Dual recognition of phosphoserine and
phosphotyrosine in histone variant H2A.X
by DNA damage response protein MCPH1
Namit Singha,1, Harihar Basnetb,1, Timothy D. Wiltshirec, Duaa H. Mohammadd,e, James R. Thompsonf, Annie Hérouxg,
Maria Victoria Botuyana, Michael B. Yaffed,e, Fergus J. Couchc, Michael G. Rosenfeldb,2, and Georges Mera,2
Departments ofaBiochemistry and Molecular Biology,cLaboratory Medicine and Pathology, andfPhysiology and Biomedical Engineering, Mayo Clinic,
Rochester, MN 55905;bHoward Hughes Medical Institute and Graduate Program in Biomedical Sciences, University of California at San Diego, School of
Medicine, La Jolla, CA 92093; Departments ofdBiology andeBiological Engineering, Massachusetts Institute for Technology, Cambridge, MA 02139; and
gBiology Department, Brookhaven National Laboratory, Upton, NY 11973
Contributed by Michael G. Rosenfeld, July 22, 2012 (sent for review April 19, 2012)
Tyr142, the C-terminal amino acid of histone variant H2A.X is phos-
phorylated by WSTF (Williams-Beuren syndrome transcription
factor), a component of the WICH complex (WSTF-ISWI chroma-
tin-remodeling complex), under basal conditions in the cell. In
response to DNA double-strand breaks (DSBs), H2A.X is instanta-
neously phosphorylated at Ser139 by the kinases ATM and ATR
and is progressively dephosphorylated at Tyr142 by the Eya1 and
Eya3 tyrosine phosphatases, resulting in a temporal switch from
a postulated diphosphorylated (pSer139, pTyr142) to monophos-
phorylated (pSer139) H2A.X state. How mediator proteins inter-
pret these two signals remains a question of fundamental interest.
We provide structural, biochemical, and cellular evidence that
Microcephalin (MCPH1), an early DNA damage response protein,
can read both modifications via its tandem BRCA1 C-terminal
(BRCT) domains, thereby emerging as a versatile sensor of H2A.X
phosphorylation marks. We show that MCPH1 recruitment to sites
of DNA damage is linked to both states of H2A.X.
exogenous insults can trigger signaling cascades relayed by
posttranslational modifications culminating in the activation of
the cell cycle checkpoint and initiation of repair. One of the key
initiating events in this process is the phosphorylation at serine
139 (pSer139) of histone H2A.X, a chromatin-bound histone
variant (1, 2). Phosphorylated H2A.X (γH2A.X) serves as a
platform for the recruitment of downstream mediator proteins as
well as chromatin modifying proteins to the affected site (3–5).
Recent studies have added a new dimension to the recognition of
γH2A.X with the identification of a new phosphorylation site
in H2A.X, Tyr142. In response to DNA damage, Tyr142 was
found to transition from a phosphorylated (pTyr142) to a non-
phosphorylated state in an Eya1/3 phosphatase-dependent man-
ner (6–8). Whereas the dephosphorylation of pTyr142 is gradual,
the phosphorylation of Ser139 is prompt, and the overlap in the
two processes is thought to give rise to the doubly phosphorylated
(pSer139, pTyr142) H2A.X state (di-γH2A.X) following geno-
toxic insult (6, 7). The existence of di-γH2A.X and proteins that
recognize this state remain open questions in the field.
The protein MDC1 (mediator of DNA damage checkpoint 1)
has emerged as an interacting partner of γH2A.X. MDC1 was
found to directly sense pSer139 and to mobilize the downstream
response by virtue of its tandem BRCA1 C-terminal (BRCT)
domains (4, 5). An important question therefore is how MDC1,
an established γH2A.X binder, responds to the presence of
diphosphorylated H2A.X. Interestingly, several groups have in-
dependently found that MDC1 does not interact with di-γH2A.X
(6, 7, 9). Indeed, efforts to identify binding partners of a di-γH2A.
