Genetics- chromosome instability

Genetics- chromosome instability

Read the paper “Chaos” and write a 2 pages article (Examples and instructions attached). No copy-paste. Cite if needed.
A viable allele of Mcm4 causes chromosome instability
and mammary adenocarcinomas in mice
Naoko Shima1–3, Ana Alcaraz2, Ivan Liachko4, Tavanna R Buske1, Catherine A Andrews1, Robert J Munroe2,3,
Suzanne A Hartford2,3, Bik K Tye4 & John C Schimenti2,3
Mcm4 (minichromosome maintenance–deficient 4 homolog)
encodes a subunit of the MCM2-7 complex (also known as
MCM2–MCM7), the replication licensing factor and
presumptive replicative helicase. Here, we report that the
mouse chromosome instability mutation Chaos3 (chromosome
aberrations occurring spontaneously 3), isolated in a forward
genetic screen, is a viable allele of Mcm4. Mcm4Chaos3 encodes
a change in an evolutionarily invariant amino acid (F345I),
producing an apparently destabilized MCM4. Saccharomyces
cerevisiae strains that we engineered to contain a
corresponding allele (resulting in an F391I change) showed a
classical minichromosome loss phenotype. Whereas
homozygosity for a disrupted Mcm4 allele (Mcm4–) caused
preimplantation lethality, McmChaos3/– embryos died late in
gestation, indicating that Mcm4Chaos3 is hypomorphic. Mutant
embryonic fibroblasts were highly susceptible to chromosome
breaks induced by the DNA replication inhibitor aphidicolin.
Most notably, 480% of Mcm4Chaos3/Chaos3 females succumbed
to mammary adenocarcinomas with a mean latency of
12 months. These findings suggest that hypomorphic alleles
of the genes encoding the subunits of the MCM2-7 complex
may increase breast cancer risk.
Maintenance of genomic integrity is a complicated process, requiring
the cell to precisely regulate DNA replication and chromosomal
division and to recognize and repair damaged DNA. Defects in any
of these activities may cause genomic instability, potentially leading to
cancer. To identify genes or alleles that predispose to cancer from such
defects, we have previously conducted an N-ethyl-N-nitrosourea
(ENU) mutagenesis screen for mice showing chromosome instability,
as assessed by micronucleus levels in erythrocytes1.
One mutation we identified in this screen is Chaos3. Heterozygotes
show mildly elevated (two- to fivefold) micronucleus frequencies
compared with wild-type animals (Fig. 1a), whereas homozygotes
show a marked (B20-fold) increase; over 7% of erythrocytes in these
animals contain micronuclei. Other than the micronucleus phenotype,
young Chaos3 heterozygotes and homozygotes of both sexes are fertile
and overtly indistinguishable from normal littermates.
We genetically mapped Chaos3 to a 1.3-Mb region between
D16Mit56 and D16Jcs61 (Fig. 1b) containing 11 RefSeq genes,
including Mcm4 (see Methods). We sequenced Mcm4 cDNA derived
from mice homozygous for Chaos3 and C57BL/6J control mice, and
we identified a de novo T-A transversion at nucleotide 1033 of the
coding region (Fig. 1c). This substitution creates an amino acid
change from phenylalanine to isoleucine at residue 345 (F345I), 15
amino acids downstream from a zinc finger motif (Fig. 1d). This
residue is conserved throughout eukaryotes (Fig. 1e) and is important
for interaction with other MCMs2. Based on estimated ENU mutation
frequencies, the presence of confounding mutations in such a small
critical region is exceedingly unlikely3. Nevertheless, we sequenced the
other genes in this region and did not find any other mutations
(Supplementary Table 1 online).
The genes encoding the MCM2-7 complex were identified in a
genetic screen in budding yeast for DNA replication mutants particularly
defective at initiation4. Null mutations of these genes are lethal,
and hypomorphic or temperature-sensitive alleles are defective in
maintaining minichromosomes with a single replication origin5
. The
structurally related MCM2-7 proteins form a heterohexamer that is
recruited to replication origins as an essential component of the
prereplication complex, which is assembled from late M to early G1
phase of the cell cycle6–8. This process is termed origin ‘licensing’,
which ensures a single initiation of DNA synthesis from MCM-bound
origins in the subsequent S phase, restricting genome replication to
once per cell cycle. Accumulating evidence also indicates that the
MCM2-7 complex is the replicative helicase9,10.
To test whether F345I has a functional consequence, we created
the corresponding allele (resulting in an F391I change) in budding
yeast11. Three independently isolated yeast Chaos3 mutant lines
showed a minichromosome loss phenotype (Fig. 2a) that was
specific to a replication defect–sensitive origin, ARS1 (autonomously
replicating sequence 1), but not to a robust origin, ARS120 (refs. 4,5).
This ARS-specific defect suggests that the F391I change disrupts
Received 28 August; accepted 3 November; published online 3 December 2006; doi:10.1038/ng1936
1Department of Genetics, Cell Biology and Development, College of Biological Sciences, University of Minnesota, Minneapolis, Minnesota 55455, USA. 2Department of
Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA. 3The Jackson Laboratory, Bar Harbor, Maine 04609, USA. 4Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA. Correspondence should be addressed to N.S. (shima023@umn.edu)
or J.C.S. (jcs92@cornell.edu).
