Molecular Cell, Vol. 17, 331–339, February 4, 2005, Copyright ©2005 by Elsevier Inc.DOI 10.1016/j.molcel.2005.01.008
The Deubiquitinating Enzyme USP1
Regulates the Fanconi Anemia Pathway
tion. MMC and DEB hypersensitivity is a hallmark of FA
and is used as a diagnostic test in the clinic (Auerbach
et al., 1989).
The genetic basis for FA is diverse, and evidence
exists for at least 11 complementation groups (Levitus
et al., 2004). FA is clinically related to various other
hereditary chromosomal instability syndromes, and re-
cent work has shown that the protein products mutated
in Bloom Syndrome, Nijmegen Breakage Syndrome
(NBS), Ataxia Telangiectasia (ATM), and Seckel Syn-
drome (ATR) functionally intersect with the FA signaling
pathway (Andreassen et al., 2004; Meetei et al., 2003b;
Nakanishi et al., 2002; Taniguchi et al., 2002b). Further-
more, hypomorphic mutations in the BRCA2 gene make
up the Fanconi complementation group D1 (FANCD1)
(Howlett et al., 2002).
Based on clinical, biochemical, and cellular pheno-
types, the FA proteins appear to function in a common
cellular signaling network. At least seven of these pro-
teins, FANCA, FANCB, FANCC, FANCE, FANCF, FANCG,
and the ubiquitin E3 ligase FANCL form a nuclear multi-
subunit complex that is critical for the monoubiquitina-
tion of the FANCD2 protein (de Winter et al., 2000; Gor-
don and Buchwald, 2003; Meetei et al., 2003a; Meetei
et al., 2004; Pace et al., 2002). Indeed, functional loss
of any of these FA proteins abrogates S phase and DNA
damage-induced FANCD2 ubiquitination.
izes to nuclear DNA damage foci, where it binds to
BRCA1 and the RAD51 recombinase and colocalizes
with FANCD1/BRCA2 (Taniguchi et al., 2002a; Wang et
al., 2004). It is thought that these nuclear foci mark the
sites of DNA damage-induced double-strand breaks
recombination. A role for FANCD2 and BRCA1 in ho-
mologous recombination is also suggested by their
presence at sites of meiotic recombination in spermato-
genesis (Garcia-Higuera et al., 2001). Although the
monoubiquitination of FANCD2 appears to be a critical
event in efficient DNA repair, the exact molecular func-
tion of FANCD2 is poorly understood.
As mentioned, FANCD2 is also monoubiquitinated
during S phase, and this event is required for normal
quitinated form of FANCD2 (FANCD2-L) disappears
when cells exit S phase and is transiently present in
cells that have been exposed to DNA damage (Garcia-
Higuera et al., 2001; Taniguchi et al., 2002a). Both forms
of FANCD2 are stable and not subject to proteasomal
degradation, indicating that the monoubiquitination
does not serve to target FANCD2-L for degradation.
