The Histone Mark H3K36me3 Regulates
Human DNA Mismatch Repair
through Its Interaction with MutSa
Feng Li,1Guogen Mao,1Dan Tong,2,3Jian Huang,2Liya Gu,1,* Wei Yang,4and Guo-Min Li1,3,*
1Graduate Center for Toxicology, Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY 40506, USA
2College of Life Sciences, Wuhan University, Wuhan 430072, China
3Tsinghua University School of Medicine, Beijing 10084, China
4Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda,
MD 20892, USA
*Correspondence: firstname.lastname@example.org (L.G.), email@example.com (G.-M.L.)
DNA mismatch repair (MMR) ensures replication
fidelity by correcting mismatches generated during
DNA replication. Although human MMR has been
reconstituted in vitro, how MMR occurs in vivo is
unknown. Here, we show that an epigenetic histone
mark, H3K36me3, is required in vivo to recruit the
mismatch recognition protein hMutSa (hMSH2-
hMSH6) onto chromatin through direct interactions
with the hMSH6 PWWP domain. The abundance of
H3K36me3 in G1 and early S phases ensures that
hMutSa is enriched on chromatin before mispairs
are introduced during DNA replication. Cells lacking
microsatellite instability (MSI) and an elevated spon-
taneous mutation frequency, characteristic of MMR-
deficient cells. This work reveals that a histone mark
regulates MMR in human cells and explains the long-
detectable mutations in known MMR genes.
DNA mismatch repair (MMR) maintains genome stability pri-
marily by correcting base-base and small insertion-deletion (ID)
mispairs generated during DNA replication (Kolodner, 1996;
Kunkel and Erie, 2005; Li, 2008; Modrich and Lahue, 1996). In
human cells, these mispairs are recognized by hMSH2-hMSH6
(hMutSa) and hMSH2-hMSH3 (hMutSb). Normally, cells express
more hMSH6 than hMSH3, leading to a hMutSa:hMutSb ratio of
?10:1 (Drummond et al., 1997; Marra et al., 1998). Despite their
redundant activities in mismatch recognition, both complexes
are required for MMR, and defective or abnormal expression of
hMSH6 or hMSH3 leads to a mutator phenotype (Drummond
et al., 1995, 1997; Harrington and Kolodner, 2007; Marsischky
et al., 1996). Previous studies have shown that genetic and
epigenetic modifications that impair the expression of these
and other MMR genes, especially hMSH2, hMSH6, and
hMLH1, cause susceptibility to certain typesof cancer, including
hereditary nonpolyposis colorectal cancer (HNPCC) (Fishel and
Kolodner, 1995; Kane et al., 1997; Modrich and Lahue, 1996).
At the cellular level, defects in MMR cause a mutator pheno-
type, which can be readily detected in eukaryotic cells as insta-
bility in simple repetitive DNA sequences called microsatellites.
Thus, microsatellite instability (MSI) is regarded as a hallmark
of MMR deficiency (Kolodner, 1996; Kunkel and Erie, 2005; Li,
2008; Modrich and Lahue, 1996). However, a significant fraction
of MSI-positive colorectal cancers express MMR genes at
normal levels and do not carry a detectable mutation in or hyper-
methylation of known MMR genes (Peltoma ¨ki, 2003). Similarly,
certain noncolorectal cancer cells with MSI also appear to
be proficient in MMR (Gu et al., 2002; Wang et al., 2011). The
molecular mechanism underlying MSI in these cases is obscure.
The MMR capacity of mammalian cells has typically been
evaluated using a functional assay that measures in vitro repair
of a naked model DNA heteroduplex (Holmes et al., 1990;
Thomas et al., 1991; Zhang et al., 2005). This assay has helped
identify MMR defects in HNPCC and other MSI-positive cancers
(Parsons et al., 1993; Umar et al., 1994) and has been invaluable
in characterizing the MMR pathway in human cells in great
molecular detail (reviewed by Li, 2008). However, increasing
evidence suggests that a mismatch assembled into nucleo-
somes is a poor substrate for the in vitro MMR system. Li et al.
(2009) showed that nucleosomes derived from recombinant his-
tones and a mismatch-containing DNA diminished the mismatch
binding and ATPase and DNA sliding activities of hMutSa, which
are required for MMR. Scho ¨pf et al. (2012) demonstrated that
hMutSa failed to restore MMR to an hMSH6-deficient nuclear
extract when DNA heteroduplexes were assembled into nucleo-
somes by preincubating with the extract. These observations
suggest that additional factors and/or mechanisms are needed
for MMR in vivo, possibly by disrupting nucleosomes or the
timely recruiting of MMR proteins, or both. Consistent with this
hypothesis, histone modifications and chromatin remodeling
factors have been implicated in MMR (Javaid et al., 2009;
Kadyrova et al., 2011), and MMR has been show to couple
with DNA replication (Hombauer et al., 2011a; Simmons
590 Cell 153, 590–600, April 25, 2013 ª2013 Elsevier Inc.
et al., 2008), during which nucleosomes are disrupted. More
strikingly, the hMSH6 subunit of hMutSa contains a Pro-Trp-
Trp-Pro (PWWP) domain (Laguri et al., 2008), and this domain,
which is present in many chromatin-associated proteins, was
3 (H3K36me3) (Dhayalan et al., 2010; Vermeulen et al., 2010;
Vezzoli et al., 2010). However, it is not yet known whether the
H3K36me3 mark plays a role in MMR.