X peptide were redirected at well-established pTyr-binding SH2/
PTB domains and led to the second PTB domain of Fe65 as a
possible target (7). Is this inability to bind Tyr142-phosphorylated
reserving genomic integrity is vital to the fitness of an or-
ganism. Damage to DNA emanating from endogenous and
H2A.X reflective of the inherent limitations of tandem BRCT
domains, previously identified as pSer or pThr binding domains,
to recognize the pTyr state?
Microcephalin (MCPH1), the first gene identified as causative
for primary autosomal microcephaly (10), has been linked to
various cellular processes including the DNA damage checkpoint,
DNA repair by homologous recombination, and DNA tran-
scription (11–15). Previous studies have established that MCPH1
is an early responder in the DNA damage response (DDR) and
regulates the recruitment of several downstream mediator pro-
teins, a characteristic shared with MDC1. Similarities between
MCPH1 and MDC1 are inescapable. Both proteins include tan-
dem BRCT domains at their C terminus that are necessary for
irradiation-induced foci (IRIF) formation in an H2A.X pSer139
phosphorylation-dependent manner (16, 17). MCPH1 and MDC1
can bind a peptide that encompasses phosphorylated Ser139
(16, 18) and in vivo mutation of this residue results in loss of
IRIF (16). Because MCPH1 IRIF formation necessitates γH2A.
X, presumably because of direct interaction with γH2A.X, we
asked whether MCPH1 could also interact with di-γH2A.X.
The discovery that H2A.X Tyr142 can be phosphorylated has
raised several interesting points. First, whereas the di-γH2A.X
species has been suggested, a deeper characterization of its ki-
netics and role in DNA damage remains to be attempted. Sec-
ond, it remains to be determined whether a relationship exists
between di-γH2A.X and γH2A.X. Third, it is not known whether
MCPH1, a previously described γH2A.X interactor, can also
recognize di-γH2A.X. Here, via the generation of an antibody
that selectively recognizes a di-γH2A.X peptide (di-pH2A.X), we
demonstrate the existence of di-γH2A.X and monitor its dy-
namics following DNA damage. Furthermore, we show that
there exists a direct relationship between pTyr142 and pSer139,
whereby the former affects the latter. Most importantly, our
work identifies MCPH1 as a BRCT protein that directly interacts
with di-pH2A.X and explains the structural basis for this rec-
ognition. Hitherto, BRCT domains were thought to function
within the realm of pSer or pThr recognition (19–21).
Author contributions: N.S., H.B., M.G.R., and G.M. designed research; N.S., H.B., T.D.W.,
D.H.M., J.R.T., A.H., M.V.B., M.B.Y., F.J.C., and G.M. performed research; N.S., H.B., M.G.R.,
and G.M. analyzed data; and N.S., H.B., and G.M. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
Data deposition: The atomic coordinates for the structures of MCPH1–pH2A.X and
MCPH1–di-pH2A.X complexes have been deposited in the Protein Data Bank, www.
pdb.org (PDB ID codes 3SZM and 3U3Z).
1N.S. and H.B. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| September 4, 2012
| vol. 109
| no. 36
(NLS) at Brookhaven National Laboratory, respectively. Data collection and
refinement statistics are presented in Table 1.
ACKNOWLEDGMENTS. We are grateful to Y. Kim at Advanced Photon Source
(APS) for assistance with X-ray data collection, Z. Dauter for insightful
comments regarding structure refinement, and A. Nussenzweig for providing
the H2a.x−/−mouse embryonic fibroblasts. The MCPH1 construct used for pep-
tide library screening was a kind gift of R. Chahwan and S. P. Jackson, Univer-
sity of Cambridge, Cambridge, United Kingdom. This work was supported by
National Institutes of Health (NIH) Grants CA132878 (to G.M.), CA097134,
DK018477, DK39949, and NS034934 (to M.G.R.), CA116167 (to F.J.C.), and
CA112967 and ES015339 (to M.B.Y.). M.G.R. is a Howard Hughes Medical In-
stitute investigator. We acknowledge the use of beamline 19-ID at APS and
beamline X29 at National Synchrotron Light Source (NLS). APS is operated by
University of Chicago’s Argonne National Laboratory, for the US Department
of Energy under Contract DE-AC02-06CH11357. Funding for NLS comes from
of the US Department of Energy and from NIH Grant P41RR012408.
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