Supplemental Materials: 1. Table S1. Primers used in the...

1

Supplemental Materials: 1. Table S1. Primers used in the study 2. Supplemental Figure legends 3. Supplemental Figures

2

Table S1. Primers used in the study.

Mouse genotyping primers Genotype Primer sequence Amplicon size (bp)

Fan1+ 5’-AAGGTCTGTGGTCATCGTGTCAG-3’ 5’-AAACGATCCCTGTCTGGCTGA-3’ 292

Fan1- 5’-TGAGACAGCTCAACTGGCACT-3’ 5’-AAACGATCCCTGTCTGGCTGA-3’ 543

Fancd2+ 5’-TCAGCCTCACATGGAGTTTAACG-3’ 5’-AATTCGCCAATGACAAGACGC-3’ 300

Fancd2- 5’-TCAGCCTCACATGGAGTTTAACG-3’ 5’-CAGGGATGAAAGGGTCTTACGC-3’ 550

Fanca+ 5’-TTCCTTCAAAGCTGCTGGGG-3’ 5’-CAGTGACATCTTCCTTCCTAACTCC-3’ 350

Fanca- 5’- TTCCTTCAAAGCTGCTGGGG-3’ 5’- GGTGAACGTTACAGAAAAGCAGGCT-3’ 600

Slx4+ 5’-CACTGAGCCATCTCACCAGC-3’ 5’-GGAGCCCAGTCTGGGACTCTG-3’ 475

Slx4f3 5’-CACTGAGCCATCTCACCAGC-3’ 5’-TCGTGGTATCGTTATGCGCC-3’ 298

Mus81+ 5’-CTAGCCGCTTGCGTTCCACAATGT-3’ 5’-GGAGCTAAGGCCTAGCGAGTACAG-3’ 412

Mus81- 5’-GGTGTGGCCCTGATGGAAGAG-3’ 5’-GGAGCTAAGGCCTAGCGAGTACAG-3’ 380

Mutagenesis primers Mutation Primer sequence

FAN1 C44A/C47A

5’-CAGTGCACCACCTGCTAAACTTGCAGCTTCAACTGCTCAT AAAATGGTGCCCA-3’

FAN1 L480P 5’-TCTGCTCCTGAGCCGAAAGCCCTGGCC-3’

FAN1 D963A 5’-GGGGCCTCCCAGCCTTGGTGGTGTG-3’

RT-PCR primers Gene Primer sequences

Fan1 5’-CACAGTCCTGTGTTCTGTGGA-3’ 5’-TCAAAGCCACTCTGCCTGTA-3’

Snm1a 5’-GCAAAGTCCACACACTGTTCC-3’ 5’-CTGCTGTGACGGGAAGGTAT-3'

Actin 5’-CTAAGGCCAACCGTGAAAAG-3’ 5’-ACCAGAGGCATACAGGGACA-3’

shRNA target sequences

shRNA Target sequences Snm1a.1 5’-TGACTCTTTTCTGCTTCT-3’ Snm1a.2 5’-CAGAATCTGTTGAAAAATCA-3’ Snm1a.3 5’-CATGTCCGTTCTATAAGAGA-3’

sgRNA target sequences Gene Target sequences

Mouse Snm1a 5’-CGAAGGTGCGCCCTGTTTAT-3’ Human FAN1 5’-CGTTCAAGTGGATCCAGGGC-3’

3

Supplemental Figure Legends

Supplemental Figure 1. Targeting strategy to create the Fan1 deficient mouse

(A) Targeting strategy for Fan1 deficient mouse. Coding exons 1-6 of the Fan1 genomic locus in wild type

(chromosome 7), the Fan1stop, the Fan1lox and the Fan1Δex3&4 mutant alleles generated are shown. The

wild-type Fan1 gene contains a 20.5 kb BamHI restriction fragment that hybridizes to the 5’(red) and 3’(dark

green) probes. A correctly targeted Fan1stop locus contains a 13.8 kb fragment that hybridizes to the 5’ probe

and 7.5 kb fragment that hybridizes to the 3’ probe. The neomycin cassette (pink), the FRT (blue), the LoxP

(green), the splice acceptor (SA), and the poly A (pA) sites are also shown. The cross between Fan1+/stop and

FLP deleter mice generated Fan1+/lox. The cross between Fan1+/lox and FVB/N-Tg(Ella-cre)C5379Lmgd/J

mice to derive Fan1+/-(Δex3&4) are shown. The comparison between full-length wild type FAN1 with the

domain architecture and the expected truncated FAN1 protein product expressed from Fan1Δex3&4 allele are

shown. (B, C) 5’ and 3’ Southern blot analyses of BamHI–digested genomic DNA from Fan1+/+ and

Fan1+/stop mice. CR, cross reacting band. (D) Schematic showing primer positions used for genotyping (top).

