Molecular Biology of the Cell
Vol. 18, 1044–1055, March 2007
Increased Common Fragile Site Expression, Cell
Proliferation Defects, and Apoptosis following Conditional
Inactivation of Mouse Hus1 in Primary Cultured Cells
Min Zhu and Robert S. Weiss
Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853
Submitted October 27, 2006; Revised December 21, 2006; Accepted December 29, 2006
Monitoring Editor: Orna Cohen-Fix
Targeted disruption of the mouse Hus1 cell cycle checkpoint gene results in embryonic lethality and proliferative arrest
in cultured cells. To investigate the essential functions of Hus1, we developed a system for the regulated inactivation of
mouse Hus1 in primary fibroblasts. Inactivation of a loxP site-flanked conditional Hus1 allele by using a cre-expressing
adenovirus resulted in reduced cell doubling, cell cycle alterations, and increased apoptosis. These phenotypes were
associated with a significantly increased frequency of gross chromosomal abnormalities and an S-phase–specific accu-
mulation of phosphorylated histone H2AX, an indicator of double-stranded DNA breaks. To determine whether these
chromosomal abnormalities occurred randomly or at specific genomic regions, we assessed the stability of common fragile
sites, chromosomal loci that are prone to breakage in cells undergoing replication stress. Hus1 was found to be essential
for fragile site stability, because spontaneous chromosomal abnormalities occurred preferentially at common fragile sites
upon conditional Hus1 inactivation. Although p53 levels increased after Hus1 loss, deletion of p53 failed to rescue the
cell-doubling defect or increased apoptosis in conditional Hus1 knockout cells. In summary, we propose that Hus1 loss
leads to chromosomal instability during DNA replication, triggering increased apoptosis and impaired proliferation
through p53-independent mechanisms.
Cell cycle checkpoints monitor the fidelity of chromosome
replication and segregation. In response to genome damage,
checkpoint signaling induces cell cycle arrest and promotes
DNA repair, or alternatively, it triggers apoptosis to elimi-
nate damaged cells. Mammalian DNA damage responses
are coordinated by two primary checkpoint pathways that
center on the phosphatidyl inositol kinase-like protein ki-
nases ataxia telangiectasia mutated (Atm) and Atm- and
Rad3-related (Atr) (Bakkenist and Kastan, 2004). An Atm-
dependent pathway responds to double-stranded DNA
breaks (DSBs) such as those caused by ionizing radiation,
whereas an Atr-dependent pathway is activated by a variety
of DNA lesions, including bulky DNA lesions and replica-
tion stress as well as DSBs.
Optimal Atr signaling requires its binding partner, Atrip, as
well as additional accessory factors TopBP1, Brca1, Claspin,
and the Rad9–Rad1–Hus1 (9-1-1) complex (Shechter et al.,
2004b). The 9-1-1 complex shares predicted structural similar-
ity with the sliding clamp proliferating cell nuclear antigen and
is loaded onto chromatin at damage sites by a clamp loader
Karnitz, 2003). 9-1-1 promotes the phosphorylation of Atr sub-
strates such as Chk1, Rad17, and Rad9 itself (Weiss et al., 2002;
Zou et al., 2002; Roos-Mattjus et al., 2003; Bao et al., 2004) and is
required for an intra-S cell cycle checkpoint that represses
DNA synthesis after DNA damage (Roos-Mattjus et al., 2003;
Weiss et al., 2003; Bao et al., 2004; Wang et al., 2004b). Addi-
tional evidence indicates that the 9-1-1 complex also has a
direct role in DNA repair. The 9-1-1 complex physically
associates with multiple translesion DNA polymerases (Kai
and Wang, 2003; Sabbioneda et al., 2005) as well as base
excision repair factors, including the MYH DNA glycosy-
lase, DNA polymerase ?, flap endonuclease I, and DNA
ligase I (Toueille et al., 2004; Wang et al., 2004a. 2006a; Chang
and Lu, 2005; Friedrich-Heineken et al., 2005; Smirnova et al.,
2005; Shi et al., 2006). The 9-1-1 complex additionally is
required for homologous recombinational repair (Pandita et
al., 2006; Wang et al., 2006b). Consistent with its important
roles in cell cycle control and DNA repair, impaired 9-1-1
function is associated with cellular hypersensitivity to rep-
lication inhibitors and DNA damaging agents (Weiss et al.,
2000, 2003; Kinzel et al., 2002; Roos-Mattjus et al., 2003;
Hopkins et al., 2004; Wang et al., 2004b, 2006b).
