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www.sciencemag.org/cgi/content/full/science.aad5634/DC1
Supplementary Materials for
Ubiquitinated Fancd2 recruits Fan1 to stalled replication forks to prevent genome instability
Christophe Lachaud, Alberto Moreno, Francesco Marchesi,
Rachel Toth, J. Julian Blow, John Rouse*
*Corresponding author. E-mail: [email protected]
Published 21 January 2016 on Science First Release DOI: 10.1126/science.aad5634
This PDF file includes:
Materials and Methods Figs. S1 to S6 References
2
Materials and Methods
Mouse strains and husbandry
Animals were housed under specific pathogen free conditions in accordance with UK and
EU regulations. All procedures were carried out in accordance with University of Dundee
and United Kingdom Home Office regulations.
To generate Fan1nd/nd mice, a targeting vector was constructed to replace exon 13 on the
murine Fan1 gene with a mutated exon where the codon for Lys975 was mutated to
encode an alanine, and a puromycin resistance cassette (Fig. S1A). FRT sites were
introduced so that the puromycin resistance cassette could be excised from the targeted
locus. A thymidine kinase cassette was included at the 3’ end of the targeting vector to
enable selection against non-targeting random integration. The arms for the targeting
vector - a short homology 5’ arm of 4.0 kb and a long homology arm of 6.2 kb - were
cloned by PCR using an appropriate BAC clone as template. All clones were fully
sequenced to ensure that they contained no PCR generated errors before assembly into
the final targeting vector (Fig. S1A). The linearized targeting vector was electroporated
into E14 C57Bl6 Tac embryonic stem (ES) cells, followed by positive (G418) and
negative (gancyclovir) selection using standard procedures. Resistant clones were
screened for targeted integration by southern blotting and PCR (data not shown), and six
targeted ES cell clones were isolated. These clones were injected into BALB/c
blastocysts, which were then re-implanted into recipient female mice. Male chimeric
mice showing a high percentage of ES cell contribution were bred to C57BL/6 female
mice. After recovery, 8 injected blastocysts were transferred to each uterine horn of 2.5
days post coitum, pseudopregnant NMRI females. Chimerism was measured in chimeras
(G0) by coat color contribution of ES cells to the BALB/c host (black/white). Highly
chimeric mice were bred to strain C57BL/6 females. The C57BL/6 mating partners were
mutant for the presence of a recombinase gene (Flp-Deleter), which then excised the
puromycin resistance cassette leaving just a single FRT site between exon 13 and 14 of
the Fan1 gene. Germline transmission was identified by the presence of black, strain
C57BL/6, offspring (G1). Correct targeting and removal of puroycin resistance cassette
3
were verified by southern blotting and PCR. Mice were routinely genotyped from ear
notch biopsies according to standard procedures. Genotyping with primers P1 (5’
TCTGTTTCTCCCTTAATGTTTAGC) and P2
(5’CACAGTGTTAGATTCTGTCTGATACC) rise to a 159 bp band in wild type alleles
and 234 bp in knockin alleles because of a residual FRT site in the knockin allele.
Representative genotyping results are shown in Fig. S1B. Additionally, successful
mutation of the K975 residue was verified by PCR using P3
(5’GGCAGAAGCACGCTTATCG) and P4 (5’GACAAGCGTTAGTAGGCACAT)
primers. The generated 326 bp product was then cloned and sequenced.
Fancd2-/- mice described previously were a kind gift from Alan d’Andrea. All mice were
backcrossed 4 times before analysis.
mRNA sequencing
Total RNAs were purified using RNeasy kit from Qiagen. Sequence at the interface
between exon 13 and the following intron have been amplified by RT-PCR (Takara
PrimeScript High Fidelity RT-PCR Kit) using primers P3 and P4 and clone into pSC-A
before sequencing.
Isolation and immortalization of MEFs
13.5 dpc embryos were derived from timed matings between heterozygous parents. After
decapitation, the heads were used for genotyping. The red organs were removed, the
embryo was minced and resuspended in 1 ml trypsin and incubated at 37°C for 15 min
before the addition of 10 ml growth medium. Cells were plated and allowed to attach
over night before cells were washed with fresh medium to remove debris. When cells
reached confluency they were split and re-plated and this was considered passage 1. All
MEFs were immortalized using SV40 large T antigen.