Genotyping PCR for Fan1+ and Fan1- alleles using DNA from MEFs of the indicated genotypes (bottom). (E)

Schematic of exons 9-11 of Fan1, showing exon 10 q-PCR primers used in panel (F). (F) Quantification of

Fan1 mRNA level in MEFs of indicated genotypes. (G) Survival of Fan1+/+ and Fan1-/- MEFs treated with

hydroxyurea (HU). MEFs were treated in triplicate with increasing concentrations of HU. Error bars, s.d. (H)

Survival of indicated MEFs after camptothecin (CPT) treatment (0-20 nM). MEFs were treated in triplicate with

increasing concentrations of CPT. Error bars, s.d. (I) MEFs stably transduced with vector expressing WT FAN1

or FAN1 D963A nuclease dead variant were exposed to MMC (0-100 nM). Error bars, s.d. (J) Immunoblot

showing expression of WT FAN1 or FAN1 D963A in MEFs used in (I). For all sensitivity assays, cell numbers

were determined six days after the treatment and numbers were normalized to untreated control to calculate

percent survival.

Supplemental Figure 2. FAN1 localizes to sites of psoralen damage independently of FANCD2

ubiquitination.

4

(A) Illustrative images showing accumulation of GFP-hFAN1 at the UV-induced ICL as a function of time.

U2OS cells were subjected to psoralen/ UV laser induced ICLs, fixed at different time points post ICL induction

and imaged. (B) Percentage of cells with a FAN1+ stripe as a function of time. Minimum of 50 cells were

quantified for each time point. (C) Representative images showing recruitment of endogenous FAN1 to the

crosslink stripe at different time points. PD20 cells complemented with empty vector (PD20+Vector), wild type

FANCD2 (PD20+D2) or a non-ubiquitinatable mutant FANCD2 (K561R) (PD20+D2KR) were subjected to

psoralen/laser induced ICLs, fixed at different time point post ICL induction and imaged. (D) Immunoblot

assessing expression of human FAN1 transduced in RA3087 FANCA-/- FAN1-/- clone 1 and clone 2. The

clones highlighted in red were used for the experiment outlined in Fig. 3F. (E) Blinded quantification of cells

with more than 20 γH2AX foci following MMC treatment.

Supplemental Figure 3. Fan1 deficient mice are born at Mendelian ratio, grow normally and are fertile.

(A) Fan1-/- mice are born at the expected Mendelian ratio. Viability of Fan1-/- mice was determined by

genotyping the progeny from crosses of Fan1+/- female and Fan1+/- male at 21 days of age. p-value was

calculated using the χ2 -test. (B) Weight of male and female Fan1+/+, Fan1+/-, and Fan1-/- mice from 3-12

weeks of age. (C) FAN1 deficient mice are fertile as assessed by the comparison of litter size from Fan1+/-

xFan1+/- and Fan1-/-x Fan1-/- crosses.

Supplemental Figure 4. Liver function, but not kidney function is abnormal in FAN1 deficient mice.

(A-D) Analysis of serum level of (A) blood urea nitrogen (BUN), (B) creatinine, (C) phosphorus, and (D)

magnesium to monitor kidney function of Fan1+/+ and Fan1-/- mice at indicated ages. (E) Quantification of

FISH signals per nucleus in 18 months old Fan1-/- animals. Tubular epithelial cells in the kidney, liver

hepatocytes and cells in the spleen were assessed (F-I) Analysis of serum level of liver enzymes and markers of

liver function: (F) alanine transaminase (ALT), (G) aspartate transaminase (AST), (H) albumin (ALB), and (I)

globulin (GLOB). Bars represent mean ± SD; ***p < 0.001, **p < 0.01 were calculated using F-test.

5

Supplemental Figure 5. Blood counts are normal in the majority of FAN1 deficient mice.

(A-E) Blood analysis in Fan1+/+ and Fan1-/- mice: (A) platelets (PLT), (B) white blood cells (WBC), (C) red

blood cells (RBC), (D) reticulocytes (RET), and (E) hemoglobin concentration.