Targeted disruption of components of the Atr-dependent
checkpoint pathway in mice causes embryonic lethality. De-
letion of Atr or Chk1 results in peri-implantation lethality
(Brown and Baltimore, 2000; de Klein et al., 2000; Liu et al.,
2000; Takai et al., 2000), whereas inactivation of Hus1, Rad9,
or Rad17 causes midgestational embryonic lethality (Weiss et
al., 2000; Budzowska et al., 2004; Hopkins et al., 2004). The
essential nature of these genes highlights the critical, yet
poorly understood, function of this pathway during an un-
perturbed cell cycle. In the course of a normal cell cycle, the
9-1-1 complex and other checkpoint components can be
detected in association with chromatin (Guo et al., 2000;
Hekmat-Nejad et al., 2000; Roos-Mattjus et al., 2002; You et
al., 2002; Zou et al., 2002; Jiang et al., 2003; Lee et al., 2003;
Dart et al., 2004). Even in the absence of extrinsic stress,
checkpoint signaling inhibits the cell cycle phosphatases
Cdc25A and Cdc25B, regulates origin firing, and suppresses
This article was published online ahead of print in MBC in Press
on January 10, 2007.
Address correspondence to: Robert S. Weiss (firstname.lastname@example.org).
1044© 2007 by The American Society for Cell Biology
premature entry into mitosis (Miao et al., 2003; Shechter et
al., 2004a; Sorensen et al., 2004; Niida et al., 2005; Syljuasen et
al., 2005; Schmitt et al., 2006).
The Atr-dependent checkpoint pathway is also thought to
play a critical role in stabilizing stalled replication forks and
promoting fork restart (Lopes et al., 2001; Tercero and Diff-
ley, 2001; Sogo et al., 2002; Trenz et al., 2006). Possibly due to
failure of these important processes, certain yeast checkpoint
mutants show defects in the elongation step of DNA repli-
cation and accumulate chromosomal breaks at particular,
nonrandom genomic regions (Cha and Kleckner, 2002;
Raveendranathan et al., 2006). These sites may be analogous
to vertebrate common fragile sites (CFSs), chromosomal re-
gions where gaps and breaks frequently arise in metaphase
chromosomes prepared from cells under conditions of rep-
lication stress. Recent studies indicate that several compo-
nents of the DNA damage checkpoint machinery, including
Atr (Casper et al., 2002), Chk1 (Durkin et al., 2006), Brca1
(Arlt et al., 2004), and TopBP1 (Kim et al., 2005), among
others, are essential for maintaining CFS stability. No pri-
mary sequence conservation has been identified at CFSs, but
generally these sites are relatively AT rich, highly flexible,
and late replicating (Glover et al., 2005). These properties
suggest that CFSs might be prone to form secondary structures
that inhibit the progression of replication forks, creating a
requirement for cell cycle delay and replication fork stabiliza-
tion or repair by the checkpoint machinery (Cimprich, 2003).
Understanding the molecular basis for fragile site stability has
important implications, because these regions are frequently
deleted or rearranged in cancer cells (Arlt et al., 2006).