Instead, it is more likely that a deubiquitinating enzyme
(DUB) removes the ubiquitin moiety after DNA damage
is repaired, and cells resume cycling. Like protein phos-
phorylation, ubiquitination is dynamic and reversible,
involving numerous ubiquitin-conjugating enzymes and
DUBs (Chung and Baek, 1999; D’Andrea and Pellman,
1998; Kim et al., 2003; Shackelford and Pagano, 2004;
Wilkinson, 2000). Homology searches in human genome
Sebastian M.B. Nijman,1,3Tony T. Huang,2,3
Annette M.G. Dirac,1,3Thijn R. Brummelkamp,1
Ron M. Kerkhoven,1Alan D. D’Andrea,2,*
and Rene ´ Bernards1,*
1Division of Molecular Carcinogenesis and Center
for Biomedical Genetics
The Netherlands Cancer Institute
1066 CX Amsterdam
2Department of Radiation Oncology
Dana-Farber Cancer Institute
Harvard Medical School
Boston, Massachusetts 02115
Protein ubiquitination and deubiquitination are dy-
namic processes implicated in the regulation of nu-
merous cellular pathways. Monoubiquitination of the
Fanconi anemia (FA) protein FANCD2 appears to be
critical in the repair of DNA damage because many of
the proteins that are mutated in FA are required for
FANCD2 ubiquitination. By screening a gene family
RNAi library, we identify the deubiquitinating enzyme
USP1 as a novel component of the Fanconi anemia
pathway. Inhibition of USP1 leads to hyperaccumula-
tion of monoubiquitinated FANCD2. Furthermore,
teins colocalize in chromatin after DNA damage. Fi-
nally, analysis of crosslinker-induced chromosomal
aberrations in USP1 knockdown cells suggests a role
in DNA repair. We propose that USP1 deubiquitinates
FANCD2 when cells exit S phase or recommence cy-
cling after a DNA damage insult and may play a critical
role in the FA pathway by recycling FANCD2.
tion against malignant transformation. Genetic disor-
ders that perturb the repair of DNA damage, induced
by either exogenous agents or endogenous events, of-
ten lead to increased cancer susceptibility. One such
disorder is Fanconi anemia (FA), a rare syndrome with
predisposition to a variety of malignancies (D’Andrea,
2003). Genes mutated in FA have also been implicated
in the carcinogenesis of sporadic tumors, underscoring
the broad relevance of studying rare human genetic
At the cellular level, FA is characterized by chromo-
somal instability and hypersensitivity to DNA-crosslink-
ing agents, such as mitomycin C (MMC), cisplatin, di-
poxybutane (DEB), and to alesser extent, ionizing radia-
3These authors contributed equally to this work.
coding for (putative) DUBs. Although DUBs have been
functionally linked with various pathways and pro-
cesses, surprisingly few mammalian DUB substrates
have been identified (Brummelkamp et al., 2003; Cum-
mings et al., 2004; Graner et al., 2004; Kovalenko et al.,
2003; Li et al., 2002; Trompouki et al., 2003). To study
the role of these enzymes in specific pathways, we have
constructed a library of 220 independent vectors ex-
DUBs. Using this library, we have previously identified
the familial tumor suppressor CYLD as a negative regu-
lator of TRAF2 poly-ubiquitination (Brummelkamp et al.,
2002b; Brummelkamp et al., 2003). Here, we identify the
deubiquitinating enzyme USP1 as a novel component
of the FA pathway and proposethat USP1 is the enzyme
that deubiquitinates FANCD2.
cells with the four shRNA vectors and an expression
vector containing a green fluorescent protein tagged
version of USP1 (GFP-USP1). As expected, all four
shRNA vectors efficiently suppressed GFP-USP1 ex-
pression (Figure 2C).
Tostudy endogenousUSP1 protein,a polyclonalanti-
serum directed against the N terminus of USP1 was
generated (see Experimental Procedures) and tested on
synthetic siRNA transfected or control HEK293 cells.
Cells were also treated with the S phase inhibitor hy-
droxyurea (HU) to induce monoubiquitinated FANCD2.
Two bands present in the control lanes were efficiently
downregulated in lysates derived from the USP1 siRNA-
transfected cells (Figure 2D, upper panel). As expected,
the observed USP1 downregulation correlated with the
upregulation of FANCD2-L (Figure 2D, lower panel). The
predicted molecular weight of endogenous, full-length
USP1 is 88 kDa, corresponding to the slower migrating
USP1 species detected by Western blot and consistent
with the size of ectopically expressed USP1 (see Figure
3D, lower panel). The faster migrating band is likely a
proteolytic fragment of USP1. We conclude that in our
experiments, both ectopically expressed and endoge-
nous USP1 protein are efficiently inhibited by RNA inter-
Because FANCD2 monoubiquitination is activated in
S phase, we investigated whether USP1 inhibition re-
sulted in an altered cell cycle distribution or S phase
delay. We retrovirally transduced U2-OS cells with a
knockdown vector targeting USP1. After selection with
puromycin, the cells were synchronized using a double-
thymidine block. Cells were released and samples for
FACS and protein analysis were taken at the indicated
time points. Propidium-iodide (PI) staining of nuclei and
subsequent FACS analysis indicated that cell cycle dis-
tribution and S phase progression of USP1 knockdown
cells was unaffected, suggesting that USP1 inhibition
does not activate a cell cycle checkpoint (Figure 2E).