Virus-based rescue experiments in MEFs
Viruses were produced by co-transfecting 293T cells with the relevant open reading
frames cloned into pBABE vector, and with pCMV-VSV-G and pCMV-Gag-Pol
4
expression vectors to direct viral packaging. After 48 h the virus-containing supernatant
was filtered through a 45 µm filter, mixed with polybrene (final concentration of 8
µg/ml) and added to MEFs. Three hours later the infection medium was replaced with
fresh growth medium. The infection protocol was repeated 24 h later. MEFs were stably
selected with puromycin (3µg/ml) 24 h after the second infection. Protein expression
was tested by western blotting.
Clonogenic survival analysis
MEFs were seeded in triplicates in 10 cm dishes and were allowed to attach before
treatment. Genotoxin was added to cells for 24 h before medium was replaced with fresh
growth medium. After 10 days cells were washed, fixed and stained with Giemsa. The
number of colonies with >100 cells were counted. For each genotype, cell viability of
untreated cells was defined as 100%. Data are represented as mean ± SD from three
independent experiments.
Analysis of chromosomal abnormalities
For the analysis of chromosomal abnormalities MEFs were left untreated, or were
exposed to MMC (50 ng/ml) for 24 h or MMS (50 µM or 100 µM) for 20 h. In the case
of HU, cells were treated with HU (1 mM) for 6 h followed by 6 h recovery. Colcemid
(0.1 mg/ml) was added to the cells for 30 min. Cells were then trypsinized, washed and
incubated with 75 mM KCl for 20 min at 37°C. Cells were subsequently fixed with 3:1
methanol:acetic acid at 4°C overnight before spreading. Spreads were stained with DAPI
and 20 spreads were analyzed for chromatid breaks and radial chromosomes; at least two
independent experiments were performed. Data are presented as chromosome
abnormalities per metaphase.
DNA fiber assay
Cells were incubated with CldU (20µM; Sigma C6891) for 20 min, then quickly washed
with PBS, before being given a 120 min pulse of IdU (100 µM; Sigma I7125) in the
presence of HU (0.5 mM) added at the same time as IdU. In control experiments, cells
were incubated with CldU for 20 min, followed by a 20 min pulse of IdU in the absence
5
of HU. Cells were harvested, lysed and spread to obtain single DNA molecules on
microscope slides before standard immunofluorescence with antibodies against BrdU
(Anti-BrdU Rat, BioRad OBT0030CX; Anti-BrdU Mouse, BD 357580). Images were
acquired using an Olympus IX70 deltavision deconvolution microscope. An Olympus
63x oil immersion objective was used. Images were captured using a CCD camera. 100
fields or more from each condition were analysed. Data from microscopy experiments
was analysed using Volocity 3-dimensional (3D) image analysis software (PerkinElmer).
A standard protocol was designed within this programme for measuring the IdU-tracks
length.
Cell culture
All cells were kept at 37°C under humidified conditions with 5% CO2. U2OS and 293T
cells were grown in DMEM, 10% FBS, L-glutamine and penicilin/streptomycin. For
MEFs, medium was supplemented with Na-pyruvate and non-essential amino acids.
Puromycin was used at 3 µg/ml for selection of complemented (virus-infected) MEFs, or
stably transfected U2OS cells.
Cell lysis and immunoprecipitation
Cells were lysed in ice–cold buffer: (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 270 mM
sucrose, 1% (v/v) Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.1% ß-mercaptoethanol)
with protease inhibitors (Roche) and 50 U/ml of benzonase (Novagen).
Immunofluorescence-based detection of Fan1 foci
For indirect immunofluoresence analysis, cells were plated onto sterile coverslips and
treated with either MMC (50ng/ml) or HU (10mM) for 6 hours. Coverslips were then
rinsed in PBS and fixed using 4% paraformaldehyde for 10 min at room temperature.
