, 1928 (2007);
et al. Anja Groth,
Histone Supply and Demand
Regulation of Replication Fork Progression Through
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Regulation of Replication Fork
Progression Through Histone
Supply and Demand
Anja Groth,1Armelle Corpet,1Adam J. L. Cook,1Daniele Roche,1Jiri Bartek,2
Jiri Lukas,2Geneviève Almouzni1*
DNA replication in eukaryotes requires nucleosome disruption ahead of the replication fork and
reassembly behind. An unresolved issue concerns how histone dynamics are coordinated with fork
progression to maintain chromosomal stability. Here, we characterize a complex in which the human
histone chaperone Asf1 and MCM2–7, the putative replicative helicase, are connected through a histone
H3-H4 bridge. Depletion of Asf1 by RNA interference impedes DNA unwinding at replication sites, and
similar defects arise from overproduction of new histone H3-H4 that compromises Asf1 function. These
data link Asf1 chaperone function, histone supply, and replicative unwinding of DNA in chromatin. We
propose that Asf1, as a histone acceptor and donor, handles parental and new histones at the replication
fork via an Asf1–(H3-H4)–MCM2–7 intermediate and thus provides a means to fine-tune replication fork
progression and histone supply and demand.
and recycled histones, must assemble on the
hen one parental nucleosome is dis-
rupted ahead of the moving replication
biosynthesis and DNA replication (2) ensures the
supply of new histones at the global level.
However, an additional layer of regulation must
be at play locally at individual replication forks to
ensure balanced deposition of new and parental
histone chaperones, such as Asf1 (antisilencing
function 1), that can participate in both nucleo-
some assembly and disassembly (1, 3). Human
Asf1a and Asf1b exist in two pools (4), a highly
mobile (cytosolic) pool that buffers excess soluble
histones during replication stress and a salt-
extractablepool innuclear extracts.Howthelatter
relates to other chromatin proteins and contributes
to nuclear function remains open.
We isolated and characterized in vivo com-
plexes containing Asf1a or Asf1b, using stable
HeLa S3 cell lines expressing tagged Asf1
(e-Asf1) (5). Mass spectrometry and Western blot-
ting revealed the presence of Mcm2, 4, 6, and 7 in
1Laboratory of Nuclear Dynamics and Genome Plasticity,
UMR218 CNRS/Institut Curie, 26 rue d’Ulm, 75248 Paris
cedex 05, France.2Institute of Cancer Biology and Centre
for Genotoxic Stress Research, Danish Cancer Society,
Strandboulevarden 49, Copenhagen DK-2100, Denmark.
*To whom correspondence should be addressed. E-mail:
Fig. 1. Characterization of a human Asf1–(H3-H4)–
blotting (right). Control extract without e-Asf1 (–) was
included to identify unspecific proteins (asterisks). (B)
Silver staining and Western blot analysis of nuclear
complexes containing wild-type (wt) or mutant (V94R)
e-Asf1a. (C) Fractionation scheme and analysis of Asf1
immunoprecipitates (IP) from soluble and chromatin-
bound material. The asterisk marks an unspecific band;
input is 10% of starting material. (D) Analysis of Asf1a
immunoprecipitates from asynchronous (left) and
synchronized cells (right) (see also fig. S2D). Input is
3% of starting material. Under low-stringency condi-
tions, some MCMs bind unspecifically to control beads.
21 DECEMBER 2007VOL 318
on January 3, 2008
the nuclear e-Asf1 (a and b) complexes, together
comparison, only Mcm2 was associated with cyto-
solic e-Asf1 (a and b) complexes. Antibodies
against Mcm6 coimmunoprecipitated Asf1 and
histone H3-H4 from nuclear extracts (fig. S1C),
whereas Mcm2, 4, and 7 were retrieved from both
cytosolic and nuclear fractions. Given that this set
umns (6), we tested whether Asf1 associates with
Mcm2, 4, 6, and 7 through histone H3-H4 by
isolating complexes containing e-Asf1a mutated
in the histone-binding domain, by replacement
of valine at codon 94 with arginine (V94R) (7).
e-Asf1a V94R did not bind histones H3-H4, as
the complex (Fig. 1B), which implicated histone
H3-H4 in bridging the interaction between Asf1
and MCMs. To further confirm the chromatin link
and to avoid the use of salt-extraction, which dis-
(fig. S1C), we used deoxyribonuclease (DNase I)–
solubilized chromatin (Fig. 1C and fig. S2A).
