a series of bitopic peptide constructs that encompassed
these two segments (Figure 2A). Polyethylene glycol chains of
various lengths (compounds 35−38), as well as a Gly3 peptide
(39) were used as conformationally flexible, yet defined linkers
between the two peptide segments. In a methyltransferase assay
which used mononucleosomes as substrates, we found that
bitopic display of H3(1−10) and H3(21−37) is favorable
compared to adding both peptides in trans (Figure 2C).
Moreover, short, flexible linkers (38, 39) outperformed longer
linkers (35−37) and the zero-length linker (40). The IC50 for
compound 38 was found to be 71 nM (Figure S2B), only a 15-
fold loss of potency from the full length 1−37 peptide (IC50 of 4.7
nM). These results imply a compact binding model for the two
distal H3 tail segments.
We next analyzed the trajectory of H3 binding to PRC2
through a cross-linking approach. p-Benzoyl-phenylalanine
(Bpa) residues were strategically placed within the H3 tail
(Figure 2A) at positions 1 (41), 11 (42), and 20 (43),
complementing previous experiments7 with photo-cross-linkers
installed at residues 27 and 31 which showed that this region of
the H3 tail specifically interacts with the EZH2 subunit. Peptides
41−43 proved to be potent inhibitors of PRC2 (Figure S3A).
PRC2 subunits (Figure 1D) interacting with the distinct H3 sites
were identified upon cross-linking based on either the mobility of
the cross-linked species as judged by SDS-PAGE (Figure S3B) or
upon streptavidin pulldown followed by Western blot for specific
PRC2 components (Figure 2D). All cross-linked species
comigrate with EZH2 (Figure S3B), and EZH2 is the sole
subunit identified upon isolation of cross-linked products (Figure
2D). As expected, the addition of wild-type mononucleosome
reduces the cross-linking signal by competing for PRC2 binding
(Figure S3B). Taken together with the previous cross-linking
studies,7 these results provide a map of the interaction of PRC2
with the H3 tail containing the cancer-derived K27M mutant.
EZH2 engulfs the entire mutant H3 tail, presumably in a compact
binding orientation, with key contributions from sites proximal to
residue 27 and at the N-terminus.
Through its accessory subunits, PRC2 is able to precisely sense
chromatin states and integrate such signals into its methyl-
transferase activity output.15 For instance, PRC2 is subject to a
positive feedback loop where K27me3 modifications allosteri-
cally activate EZH2 through the EED subunit.16 In addition,
trimethylation of Lys4 of H3a PTM associated with active
chromatin regionsdiminishes PRC2 activity.17 We therefore
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures, analytical data, supporting schemes,
and figures. Full citations for references 5, 6, 9, 11 and 17. This
material is available free of charge via the Internet at http://pubs.
acs.org.
AUTHOR INFORMATION
Corresponding Author
*muir@princeton.edu
■
Author Contributions
Figure 3. Histone PTMs can detoxify K27M mutants. (A) Reduction of
Lys27Nle-dependent inhibition of PRC2 activity by PTMs in vitro. (B)
Immunoblots of whole-cell extract from lentivirus-transduced
HEK293T cells expressing the indicated HA-tagged H3.3 transgenes.
The phosphorylation mimic S28E diminishes K27M-dependent PRC2
inhibition in vivo.
∥These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Lotus separations for purification of Fmoc-2-
aminoheptanoic acid, Ha-Eun Kong for help with peptide
synthesis, Dr. Lenka Bittova for providing p300, and members of
the Muir and Allis laboratories for valuable discussions. This
research was supported by the U.S. National Institutes of Health
(grants R37-GM086868 and R01 GM107047) and the STARR
foundation (grant I6-A614).
hypothesized that PRC2 inhibition may be similarly affected by
PTMs occurring at binding hotspots. To test this idea, we
synthesized peptides containing known activating PTMs within
H3. Trimethylation at Lys4 (peptide 44) reduced inhibitor
potency ∼4-fold, whereas asymmetric dimethylation at Arg26
(peptide 45)18 diminished inhibition ∼30-fold (Figures 3A, S4).
Polyacetylation of Lys27Nle inhibitor constructs (peptide 46,
acetylated on Lys residues 9, 14, 18, and 23) dramatically
impaired Prc2 inhibition, in this case over 80-fold. Notably,
K27M nucleosomes are readily acetylated by the histone
acetyltransferase p300 (Figure S4C). Finally, phosphorylation
at Ser28 (peptide 47)19 reduced the inhibitor potency ∼12-fold.
These results demonstrate that PRC2 inhibition by K27M can be
reduced through the deposition of specific histone PTMs within
the same tail, in analogy to binary switches observed in many
histone modification crosstalks.20
We were intrigued by the possibility that histone PTMs may
also be able to detoxify the K27M mutation in vivo. This
hypothesis is most easily tested for phosphoserine since this
residue can be mimicked by a Ser to Glu mutation. In vitro, the
K27Nle/S28E double mutant (peptide 48) displayed reduced
inhibitor potency compared to the K27Nle mutant (Figures 3A
and S4B). To extend these results in vivo, we transduced HEK
293T cells with lentiviruses containing wild-type histone H3.3,
K27M, S28E, or the double mutant K27M/S28E and compared
the level of K27me2 and K27me3 in the resulting cell lines
(Figures 3B and S4D). In agreement with our in vitro
measurements, the phosphoserine-mimetic S28E partially
rescued the formation of K27me2 and K27me3 modifications
that are suppressed by the presence of K27M. These results argue
that PTMs can strongly attenuate K27M-dependent loss of K27
methylation, perhaps contributing to the existence of K27me3-
rich chromatin regions in K27M-containing tumors.
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