Supplemental Figure 6. Bone marrow function is intact without exogenous stress in FAN1 deficient mice

but the mice are sensitive to mitomycin C.

(A) Representative FACS profiles of HSCs isolated from bone marrow of Fan1+/+ and Fan1-/- mice,

indicating LSK and LK population. (B) Analysis of LT-HSC, ST-HSC and MPP isolated from femurs and

tibiae of Fan1+/+ and Fan1-/- mice at indicated ages assessed by FACS. Error bars, s.d., n = 3 per genotype. (C)

Analysis of MEP (megakaryocyte/erythroid progenitors), GMP (granulocyte/monocyte progenitors), and CMP

(common-myeloid progenitors) isolated from femurs and tibiae of Fan1+/+ and Fan1-/- mice at indicated ages

assessed by FACS. Error bars, s.d., n = 3 per genotype. (D) Analysis of LT-HSC, ST-HSC and MPP isolated

from femurs and tibiae of 6 months old WT, Fan1-/-, Fancd2-/-, and Fan1-/-Fancd2-/- mice assessed by FACS.

Error bars, s.d., n = 3 per genotype. (E) Analysis of MEP, GMP, and CMP isolated from femurs and tibiae of 6

months old WT, Fan1-/-, Fancd2-/-, and Fan1-/-Fancd2-/- mice assessed by FACS. Error bars, s.d., n = 3 per

genotype.

Supplemental Figure 7. FAN1 deficient mice are sensitive to mitomycin C.

(A) Weight monitoring of mice after treatment with MMC. Following intraperitoneal injection of 10 mg of

MMC per kg, Fan1+/+, Fan1+/-, and Fan1-/- mice were weighed every 2 days for 20 days or until death.

Weight is expressed as % of original weight on the day of MMC injection. (B) Number of nucleated cells per

femur of Fan1+/+ and Fan1-/- mice untreated or treated 1 week prior with 10 mg/kg MMC. Bars represent

mean ± SD; **p < 0.01 were calculated using unpaired t-test. (C) Representative FACS profile of bone marrow

cells that were enriched for c-Kit positive population, determined by FSC and 7-ADD (7-amino-actinomycin D)

viability gate. The bone marrow cells were isolated from Fan1+/+ and Fan1-/- mice untreated or treated 1 week

prior with 10 mg of MMC per kg. (D) Red blood cell (RBC) and reticulocyte (RET) counts of Fan1+/+ and

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Fan1-/- mice untreated or treated 1 week prior with 10 mg of MMC per kg. Bars represent mean ± SD; *p <

0.05 was calculated using unpaired t-test.

Thongthip et al. Supplemental Fig. 1

A CB

Fan1

+/st

opFa

n1+/

+Fa

n1+/

stop

Fan1

+/+

Fan1

+/st

op

23 kb 9 kb 7 kb

3’ southern blot

7.5 kb

20.5 kbCR

5’ southern blot

23 kb 9 kb 7 kb

Fan1

+/st

opFa

n1+/

+Fa

n1+/

stop

Fan1

+/+

Fan1

+/st

op

13.8 kb20.5 kb

Fan1+/stop x FLP deleter

Ex1 Ex2 Ex5 Ex6

p.Ala415Glufs*29

100830 67 469 509 893

NUCUBZ SAPWT414

UBZ

443

Fan1+/lox x FVB/N-Tg(Ella-cre)C5379Lmgd/J

Fan1- (Δex3&4)

Ex1Ex2

Ex1Ex2

Wild type Fan1+

Fan1stopFRT FRTLoxP Ex3Ex4 LoxPLoxP Ex5 Ex6

Ex3Ex4 Ex5 Ex6

neo

SA pA

BamHI

BamHI

BamHI

BamHI

BamHI BamHI

5’probe

5’probe 3’probe

3’probe

20.5 kb

13.8 kb 7.5 kb

Fan1loxEx1Ex2 Ex3Ex4 LoxPLoxP Ex5 Ex6

FRT

Protein expression

Fan1Ex9 Ex10 Ex11

forward reverse

E F

0

0.2

0.4

0.6

0.8

1

Fan1+

/+

Fan1+

/-

Fan1-/

-

Fan1

mR

NA

leve

l re

lativ

e to

Fan

1+/+

cel

lsEx1Ex2 Ex5 Ex6

Ex1Ex2 Ex3Ex4 Ex5 Ex6

Fan1- (Δex3&4)