NATURE GENETICS VOLUME 39 [ NUMBER 1 [ JANUARY 2007 9 3
LETTERS
© 2007 Nature Publishing Group http://www.nature.com/naturegenetics
the early stages of DNA replication such as prereplication
complex assembly.
To further confirm that F345I is causative for the Chaos3 phenotype
and to explore the nature of this allele, we generated mice that carry a
disruption allele of Mcm4 (referred to as Mcm4–). Mcm4– heterozygotes
appeared normal and did not show a higher level of spontaneous
micronuclei, unlike Mcm4Chaos3 heterozygotes (data not
shown). Mcm4– is derived from BayGenomics embryonic stem cell
line RRE056 (see Methods) that contains a gene trap vector inserted
between exons 12 and 13 of Mcm4, disrupting the highly conserved
MCM domain (Supplementary Fig. 1 online). Intercrosses between
Mcm4– heterozygotes did not produce any postimplantation homozygotes.
We then cultured blastocysts to assess the ability of inner cell
masses to proliferate. Consistent with the essential nature of Mcm4,
none of 36 embryos that outgrew was a homozygote, whereas wildtype
embryos (n ¼ 9) and heterozygotes (n ¼ 23) were present (P o
0.005; genotype was unknown for four blastocysts). To assess complementation,
we crossed heterozygotes for each allele to each other,
but we did not recover any live-born Mcm4Chaos3/– offspring. We
then conducted a series of timed matings to define the timing of
the presumed embryonic lethality (Table 1). We observed that
Mcm4Chaos3/– embryos were present until 14.5 d post-coitum (dpc),
but the majority were growth retarded in the C57BL/6J congenic
background (Fig. 2b). However, certain genetic backgrounds extended
the survival of Mcm4Chaos3/– embryos (Supplementary Fig. 2 online).
These results lead us to conclude that (i) the Mcm4– allele is probably
a null, (ii) the Mcm4Chaos3 allele is a hypomorph, as it could partially
rescue early embryonic lethality of Mcm4–, and (iii) the Mcm4Chaos3
allele also has a dominant component with respect to chromosome
instability in animals, as heterozygotes have an elevated level of
micronuclei, but Mcm4–/+ animals do not. In sum, the evidence
presented here (and below) demonstrates that Chaos3 is a viable,
defective allele of Mcm4. Both the Mcm4Chaos3 and Mcm4– alleles are
the first reported functional mutations of any Mcm gene in mammals.
To investigate cellular phenotypes associated with these two alleles,
we generated embryonic fibroblasts (MEFs). Cell cycle progression
profiles of Mcm4Chaos3/Chaos3 and Mcm4Chaos3/– MEFs were nearly
identical. They both showed a mild defect in cell proliferation, a
lower number of S phase cells and a slightly increased level of G2/M
populations relative to isogenic wild-type controls (Fig. 2c–e). Next,
we conducted cytogenetic analysis to examine chromosome instability.
In contrast to the very high number of micronuclei in erythrocytes,
Mcm4Chaos3/Chaos3 MEFs did not show a significant increase in spontaneous
chromosome aberrations. We speculate that erythroblasts
have less stringent DNA repair and checkpoint controls because the
final cell division is terminal and nuclei become lost. We therefore
treated MEFs with the DNA replication inhibitor aphidicolin (Aph),
which induces chromosome breaks at common fragile sites (specific
chromosome loci that are highly unstable under replication stress)12.
Both Mcm4Chaos3/Chaos3 and Mcm4Chaos3/– MEFs were highly susceptible
to Aph-induced chromosome breaks (Fig. 3a), indicating
that the Mcm4Chaos3 allele predisposes cells to chromosome breakage
under replication stress. Representative metaphase spreads are
shown in Figure 3b.
To assess whether the mutation affects MCM4 stability, we examined
amounts of protein in MEFs by protein blot analysis (Fig. 3c).
The amount of MCM4 in Mcm4+/– MEFs was similar to wild-type
MEFs; however, the amount of MCM4 seemed slightly reduced in
Mcm4Chaos3/Chaos3 MEFs and very markedly reduced in Mcm4Chaos3/–
MEFs. Thus, F345I appears to destabilize the MCM4 protein. Unexpectedly,
the level of MCM7 was appreciably lower in both
Mcm4Chaos3/Chaos3 and Mcm4 Chaos3/– MEFs. In yeast, reduced amounts
Figure 1 Elevated levels of micronuclei in
Chaos3 mutant mice and positional cloning of
an Mcm4 mutant allele. (a) Representative flow
cytometric plots of wild-type (top), Mcm4Chaos3/+
(middle) and Mcm4Chaos3/Chaos3 (bottom)
erythrocytes. Spontaneous micronuclei were
measured in normochromatic erythrocytes (NCE),
an older erythrocyte population (the lower
quadrants). Micronucleated NCEs (MN-NCE)
reside in the lower right quadrant, which
represents a population positive for propidium
iodide but not the CD71 cell surface antigen.