Cells were permeabilized with 0.2% Triton X–100 in PBS for 10 min at room
temperature, washed several times in PBS and incubated in blocking solution (PBS
containing 5% donkey serum, 0.1% fish skin gelatine and 0.05% Tween-20). Coverslips
were incubated with primary antibodies against GFP (Abcam) in blocking solution for 1
h at room temperature. After washing in PBS, coverslips were incubated with the
appropriate 488-conjugated secondary antibodies (Life Technologies) for 90 min. The
6
nuclei were stained with DAPI for 5 min and coverslips mounted on glass slides. Cells
were imaged using a Zeiss 710 confocal microscope equipped with a 37°C environmental
chamber and supplied with humidified 5% CO2, a Zeiss Plan-Apochromat 63x 1.4NA oil
immersion objective lens.
Detection of G2 arrest by flow cytometry
Low passage, low confluence cultures of asynchronously growing MEFs were treated
with MMC (50ng/ml) (Sigma-Aldrich) for 48 h. Following trypsinization, cells were
collected by centrifugation, washed in PBS and fixed in ice-cold 70% ethanol. Cells were
centrifuged, washed in PBS and treated for 15 min at 37°C with 50 µg/ml RNase A. Cells
were stained at room temperature with propidium iodide (PI) solution (2 mM MgCl2, 10
mM PIPES buffer, 0.1 M NaCl, 0.1% Triton X-100, 0.01 mg/ml PI) and analysed on a
FACS Calibur flow cytometer (BD Biosciences). Cell cycle profiles were created using
FlowJo analysis software (Tree Star Inc., Ashland OR).
Edu incorporation detection by flow cytometry
Low passage, low confluence cultures of asynchronously growing MEFs were treated
with HU (1mM, 2 h) prior treatment with Edu (0.5 mM, 2 h). Following trypsinization,
cells were collected by centrifugation, washed in PBS and fixed in ice-cold 70% ethanol.
Cells were centrifuged, washed in PBS and treated for 15 min at 37°C with 50 µg/ml
RNase A. Cells were stained at room temperature with propidium iodide (PI) solution (2
mM MgCl2, 10 mM PIPES buffer, 0.1 M NaCl, 0.1% Triton X-100, 0.01 mg/ml PI) and
analysed on a FACS Calibur flow cytometer (BD Biosciences). Cell cycle profiles were
created using FlowJo analysis software (Tree Star Inc., Ashland OR).
Histopathology
Animals were euthanased by means of CO2 asphyxiation and a complete post mortem
evaluation and dissection was performed. The following tissues were collected and fixed
in 10% neutral buffered formalin: brain, salivary glands and mandibular lymph nodes,
thymus, heart, lungs, stomach, pancreas, small and large intestine, pancreatic and
mesenteric lymph nodes, spleen, liver, kidney, female reproductive system (ovaries and
7
uterus), male reproductive system (testes, epididymides, seminal vesicles and prostate),
femur, with stifle joint and proximal tibia.
Following 48 hour fixation the tissues were trimmed and samples were processed to
paraffin blocks. Sections (3 µm-thick) were stained with haematoxylin and eosin (HE)
and evaluated by a veterinary pathologist unaware of the genotype of the animals.
Antibodies
Antibodies against mouse Fan1 were raised in sheep against the 300 first amino acid of
mouse Fan1 fused to GST. Antibodies were affinity purified using immobilized antigen
(S778D) and used at 0.5 mg/ml for 1h. Antibodies against mouse Fancd2 were raised in
sheep against the full human FancD2 fused to GST (S099D) and used at 0.5 mg/ml for
12h at 4oC. Anti-GAPDH (14C10) antibodies were purchased from Cell Signalling. All
secondary antibodies were purchased from Pierce. Anti-GFP and anti-mCherry antibodies
were purchased from Abcam. Commercial antibodies were used according to the
manufactures instructions.
DNA constructs
The full-length coding regions for the relevant human proteins were generated by PCR
using IMAGE consortium EST clones. Full-length coding regions for the relevant mouse
proteins were generated by RT-PCR (Takara PrimeScript High Fidelity RT-PCR Kit).