Fan1+

D

+ Fan1+ ++ + Fan1-

Fan1+/+ Fan1+/- Fan1-/-

0

20

40

60

80

100

0 20 40 60 80 100

Sur

viva

l (%

of u

ntre

ated

)

MMC (nM)

Fan1+/+ Fan1-/- Fan1-/- + WT FAN1 Fan1-/- + FAN1 D963A

I

mFAN1

Fan1

-/-

Fan1

+/+

α tubulin

WT

FAN

1FA

N1

D96

3A

Fan1-/-J

G

0 20 40 60 80 100HU (mM)

0

20

40

60

80

100

Sur

viva

l (%

of u

ntre

ated

) Fan1+/+

Fan1-/-

H

0 5 10 15 20CPT (nM)

0

20

40

60

80

100

Sur

viva

l (%

of u

ntre

ated

)

WTFan1-/- Slx4-/-Mus81-/-

Thongthip et al. Supplemental Figure 2

BA

FAN1DAPI

1m 2m 3m

4m 10m

30m0

0.2

0.4

0.6

0.8

1

1.2

0 1-5 6-10 10-15 15-30

Frac

tion

of c

ells

with

FA

N1+

str

ipe

15m

Time (min)

FAN1DAPI+

Vec

tor

1m 5m 15m

+ D

2

1m 5m 15m

PD

20+

D2K

R

1m 5m 15m

C

RA

3087

FA

NC

A-/-

A11

70 F

AN

1-/-

RA3087 FANCA-/-sgFAN1.1 +HA-hFAN1

2 3 4 5 6 7 8 9

α-tubulin

hFAN1***

RA3087 FANCA-/-sgFAN1.2 +HA-hFAN1

3 4 5 6 7 8 9 10RA

3087

FA

NC

A-/-

sgFA

N1.

1

RA

3087

FA

NC

A-/-

sgFA

N1.

2

1 2 11

D E

0

20

40

60

80

100

Untreated 8hr 24hr 48hr

% o

f cel

ls w

ith >

20

H2A

X fo

ci

Fan1+/+shcon

Fan1+/+shSnm1a

Fan1-/-shcon

Fan1-/-shSnm1a

Post 3 μM MMC

Genotype # of pups Observed ratio

Expected Ratio

Fan1+/+ 49 0.24 0.25

Fan1+/- 107 0.51 0.50

Fan1-/- 51 0.25 0.25

Total number 207 p-value 0.8714

Thongthip et al. Supplemental Figure. 3

B

C

A

Maternal genotype

Paternal genotype

Number of Mating tested

Number of litters

Number of pups

Pups/litters

Fan1+/- Fan1+/- 15 15 110 7.3

Fan1-/- Fan1-/- 8 8 65 8.1

5

10

15

20

25

30

3 4 5 6 7 8 9 10 11 12

Wei

ght (

g)

Age (weeks)

Fan1+/+ (n=12)Fan1+/- (n=12)Fan1-/- (n=10)

5

10

15

20

25

30

3 4 5 6 7 8 9 10 11 12

Wei

ght (

g)

Age (weeks)

Fan1+/+ (n=10)Fan1+/- (n=10)Fan1-/- (n=10)

Male Female


BA

C D

F G

H I

Fan1+

/+

Fan

1-/-

Fan1+

/+

Fan

1-/-

Fan1+

/+

Fan

1-/-

Fan1+

/+

Fan

1-/-

0

1

2

3

4

5

Mg

(mg/

dL)

3 months 6 months 12 months 18 monthsFan

1+/+

Fan

1-/-

Fan1+

/+

Fan

1-/-

Fan1+

/+

Fan

1-/-

Fan1+

/+

Fan

1-/-

0

5

10

15

P (m

g/dL

)

3 months 6 months 12 months 18 months

Fan1+

/+

Fan

1-/-

Fan1+

/+

Fan

1-/-

Fan1+

/+

Fan

1-/-

Fan1+

/+

Fan

1-/-

0.0

0.1

0.2

0.3

0.4

0.5

CR

EA (m

g/dL

)


Fan1+

/+

Fan

1-/-

Fan1+

/+

Fan

1-/-

Fan1+

/+

Fan

1-/-

Fan1+

/+

Fan

1-/-

0

10

20

30

40

50

BU

N (m

g/dL

)


BUN CREA

P Mg

Fan1+

/+

Fan

1-/-

Fan1+

/+

Fan

1-/-

Fan1+

/+

Fan

1-/-

0

500

1000

AST

(U/L

)