Spontaneous micronuclei in CD71-positive
reticulocytes (upper right quadrant) are rare in
general, as reticulocytes constitute only a small
percentage of total erythrocytes. (b) Genetic
mapping of Chaos3 to a 1.3-Mb region of
chromosome 16 containing Mcm4. Coordinates
in Mb are from the National Center for
Biotechnology Information (NCBI) m36 assembly.
Genes with known functions are shown. Shown
are five recombinants, recovered from 971
meioses, that define the Chaos3 critical region.
(c) Sequence traces showing the T-A
transversion (arrows) identified in the Mcm4
allele of Chaos3 mice. (d) F345I change
identified in MCM4 (shown as a rectangle with
corresponding amino acid residue numbers). The
zinc finger motif and the highly conserved MCM box are also shown. (e) Sequence alignment of Methanobacterium thermoautotrophicum MCM (MtMCM) and
eukaryotic MCM4 proteins. M. thermoautotrophicum has a single MCM protein, which is used as a model for eukaryotic MCMs. Mm, Mus musculus; Hs,
Homo sapiens; Xt, Xenopus tropicalis; Sc, Saccharomyces cerevisiae. The phenylalanine mutated in Mcm4Chaos3 mice (in red) is conserved between MtMCM
and eukaryotic MCM4 proteins. Other conserved residues are in boldface.
104
103
102
101
100
100 101 102 103 104
104
103
102
101
100
101 100 102 103 104
104
103
102
101
100
Propidium iodide
100 101 102 103 104
CD71-FITC
15.27
15.83
16.55
16.85
MMU16 Mb Gene Marker Number of
recombinants
Ube2v2
Mcm4
Prkdc
Pkp2
Yars2
Dmn1l
Fgd4
D16Mit56
D16Mit34
D16Jcs61
D16Mit74
3/971
2/971
Chaos3 critical
region
MtMCM
MmMCM4
HsMCM4
XtMCM4
ScMCM4
158
330
331
331
376
185
359
360
360
405
WT
Mutant
MCM4
1
Zn
444 769
862
MCM box
F345I
a b
c
e
d
MN-NCE
7.51 %
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© 2007 Nature Publishing Group http://www.nature.com/naturegenetics
of MCM proteins have been reported to compromise normal licensing
and genome stability13,14. A recent study proposed that a reduced
number of licensed origins could have a profound effect on DNA
replication, especially under replication stress15. Given the low levels of
MCM4 and MCM7, Mcm4Chaos3/Chaos3 cells may start replication with
fewer licensed origins, which could explain the hypersensitivity to
Aph-induced chromosome breakage.
To determine if the genomic instability in Mcm4Chaos3 mice predisposes
to cancer, we conducted a preliminary study of mice in a mixed
genetic background. This gave a strong indication that homozygous
females were prone to tumors (Supplementary Table 2 online).
Therefore, we rendered the mutation congenic in the C3HeB/FeJ
background to study tumor latency and spectrum in a defined genetic
background. Whereas homozygous males did not show any significant
increase in tumor incidence (data not shown), homozygous females
(the majority of which were virgins) were highly and exclusively prone
to mammary tumors. Thirteen out of sixteen homozygous females
developed mammary tumors with a mean latency of 12 months
(Fig. 4a–c), and five females had more than one tumor. As summarized
in Table 2, the thoracic mammary gland was the most commonly
affected site (9/18 tumors), followed by the cervical mammary gland
(7/18 tumors). Two tumors metastasized to the lung. Most of the
neoplastic lesions were solid, pale, palpable subcutaneous nodules.
A few had soft centers. The histological characteristics of all tumors
were consistent with adenocarcinomas, varying from solid homogeneous
to papillary patterns (Fig. 4d).
As MCMs are present at high levels in cycling cells but not in
quiescent cells, they have emerged as a diagnostic precancer marker16.
However, our data provide the first evidence that defects in MCM
proteins themselves can cause cancer and that their proper function is
required for tumor suppression. It has been suggested that the MCM
proteins may have additional roles beyond replication such as cellcycle
checkpoint responses and transcriptional regulation17,18. Notably,
an unusually high fraction of breast epithelial cells express MCM2
despite their nonproliferating status19,20, which may have implications
for the tumor specificity of the Mcm4Chaos3 mutation.
Genomic instability is a hallmark of cancer cells, although the extent
to which it has a causative role in cancer is a source of debate. However,
recent studies have demonstrated common fragile site instability
in precancerous lesions, suggesting that replication stress–induced
chromosome instability may contribute to cancer development21,22.
Considering that key genes involved in the G1-to-S transition are
mutated or deregulated in most human cancers23, premature entry to
S phase might create a situation leading to chromosome instability24
by inhibiting chromatin loading of the MCM2-7 complex25,26.
In this context, the mechanism of chromosome instability in
Mcm4Chaos3/Chaos3 cells might be relevant to other cancer cells. Detailed
analyses of S phase dynamics, particularly origin licensing, might
illuminate the nature of chromosome instability in cancer.
ARS120

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