Again, Mcm2, 4, 6, and 7 coimmunoprecipitated
with Asf1 (Fig. 1, C and D), and Mcm6 antibodies
retrieved Asf1 (a and b) (fig. S2B). Under these
conditions, which preserve the hexameric MCM2–
7 complex (8) (fig. S2B), Mcm3 and Mcm5 co-
immunoprecipitated with Asf1 (Fig. 1D), which
was also confirmed by epitope tag purification of
1D), which suggested a role in DNA replication.
S-phase defects have been reported in var-
ious systems upon interference with Asf1 function
(4, 9, 10). Human cells depleted of Asf1 (a and b)
accumulated in S phase (Fig. 2A) with reduced
5-bromo-2′-deoxyuridine (BrdU) incorporation (fig.
S3C). However, the appearance and distribution of
replication factories marked by proliferating cell nu-
clear antigen (PCNA) and the pattern of chromatin-
bound Mcm2 were unchanged (fig. S3), which
was consistent with findings in Drosophila (10).
replicative helicase (11), we wondered whether in-
efficient replication could reflect problems of un-
level of single-stranded DNA (ssDNA) at repli-
replication sites, we used two markers, replication
RPA and PCNA showed characteristic replication
patterns (Fig. 2A and fig. S4A). Although PCNA
patterns were unchanged in Asf1-depleted cells,
RPA replication patterns were barely detectable.
Some nonextractable RPA localized to bright nu-
clear foci, which we identified as promyelocytic
leukemia (PML) nuclear bodies, clearly distinct
thatAsf1depletion did not affect RPA expression
(fig. S3A) or its ability to bind ssDNA (fig. S5).
Thus, absence of RPA replication profiles is con-
sistent with the hypothesis of a helicase defect.
To examine helicase function, we analyzed
DNA unwinding in the absence of polymerase
progression by treating cells with hydroxyurea
(HU) to deplete the nucleotide pool, which inhibits
the DNA polymerase and leads to formation of
ssDNA (12). In Xenopus, this response is de-
pendent on MCM2–7 function (13). We measured
formation of ssDNA ahead of the polymerase by
detection of BrdU-substituted DNA and acute
accumulation of RPA at replication sites (fig. S6).
In control cells treated with HU, 75% of cells in S
Fig. 2. Asf1depletionimpairsDNAunwinding.(A)(Left)CellcycleprofileofU-2-OScellstreatedwithsmall
interfering RNAs (siRNAs) against Asf1a and Asf1b or control siRNA against GFP (siGFP). (Right) RPA and
PCNA replication profiles in preextracted siRNA-treated cells. Images representative of five experiments
show early S-phase cells with enlargements (4×). Cells in mid and late S phase showed similar defects (fig.
S4A). Scale bars, 10 mm and 1 mm. (B) RPA accumulation (left) and ssDNA formation (right) after 1 hour of
HU treatment (3 mM). For ssDNA analysis, BrdU was detected without double-stranded DNA (dsDNA)
nucleosomal histones from pellet material (1× corresponds to same cell numbers as the nuclear extract).
VOL 31821 DECEMBER 2007
on January 3, 2008
of RPA to these ssDNA patches at replication sites
(Fig. 2B). This response was dramatically reduced
when we depleted Asf1 (a and b) (Asf1 knock-
down), which indicated that impaired replication
reflects a DNA unwinding defect and implied that
the replicative helicase. This could reflect a direct
effect of Asf1 on DNA unwinding and fork pro-
at the replication fork, replisome collapse, and/or
checkpoint signaling. However, we found no evi-
dence of DNA damage or checkpoint activation
checkpoint abrogation by caffeine did not rescue
the unwinding defect (fig. S7B). Instead, induction
of g-H2AX (phosphorylation of a histone 2A
variant) in response to HU treatment was impaired
in Asf1-depleted cells (fig. S7D), which was con-
(12, 14). Furthermore, expression and chromatin as-
sociation of several key replication factors remained
unchanged upon Asf1 knockdown (fig. S7C).