3 months 6-9 months 12-18 months

***AST

Fan1+

/+

Fan

1-/-

Fan1+

/+

Fan

1-/-

Fan1+

/+

Fan

1-/-

0

200

400

600

800

ALT

(U/L

)


**

Fan1+

/+

Fan

1-/-

Fan1+

/+

Fan

1-/-

Fan1+

/+

Fan

1-/-

0

1

2

3

4

ALB

(g/d

L)


***

ALT

ALB

Fan1+

/+

Fan

1-/-

Fan1+

/+

Fan

1-/-

Fan1+

/+

Fan

1-/-

0

1

2

3

4

Glo

bulin

(g/d

L)


***

GLOB

E

0

10

20

30

40

50

0 1 2 3 4 5 6

% o

f cel

ls

Number of copies per genomic locus

12qA1.1

16qA

17qA1

Kidney

0

5

10

15

20

25

0 1 2 3 4 5 6

% o

f cel

ls


12qA1.1

16qA

17qA1

Liver

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

% o

f cel

ls


12qA1.1

16qA

17qA1

Spleen

Fan1+

/+

Fan1-/

-

Fan1+

/+

Fan1-/

-0

1000

2000

3000

PLT

(K/u

L)

3 months 18 months

Fan1+

/+

Fan1-/

-

Fan1+

/+

Fan1-/

-0

5

10

15

RBC

(M/u

L)

3 months 18 months

Fan1+

/+

Fan1-/

-

Fan1+

/+

Fan1-/

-0

5

10

15

WBC

(K/u

L)

3 months 18 months

Fan1+

/+

Fan1-/

-

Fan1+

/+

Fan1-/

-0

1000

2000

RET

# (K

/uL)

3 months 18 months

Fan1+

/+

Fan1-/

-

Fan1+

/+

Fan1-/

-

5

10

15

HG

B (g

/dL)

3 months 18 months

A B C

D E


PLT WBC RBC

RET HGB

LT-HSC ST-HSC MPP0

50

100

150

Freq

uenc

y (%

of W

T)

WTFan1-/-Fancd2-/-Fan1-/-Fancd2-/-

MEP GMP CMP0

50

100

150Fr

eque

ncy

(% o

f WT)

WTFan1-/-Fancd2-/-Fan1-/-Fancd2-/-

MEP GMP CMP0

50

100

150

Freq

uenc

y (%

of F

an1+

/+)

Fan1+/+Fan1-/-

B

C

D E

LT-HSC ST-HSC MPP0

50

100

150

Freq

uenc

y (%

of F

an1+

/+)

Fan1+/+Fan1-/-

3 months old

3 months oldMEP GMP CMP

0

50

100

150

Freq

uenc

y (%

of F

an1+

/+)

Fan1+/+Fan1-/-

18 months old

LT-HSC ST-HSC MPP0

50

100

150

Freq

uenc

y (%

of F

an1+

/+)

Fan1+/+Fan1-/-

18 months old


A

Fan1+/+ Fan1-/-

50

70

90

110

1 3 5 7 9 11 13 15 20

% o

f wei

ght o

n th

e da

y of

MM

C in

ject

ion

Days post 10mg/kg MMC injection

WT1WT2WT3WT4WT5WT6WT7WT8WT9WT10Fan1+/-1Fan1+/-2Fan1+/-3Fan1+/-4Fan1+/-5Fan1+/-6Fan1+/-7Fan1+/-8Fan1+/-9Fan1+/-10Fan1-/-1Fan1-/-2Fan1-/-3Fan1-/-4Fan1-/-5Fan1-/-6Fan1-/-7Fan1-/-8Fan1-/-9Fan1-/-10

A

Thongthip et al. Supplemental Fig. 7 Revised

B

Forward Scatter (FSC)

7-AA

D

30.4%Live cells


7-AA

D

2.7%Live cells


7-AA

D

24.9%Live cells


7-AA

D

32.8%Live cells

Untreated + MMC

Fan1

+/+

Fan1

-/-

RET

(K/u

L)

0

150

300

450

RBC

(M/u

L)0

5

10

Fan1+

/+

Fan1-/

-

Fan1+

/+

Fan1-/

-

Untreated +MMCFan

1+/+

Fan1-/

-

Fan1+

/+

Fan1-/

-

Untreated +MMC

D

0

2

4

6

8

10

12

Nuc

leat

ed c

ells

per

fem

ur (x

106 )

Fan1+

/+

Fan1-/

-

Fan1+

/+

Fan1-/

-

Untreated +MMC

C

*

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