Point mutations were introduced by Quickchange mutagenesis. The sequence integrity
was confirmed by sequencing analysis. Human and mouse cDNA were respectively
cloned into pcdna5 FRT/TO.puro vector and pBabe.puro vectors.
Statistical analysis
Statistical significance calculated using one-way ANOVA (****, p < 0.0001) and
Bonferroni’s Multiple Comparison Test.
8
Reagents
All cDNA plasmids, antibodies and recombinant proteins generated for this study are
available to request through our reagents website (https://mrcppureagents.dundee.ac.uk/).
9
Supplementary figures
Lachaud_Fig.S1
GFP-Fan1UBZ*
GFP-Fan1
untreated HU MMC
0
20
40
60
80
cells
with
>5
foci
(%) mock
MMC HU
C.
A.
D.
U2OS MEF
GFP-Fan1wild-type UBZ*GFP
B.
Fan1
GFP
Fan1
GapdhGapdh
GFP
blot: blot:GFP-Fan1
endo. Fan1
Fan1nd/nd
GFP
GFP
-Fan
1
GFP
-Fan
1UBZ*
Fan1-/-Fan1
-/-
Fan1
+/+
GFP
GFP
-Fan
1G
FP-F
an1UB
Z*
GFP
-Fan
1M50
R
GFP
GFP-Fan1endo. Fan1
10
Figure S1: Murine Fan1 UBZ mutation abolishes Fan1 foci
A. Extracts of U2OS Fan1-/- cells stably expressing GFP or GFP-tagged forms of Fan1,
Fan1-UBZ* (C44A+C47A) or Fan1 M50R, and extracts of parent U2OS cells (Fan1+/+)
expressing GFP were blotted with the antibodies indicated. B. Extracts of Fan1nd/nd MEFs
stably expressing GFP or GFP-tagged forms of Fan1 or Fan1-UBZ* (C44A+C47A) were
blotted with the antibodies indicated. C. Representative images of foci formed by GFP-
tagged murine Fan1 or the Fan1 C44A+C47A (UBZ*) mutant in U2OS cells 6h after 6 h
exposure to the genotoxins indicated. D. The proportion of cells with greater than five
Fan1 foci per cell was counted in 100 cells, in three independent experiments.
11
targeted allele
(after homologous recombination)
1 2 3 4 5 6FAN1 locus 7 8 9 10 11 12 13 14
Targeting vectorPuroR
23TK
13 14
K975A
SHA: 4.0 kb LHA: 6.2 kb
PuroR
1 2 3 4 5 9 10 11 12 13 14
knockin allele
(after Flp recombination)
3 4 5 9 10 11 12 13 14
Fan1 coding exon Fan1 targeted exon
Fan1 untranslated region
Mouse genomic region
FRT site SHA: Short homolgy ArmLHA: Long Homology Arm
11 12 13 14 11 12 13 14
11-14
genomic DNA
mRNA
S H H C LK V VE
exon_13 exon_14
AGCCACCATTGCAAGCTGGTGGAGGTG
TCGGTGGTAACGTTCGACCACCTCCAC
S H H C VA S ER
exon_13 Intron
AGCCACCATTGCGCCGTCAGTAGAGAACACCATTTGTAA
TCGGTGGTAACGCGGCAGTCATCTCTTGTGGTAAACATT
H H L *
975
C.
D.
A.
100-200-300-400-500-
1000-3000-
+/+ +/nd nd/nd
nd alleleWT allele
Fan1
GAPDH
115-
39-
+/+E.
B.