Previous attempts at molecular analysis of the essential
functions of Hus1 by using a conventional gene targeting
approach were complicated by severe phenotypes, includ-
ing midgestational lethality in embryos and proliferative
arrest in mouse embryonic fibroblasts (MEFs) (Weiss et al.,
2000). Successful culturing of Hus1-deficient cells from a
constitutive knockout mouse model additionally required
deletion of the checkpoint genes p21 or p53 (Weiss et al.,
2000; our unpublished data). Furthermore, because embryos
lacking both Hus1 and either p21 or p53 remained under-
sized and developmentally delayed, sufficient numbers of
cells for experimental analysis could be obtained only with
immortalized cultures. In this report, we describe a system
for the regulated deletion of Hus1 in primary cultured cells,
for use in dissecting the immediate consequences of Hus1
inactivation. By infecting primary MEFs containing a loxP
site-flanked conditional Hus1 allele with a cre-expressing
recombinant adenovirus (Ad-cre), we generated and ana-
lyzed large populations of Hus1-deficient and control cells in
vitro. Our results indicate that Hus1 inactivation results in
impaired cell proliferation and apoptosis associated with
CFS expression and S-phase–specific DSB accumulation.
MATERIALS AND METHODS
Mouse Strains and Cell Culture
Previously described Hus1floxand Hus1?1mice were maintained on an 129S6
inbred genetic background (Weiss et al., 2000; Levitt et al., 2005). p53?/?mice
harboring the Trp53tm1Tyjallele were maintained on a C57BL/6J background
(Jacks et al., 1994). Mice were housed in accordance with institutional animal
care and use guidelines. MEFs were prepared from 13.5 dpc embryos from
timed matings between Hus1flox/floxand Hus1?/?1mice or from Hus1flox/flox
p53?/?and Hus1?/?1p53?/?mice. Briefly, embryos were dissected from the
deciduum, mechanically disrupted, and cultured in DMEM supplemented
with 10% fetal bovine serum, 1.0 mM l-glutamine, 0.1 mM minimal essential
medium nonessential amino acids, 100 ?g/ml streptomycin sulfate, and 100
U/ml penicillin. The initial plating was defined as passage zero (p0). MEFs at
p1 or p2 were used for all experiments.
Ad-cre, an adenovirus that expresses Cre from the cytomegalovirus promoter
(University of Iowa Gene Transfer Vector Core, Iowa City, IA) (Stec et al.,
1999), was prepared in 293 cells. Briefly, cells were harvested at 48–72 h
postinfection, and viral lysate was subjected to CsCl gradient ultracentrifu-
gation at 63,000 rpm at 14°C for 7 h. Virus was further purified with a PD-10
desalting column (GE Healthcare, Little Chalfont, Buckinghamshire, United
Kingdom), and virus titer was estimated by spectrophotometry according to
the formula: 1 OD280? 1012virus particles/ml. For infections, 1 ? 106MEFs
were plated into a 10-cm culture dish and grown for 1 d. Cells were infected
with 1.95 ? 1011Ad-cre particles in 2.5 ml of culture medium at 37°C for 6 h,
after which time the virus was removed and fresh medium was added. Unless
otherwise specified, cells were passaged at 1 d postinfection (dpi) and then
maintained on a 3T3 culture schedule in which 1 ? 106cells were passaged
onto a 10-cm culture dish every 3 d (Todaro and Green, 1963).
Southern and Northern Blotting
Genomic DNA for Southern blotting was isolated from MEFs by proteinase K
digestion and precipitation with ethanol. DNA was digested with NheI, run
through a 0.8% agarose gel, transferred to a nylon membrane, and hybridized
with a32P-labeled 190-base pair EagI fragment from plasmid pCR2.1-5?UTR-
?2,3 (Levitt et al., 2005). For Northern blotting, total RNA was prepared from
MEFs by using RNA STAT-60 reagent (Tel-Test, Friendswood, TX), and
poly(A)?mRNA was isolated with biotinylated oligo(dT) (Promega. Madi-
son, WI). Purified mRNA was resolved on a 1% agarose/formaldehyde gel,
transferred to a nylon membrane, and hybridized with a32P-labeled cDNA
probe containing the entire mouse Hus1 open reading frame as described
previously (Weiss et al., 1999). After stripping, the membrane was hybridized
to a32P-labeled mouse Gapdh cDNA probe.