To explore whether a direct role of Asf1 in
facilitating DNA unwinding could involve interac-
tion with histones and MCM2–7, we followed the
Asf1–(H3-H4)–MCM complex when helicase
progression is uncoupled from the polymerase.
Nuclear Asf1 bound significantly more Mcm2, 4,
6, and 7 and histone H3-H4 in HU-treated cells
putative targets of ATR (15) and Cdc7-Dbf4 (16)],
which underlined a connection to replication
control. During HU treatment, continued unwind-
DNA synthesis creates a situation where displaced
parental histones cannot immediately be recycled.
The accumulation of Asf1–(H3-H4)–MCM com-
plexes under such conditions suggests that this
complex could be an intermediate in parental
histone transfer. Within these complexes, we could
detect histone modifications, H4 with acetylated
lysine 16 (H4K16Ac) and H3 with trimethylated
lysine 9 (H3K9me3) (Fig. 2C). This further
substantiates our hypothesis, as these chromatin
Our results suggest that Asf1 coordinates his-
tagged histoneH3.1 and H4 (Fig.3A).About50%
of the cells expressed H3.1-H4 when tetracycline
was removed, and Asf1 bound the exogenous
histones (Fig. 3A). After induction, the nonnucleo-
somal histone pool increased two- to threefold (fig.
S8A), a range that is comparable to histone
overload during a replication block (4). Increasing
and caused acute accumulation of H3.1-H4 over-
expressing cells in S phase [tracked by the green
24 hr -Tet
48 hr -Tet
HA GFP Merge + dapi
-Tet = ON
+Tet = OFF
Dapi GFPPCNA RPAMerge
PCNA positive cells with
RPA in replication pattern (%)
old / new
+ / +
+ / +++
24 hr -Tet
48 hr -Tet
-Tet 40 hr, H4-GFP+
24 hr -Tet
Fig. 3. Histone H3-H4 excess impairs DNA unwinding. (A) Cell line for conditional coexpression of H3.1-HA-Flag
and H4-GFP from a bidirectional promoter in the U-2-OS Tet-Off system. Cells cultured with or without tetracycline
(Tet) were analyzed by immunofluorescence (left) or Asf1 immunoprecipitation (right). (B) Fluorescence-activated
cell sorting (FACS) analysis of DNA content and H4-GFP expression with profiles of GFP-negative (–) and GFP-
positive cells (+) from the same samples. (C) RPA localization as in Fig. 2A. (Right) Quantification of PCNA-positive
cells with RPA replication patterns. Error bars indicate standard deviation in three experiments (n > 130). (D) Cell
cycle profiles of Myc-Asf1–expressing and control cells transiently transfected with H3.1-Flag-HA/H4-GFP. (E) Cell
cycle profile of siRNA-transfected cells after 40 hours of histone induction [(as in (B)].
Fig. 4. Model for Asf1 function in replication as
a histone acceptor and donor.
21 DECEMBER 2007VOL 318
on January 3, 2008
fluorescent protein (GFP) tag on H4] (Fig. 3B). At Download full-text
later time points,the majority of GFP-positive cells
arrested in late S/G2. We focused on the S-phase
defect to address whether H3-H4 excess mimicked
Asf1 depletion. The moderate increase in H3-H4
expression did not cause DNA damage monitored
H4 induction) as an internal control for proper
localization (Fig. 3C). Again, as in Asf1-depleted
cells, RPA replication patterns in histone-over-
expressing cells were barely visible, with some
RPA localized to bright nuclear foci mainly
corresponding to PML bodies (Fig. 3C and fig.