Furthermore, although FANCD2-L levels decreased sig-
nificantly in the control cells about 4 hr after release,
this decrease appeared to be strongly delayed in the
USP1 knockdown cells (Figure 2F). USP1 inhibition did
lation was not an indirect effect of DNA damage.
was dependent on a functional FA core complex. We
transfected a FANCA-deficient (FA-A) cell line or a
knockdown vector. Subsequently, HU-stimulated or
-untreated cells were analyzed for FANCD2 ubiquitina-
tion (Figure 2G, upper panel). USP1 knockdown did not
result in FANCD2-L accumulation in the FA-A cell line.
cells, indicating that the ability of USP1 to affect
FANCD2 monoubiquitination is dependent on a func-
tional FA signaling pathway.
To investigate whether USP1 requires its protease
activity to affect FANCD2 ubiquitination, we generated
a catalytically inactive USP1 mutant in which the active
site cysteine is replaced by a serine residue (GFP-USP1
C/S) (Papa and Hochstrasser, 1993). Overexpression of
Identification of USP1 as a Regulator
of FANCD2 Monoubiquitination
Previous experiments have indicated that during normal
cell cycle progression, FANCD2 ubiquitination is dy-
namic (Taniguchi et al., 2002a). Therefore, we reasoned
that inhibition of a DUB that cleaves the ubiquitin moiety
from FANCD2 would lead to an overall increase of
FANCD2-L (the monoubiquitinated isoform of FANCD2)
in asynchronous cycling cells. To identify DUBs that
generated in our laboratory (Brummelkamp et al., 2003).
The library currently consists of 55 pools of 4 indepen-
dent shRNA-encoding plasmids targeting 55 DUBs for
suppression by RNA interference (Figure 1A and see
Supplemental Table S1 at http://www.molecule.org/cgi/
content/full/17/3/331/DC1/). We electroporated each
pool of DUB knockdown vectors separately in U2-OS
cells and selected for shRNA expression. After 72 hr,
we analyzed cell lysates by Western blot with an anti-
FANCD2 antibody. As shown in Figure 1B, the pool tar-
geting the ubiquitin-specific protease 1 (USP1, pool 47)
significantly increased the FANCD2-L fraction (Fujiwara
et al.,1998). Theincrease inFANCD2-L wascomparable
to the levels observed in MMC-treated cells (Figure 1B).
Further validation showed that only the pool targeting
USP1 reproducibly had this effect on FANCD2 (Figure
2A and data not shown).
Next, we tested the four independent USP1 shRNA
vectors (A–D) present in the original pool for their ability
to induce FANCD2-L accumulation (Figure 2A). Both
MMC-treated and -untreated cells displayed enhanced
FANCD2 monoubiquitination upon transfection of all
four vectors (A–D). However, vectors A and C were more
potent in inducing FANCD2-L than vectors B and D.
Retroviral delivery of an shRNA targeting USP1 by
using the pRetroSuper vector (pRS) also enhanced
Compared to control cells, retrovirally transduced USP1
knockdown cells displayed enhanced FANCD2 ubiquiti-
nation when stimulated with MMC or left untreated (Fig-
To verify that USP1 expression was indeed inhibited
by the knockdown vectors, we cotransfected HEK293
USP1 Regulates FANCD2
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