Lachaud_Fig.S2
Fan1:
+/nd
knockin Fan1 allelewild type Fan1 allele
nd/nd
GAPDH
Fan1Fan
1nd/n
d
Fan1+
/+
Fan16
Fan1F
L
MEFsU2OS Fan1-/-
+ plasmid
P1 P2
Fan1F
L
Fan1+
/+
F.
stop
stop
stop
stop
975
11-13 14
P3P4
12
Figure S2: Generating Fan1 nuclease-defective mice
A. Generation of Fan1 nuclease-defective mice (see Methods below). A strategy was
devised to introduce a mutation in exon 13 of the murine Fan1 gene that would replace
Lys975 with an alanine residue. The black/grey rectangles represent exons and the green
triangles represent FRT sites. The knock-in allele containing the K975A mutation in exon
13 is illustrated as a red rectangle. The knock-in allele can be detected by genotyping
using PCR primers P1 and P2, which are represented by black arrows. B. Genomic DNA
extracted from tail biopsies were analyzed by PCR with the oligonucleotides P1 and P2
indicated in A. Results are displayed as an electronic gel picture. PCR with primers P1
and P2 yields a 159 bp band with the wild type Fan1 allele, and 234 bp with the knockin
allele because of a residual FRT site in the knockin allele. C. Effect of Fan1 knockin
mutation on Fan1 pre-mRNA splicing. D. The Fan1 knockin mutation changed K975 to
A, but also inhibited splicing of exon 13 with exon 14 (the last Fan1 exon), resulting in
retention of the intervening intron in the mRNA. As a result A975 in the knockin allele is
followed by seven intron-encoded amino acids and a stop codon, concomitant with
deletion of the last 41 amino acids of Fan1. The 41 amino acids deleted includes several
residues essential for catalysis such as Asp 981 and Arg 982 (4). E. Western blot of MEF
extracts from mice of the indicated genotypes. F. Human U2OS Fan1-/- cells were
transfected with full length (FL) murine Fan1, or a truncated form of Fan1 (Fan1Δ)
corresponding to the truncated protein that results from inhibition of splicing of exon 13
(see D). Extracts were subjected to SDS-PAGE and western blotting, in parallel with
extracts of Fan1+/+ and Fan1nd/nd MEFs.
13
Lachaud_Fig S3 A. Genomic DNA sequence: FAN1+/+ CTGTCCTCAGTGGTGTGTGCAGGCGCCTGGCTGCTGACTTTCGGCACTGCCGAGGGGGCCTCCCAGACTTGGTGGTGTGGAATTCTCAGAGCCACCATTGCAAGGTCAGTAGAGAACACCATTTGTAATTTCAGCAGCAGCTATGTCACAGTTCACGTGCTGGACATTTTCTTCTGACTGTTGGAAGGATCAAATTCCTTGATAGCCACCAGGCTGAGGAGCTGGGGTGCAAGCAAGACAGGCAGTCTCATGTCAGCCCATCATGGAGGTGATAGTCGACCAGTACAACATTCTGTTACTAGAACAAGCCACTAGCTGCGAGGCAC Genomic DNA sequence: FAN1nd/nd CTGTCCTCAGTGGTGTGTGCAGGCGCCTGGCTGCTGACTTTCGGCACTGCCGAGGGGGCCTCCCAGACTTGGTGGTGTGGAATTCTCAGAGCCACCATTGCGCCGTCAGTAGAGAACACCATTTGTAATTTCAGCAGCAGCTATGTCACAGTTCACGTGCTGGACATTTTCTTCTGACTGTTGGAAGGATCAAATTCCTTGATAGCCACCAGGCTGAGGAGCTGGGGTGCAAGCAAGACAGGCAGTCTCATGTCAGCCCATCATGGAGGTGATAGTCGACCAGTACAACATTCTGTTACTAGAACAAGCCACTAGCTGCGAGGCAC B. RT-PCR analysis of mRNA from Fan1nd/nd mouse kidney, using Fan1 primers P3 and P4 (see Fig. S1A) CTGTCCTCAGTGGTGTGTGCAGGCGCCTGGCTGCTGACTTTCGGCACTGCCGAGGGGGCCTCCCAGACTTGGTGGTGTGGAATTCTCAGAGCCACCATTGCGCCGTCAGTAGAGAACACCATTTGTAATTTCAGCAGCAGCTATGTCACAGTTCACGTGCTGGACATTTTCTTCTGACTGTTGGAAGGATCAAATTCCTTGATAGCCACCAGGCTGAGGAGCTGGGGTGCAAGCAAGACAGGCAGTCTCATGTCAGCCCATCATGGAGGTGATAGTCGACCAGTACAACATTCTGTTACTAGAACAAGCCACTAGCTGCGAGGCAC