S8D). Furthermore, as for Asf1 knockdown, an
excess of new H3-H4 histones impaired ssDNA
(fig. S9, A and B), as well as checkpoint activation
in response to HU (figs. S9C and S8C). Together,
these data indicate that overproduction of histone
H3-H4 impairs replication by impeding DNA
unwinding. Consistent with the possibility that this
results from interference with Asf1 function, we
found that ectopic expression of Asf1a partially
alleviated the inhibitory effect of histone excess on
S-phase progression (Fig. 3D). Moreover, Asf1
depletion aggravated the S-phase defect resulting
G2was delayed even further (Fig. 3E).
Together, our results show that replication fork
progression is dependent on the histone H3-H4
chaperone, Asf1, and on an equilibrium between
histone supply and demand. This dependency
could ensure that replication only proceeds when
of the parental chromatin template in coordination
with nucleosome assembly on daughter strands.
Nucleosome disruption during replication fork
of the MCM2–7 complex and transfer of parental
intermediate, followed by their deposition onto
daughter strands. In parallel, Asf1 would provide
the additional complement of histones through its
established role as a new histone donor (4, 20, 21).
Asf1 knockdown will impair histone transfer and
an impediment to unwinding and replication fork
of Asf1, an excess of new histones will not leave
Asf1 available for parental transfer, which im-
pairs unwinding. On the basis of structural data
(H3-H4)2, like new histones (24), go through a
transientdimeric state during transfer. Furthermore,
the MCM–(H3-H4)–Asf1 connection opens new
angles to understand MCM2–7 function in chro-
matin (25). In conclusion, having Asf1 deal with
both new and parental histones could provide an
ideal means to fine-tune de novo deposition and
recycling with replication fork progression. By of-
fering a mechanism to coordinate new and pa-
rental histones during replication, our model
should pave the way to addressing key questions
transmission of histone modifications.
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Supporting Online Material
Materials and Methods
Figs. S1 to S9
9 August 2007; accepted 1 November 2007
Switching from Repression to
Activation: MicroRNAs Can
Shobha Vasudevan, Yingchun Tong, Joan A. Steitz*
AU-rich elements (AREs) and microRNA target sites are conserved sequences in messenger RNA (mRNA)
3′ untranslated regions (3′UTRs) that control gene expression posttranscriptionally. Upon cell cycle
arrest, the ARE in tumor necrosis factor–a (TNFa) mRNA is transformed into a translation activation
signal, recruiting Argonaute (AGO) and fragile X mental retardation–related protein 1 (FXR1), factors
associated with micro-ribonucleoproteins (microRNPs). We show that human microRNA miR369-3 directs
association of these proteins with the AREs to activate translation. Furthermore, we document that two
well-studied microRNAs—Let-7 and the synthetic microRNA miRcxcr4—likewise induce translation up-
regulation of target mRNAs on cell cycle arrest, yet they repress translation in proliferating cells. Thus,
activation is a common function of microRNPs on cell cycle arrest. We propose that translation regulation
by microRNPs oscillates between repression and activation during the cell cycle.
internal stimuli (1). MicroRNAs are small non-
U-rich elements (AREs) bind specific
proteins to regulate mRNA stability or
translation in response to external and
coding RNAs that recruit an Argonaute (AGO)
which results in translation repression or degrada-
tion of the mRNA (2, 3). We previously dem-
onstrated that the tumor necrosis factor–a (TNFa)
ARE can be transformed by serum starvation,
which arrests the cell cycle, into a translation
activation signal (4). AGO2 and fragile X mental
required to increase translation efficiency. Two key
questions arose. First, is binding of the AGO2-
FXR1 complex, which activates translation,
directed by a microRNA complementary to the
ARE? Second, can micro-ribonucleoproteins
(microRNPs), in general, up-regulate translation
under growth-arrest conditions, thereby switching
to the cell cycle?
A bioinformatic screen identified five micro-
supporting online material (SOM) text]. Of these,
only human miR369-3 (Fig. 1A and fig. S1) tested
positive in the following assays. Its seed sequence
potentially forms base pairs with two target sites
[seed1 and seed2, shaded in (Fig. 1A)] within the
minimal TNFa ARE needed for translation activa-
VOL 31821 DECEMBER 2007
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