Here, we demonstrate that H3K36me3 interacts specifically
with the hMSH6 PWWP domain of hMutSa in vitro and in vivo
and that the histone methyltransferase SETD2, which is respon-
sible for trimethylation of H3K36 (Edmunds et al., 2008), is
required for human MMR in vivo. Consistent with this, cells
depleted of SETD2 and H3K36me3 display a mutator phenotype
characterized by MSI and an elevated mutation frequency at the
HPRT locus. The data presented here strongly suggest that
the H3K36me3 histone mark regulates human MMR in vivo by
recruiting hMutSa onto chromatin to be replicated. We therefore
propose that the status of H3K36me3 in a specific gene or inter-
genic region could potentially influence the local mutation rate in
that region of the chromosome.
The hMSH6 PWWP Domain Interacts with H3K36me3
and Is Essential for hMutSa Binding to Chromatin
et al., 2008), and this conserved domain has recently been pro-
posed to interact specifically with H3K36me3 (Dhayalan et al.,
2010; Vermeulen et al., 2010; Vezzoli et al., 2010). Figure 1A
shows an alignment of the hMSH6 PWWP domain with five other
PWWP domains, including that of BRPF1, the only PWWP
domain for which an atomic resolution structure of the complex
with H3K36me3 is available (Vezzoli et al., 2010; Wu et al., 2011).
Interestingly, the cocrystal structures of the BRPF1 PWWP and
H3K36me3 peptide (Vezzoli et al., 2010; Wu et al., 2011) indicate
that three residues in the PWWP domain form an aromatic cage
our alignment of PWWP domains shows that the proposed
‘‘cage’’ residues are highly conserved (Figure 1A, blue dots).
Based on these data, we generated a model of hMSH6 bound
to the H3K36me3 peptide (Figure 1B, right) by superimposing
the PWWP domains of hMSH6 and BRPF1 (Laguri et al., 2008).
The above data prompted us to ask whether the H3K36me3
mark modulates the interaction between hMutSa and chromatin
and whether such an interaction involves the hMSH6 PWWP
domain. To answer these questions, a glutathione S-transferase
(GST) fusion protein including hMSH6 residues 89 to 194 was
used to pull down histone octamers isolated from HeLa cells
(carrying ‘‘native’’ histone modifications) or assembled using
recombinant histones. The results show that the hMSH6
PWWP domain efficiently pulls down histone octamers from
HeLa cells, but it pulls down recombinant histone octamers
with very low efficiency (Figure 2A). This suggests a specific
interaction between the hMSH6 PWWP domain and an epi-
genetic histone signature.
The specificity of the interaction between the hMSH6 PWWP
domain and natively modified octamers was examined by the
following experiments. First, the same pull-down assay was
performed using native histone octamers purified from HeLa
cells (Rodriguez-Collazo et al., 2009) and wild-type or a mutant
hMSH6 PWWP fusion protein in which W105 and W106 are
Figure 1. The PWWP Domain and Structural
Models for Interaction with H3K36me3
(A) Domain structure of hMSH6 and sequence
alignment of PWWP modules. Upper panel shows
the domain structure of hMSH6. PIP, PCNA-
interacting protein motif; NLS, nuclear localiza-
tion signal. Lower panel shows alignment of
representative human PWWP domains. The blue
dots indicate residues that form an aromatic
cage and bind to H3K36me3. Aligned proteins
are hMSH6, BRPF1, WHSC1_N, WHSC1_C,
ZDWPW1, and HDGF.
(B) Interactions between PWWP domains and the
H3K36me3-containing H3 peptide. The crystal
structure of BRPF1 PWWP domain bound to the
H3K36me3 peptide (Protein Data Bank [PDB] ID
code 2X4Y) is shown in the left panel. BRPF1 does
not have the Trp residues and instead contains a
the hMSH6 PWWP domain, which has the
authentic PWWP motif, was determined by
nuclear magnetic resonance (Laguri et al., 2008) in
the absence of an H3 peptide (PDB ID code
2GFU). The H3 peptide in the BRPF1 complex is
shown with human hMSH6 (right panel) after
superimposing the conserved PWWP domains of
the two proteins. The aromatic cage (colored blue)
encloses the trimethylated Lys. The rotamer con-
to adjust upon binding of the H3K36me3.
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