14
Fig. S3; Sequencing around Fan1 knockin mutation in Fan1nd/nd mice
A. Genomic DNA extracted from ear notch biopsies of Fan1+/+ and Fan1nd/nd mice was
subjected to PCR analysis with the primers P3 and P4 (shown in Fig. S1A). Primer P3
lies within exon 13 and primer P4 within the following intron. The resulting PCR
products were TA-cloned into plasmid pSC-A (Invitrogen), and sequenced backwards
and forwards. The raw DNA sequences of the cloned inserts are shown. The codon
corresponding to K975 is highlighted in red. The primer sequences are highlighted in
yellow. B. Total RNA extracted from the kidneys of Fan1+/+ and Fan1nd/nd mice was
subjected to reverse transcription followed by PCR with the primers P3 and P4. This
yielded a product of 326 bp using mRNA from Fan1nd/nd mice but not from wild-type
mice – P4 anneals to the intron after exon 13, which is spliced out of the wild-type Fan1
mRNA. The PCR product from Fan1nd/nd mice was TA-cloned into plasmid pSC-A, and
sequenced backwards and forwards. The sequence of the cloned insert is shown. The
sequences annealing to primers P3 and P4 are highlighted in yellow. Exon 13 is
highlighted in green. The codon corresponding to A975 is highlighted in red. The intron
that lies between exon 13 and 14 is highlighted in blue.
15
Lachaud_Fig.S4
0
2
4
6
8
10
12
0 50 100 0 50 100
Fan1-/-Fan1+/+
MMS (µM):
DEQRUPDOLWLHV�SHU�P·SKDVH
0.25 1.05 2 0.3 3.1 5.5
*********
nsns
mean:fold: 1 4.2 8.0 1.2 12.4 22
Figure S4: Increased chromosome abnormalities after MMS treatment of Fan1-/-
U2OS cells
Chromosome abnormalities per metaphase were counted in human U2OS Fan1-/- cells, or
parental U2OS cells (Fan1+/+) exposed to the indicated concentrations of MMS for 20 h.
16
Lachaud_Fig.S5
+/nd nd/nd +/nd nd/nd +/- +/- -/- -/-
+/nd
nd/nd
+/nd
nd/nd
+/-
+/-
-/-
-/-
Fan1 FancD2
Fan1 genotype:Fancd2 genotype:
fiber
leng
th
0
10
20
30 nsgenotypes
A.
B.
mCherry-FancD2
Gapdh
input
Empt
y
mC
he-F
ancD
2
mC
he-F
ancD
2K559
R
FancD2-/-
Empt
y
mC
he-F
ancD
2
mC
he-F
ancD
2K559
R
IP: mCherry
*
Figure S5: Fan1/Fancd2 does not affect progression of forks before stalling
A. Extracts of Fancd2-/- MEFs stably expressing mCherry, mCherry-Fancd2 or mCherry-
Fancd2-K561R were blotted with the antibodies indicated. Asterisk denotes non-specific
band. B. Dot plots of ldU replication tracks in MEFs of the genotypes indicated that were
not treated with HU. Representative CldU and IdU replication tracks are shown (right
panels). Red lines represent mean track length.
17
Lachaud_Fig.S6
Ed
U (
rela
tive
flu
ore
sce
nce
)
0 50 100 200
mock HU
Fan1
+/+
Fan1
nd/n
d
102
0
103
104
105
102
0
103
104
105
0 50 100 200
DNA content (A.U.)
Figure S6: Edu incorporation in Fan1nd/nd MEFs after HU treatment
Fan1+/+ and Fan1nd/nd MEFs were treated with HU (1 mM; 2 h) follow by EdU treatment
(30 min). After fixation, cells were stained with propidium iodide and subjected to FACS
to monitor DNA content and EdU incorporation.
18
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