Cell Proliferation Assays and Cell Cycle Analysis
For cell proliferation assays, triplicate cultures were maintained on a 3T3
culture schedule. Population doublings (PDLs) were calculated using the
formula ?PDL ? log(nf/n0)/log2, where n0is the initial number of cells and
nfis the final number of cells (Blasco et al., 1997). For cell cycle analysis, 1 ?
106cells were plated per 10-cm culture dish 24 h before analysis. The next day,
the cells were incubated with 10 ?M bromodeoxyuridine (BrdU) for 45 min,
harvested by trypsinization, washed once in phosphate-buffered saline (PBS),
and fixed in 70% ethanol at ?20°C. The cells were then incubated in 2 N HCl,
0.5% Triton X-100, washed twice with 0.1 M Na2B4O7?10H2O, pH 8.5, incu-
bated with fluorescein isothiocyanate (FITC)-conjugated anti-BrdU (BD Bio-
sciences, Franklin Lakes, NJ) for 30 min at room temperature (RT), washed,
treated with RNAse A, and stained with propidium iodide (PI). Flow cytom-
etry was performed on a FACScan flow cytometer (BD Biosciences).
Cells grown in 10-cm culture dishes were collected by trypsinization along
with floating cells in the culture medium, washed twice with PBS at 4°C, and
resuspended in 1? binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5
mM CaCl2) at a concentration of 1 ? 106cells/ml. Cells (1 ? 105) were then
incubated with Annexin V-FITC (BD Biosciences) and PI for 15 min at RT.
Flow cytometry was performed on a FACScan flow cytometer (BD Bio-
sciences) within 1 h.
Indirect Immunofluorescence Assays (IFAs)
Cells grown on coverslips were fixed in 2% paraformaldehyde in TBS for 35
min at 4°C (for ?-H2AX IFA) or in methanol at ?20°C for 30 min followed by
ice-cold acetone for two seconds (for p53 IFA). Cells were then incubated in
3% bovine serum albumin (BSA), 0.01% skim milk, 0.2% Triton X-100 in
Tris-buffered saline (TBS) for 20 min at RT. For ?-H2AX IFA, cells were
incubated with primary anti-?-H2AX antibody (JBW301; Upstate Biotechnol-
ogy, Lake Placid, NY) at 1:500 for 45 min, followed by secondary goat
anti-mouse Ig (H?L)-FITC (Southern Biotechnology Associates, Birmingham,
AL) at 1:60 for 35 min. For p53 IFA, cells were incubated with primary
anti-p53 antibody (FL393; Santa Cruz Biotechnology, Santa Cruz, CA) at 1:60
dilution at RT for 1 h, followed by secondary goat anti-rabbit Ig (H?L)-FITC
(Southern Biotechnology Associates) at 1:60 at RT for 35 min. Cells were
counterstained with 33 ng/ml 4?,6-diamidino-2-phenylindole (DAPI) for 1
Fluorescence-activated Cell Sorting (FACS) Analysis of
Cells (1 ? 106) were fixed in ice-cold 70% ethanol, incubated with 1% BSA,
0.25% Triton X-100 in TBS for 15 min on ice, and stained with primary
anti-?-H2AX antibody (JBW301, Upstate Biotechnology) at 1:500 overnight at
4°C. The next day, the cells were stained with secondary goat anti-mouse Ig
(H?L)-FITC (Southern Biotechnology Associates) at 1:400 for 30 min at RT
and counterstained with 5 ?g/ml PI containing RNAse A for 30 min at RT.
Flow cytometry was performed on an LSR II flow cytometer (BD Biosciences).
Regulated Hus1 Deletion in Primary Cells
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