UK - media.nature.com · 1 Supplementary Information for: 5-Formylcytosine can be a stable DNA modification in mammals Martin Bachman1,2, Santiago Uribe-Lewis2, Xiaoping Yang2, Heather

1

Supplementary Information for:

5-Formylcytosine can be a stable DNA modification in mammals

Martin Bachman1,2, Santiago Uribe-Lewis2, Xiaoping Yang2, Heather E

Burgess3, Mario Iurlaro3, Wolf Reik3,4, Adele Murrell2,5 & Shankar

Balasubramanian1,2*

Affiliations

1Department of Chemistry, University of Cambridge, Cambridge CB2 1EW,

UK

2Cancer Research UK Cambridge Institute, University of Cambridge,

Cambridge CB2 0RE, UK

3Babraham Institute, Babraham CB22 3AT, UK

4Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK

5Present address: Centre for Regenerative Medicine, Department of Biology

and Biochemistry, University of Bath, Bath BA2 7AY, UK

*e-mail: [email protected]

Nature Chemical Biology: doi:10.1038/nchembio.1848

2

Supplementary Results

Supplementary Fig. 1. Mass spectra (measurements of global levels).

Supplementary Fig. 2. Extracted ion chromatograms (synthetic standards).

Supplementary Fig. 3. Calibration curves.

Supplementary Fig. 4. Extracted ion chromatograms (genomic DNA samples).

Supplementary Fig. 5. Reproducibility of mass spectrometry measurements.

Supplementary Fig. 6. Global levels of 5mC, 5hmC and 5fC in all tissues.

Supplementary Fig. 7. Global levels of 5mC, 5hmC, 5fC and 5caC in CD1

mice.

Supplementary Fig. 8. Correlations between 5fC, 5mC and 5hmC.

Supplementary Fig. 9. Mass spectra (measurements of labelling ratios).

Supplementary Table 1. Masses of parent ions and base fragments.


3

Supplementary Figure 1. Mass spectra of all nucleoside base fragments

acquired at a the resolution of 70,000 and used for quantification of global

5mC, 5hmC, 5fC and 5caC levels. Theoretical and found accurate masses

are listed in Supplementary Table 1. Theoretical masses ± 5 ppm were used

to generate the extracted ion chromatograms shown in Supplementary Fig 2.

5mC

126.05 126.07 126.090

1×1006

2×1006

3×1006

m/z

5mC_IS

129.07 129.09 129.110

1×1007

2×1007

3×1007

m/z

5hmC

142.04 142.06 142.080.0

5.0×1004

1.0×1005

1.5×1005

m/z

5hmC_IS

145.06 145.08 145.100

2×1005

4×1005

6×1005

8×1005

1×1006

m/z

5fC

140.03 140.05 140.070.0

5.0×1003

1.0×1004

1.5×1004

2.0×1004

2.5×1004

m/z

5caC

156.02 156.04 156.060

1×1003

2×1003

3×1003

m/z

NH

NH+

NH2

O

H

O

NH

NH+

NH2

O

H

OHH

NH

NH+

NH2

O

H

HH

C

112.03 112.05 112.070

5×1007

1×1008

2×1008

2×1008

2×1008

m/z

Rela

tive

abun

danc

e (A

.U.)

C_IS

115.04 115.06 115.080

2×1005

4×1005

6×1005

m/z

Rela

tive

abun

danc

e (A

.U.)

NH

NH+

NH2

O

NH

NH+

NH2

O

HO

O

NH

NH+

NH2

O

D

OHD

DNH

NH+

NH2

O

D

DD

NH

NH+

15NH2

O

D

D


4

Supplementary Figure 2. Example of extracted ion chromatograms for each

analyte and their corresponding internal standards. Areas under the curve

were used for quantification. The numbers show retention times (in min)

achieved on a capillary column packed with 3 µm Hypercarb beads and a 15-

min gradient.

C

4 6 8 10 12 140

4×1008

8×1008

RT (min)

Inte

nsity

(AU)

7.07

C_IS

4 6 8 10 12 140

1×1005

2×1005

RT (min)

Inte

nsity

(AU)

7.11

5mC

4 6 8 10 12 140

5×1007

1×1008

RT (min)

7.67

5mC_IS

4 6 8 10 12 140

1×1005

2×1005

RT (min)

7.64

5hmC

4 6 8 10 12 140

1×1007

2×1007

RT (min)

7.50

5hmC_IS

4 6 8 10 12 140

1×1005

2×1005

RT (min)

7.47

5fC

4 6 8 10 12 140

5×1006

1×1007

RT (min)

9.98

5caC

4 6 8 10 12 140

2×1006

4×1006

RT (min)

11.40


5

Supplementary Figure 3. Calibration curves were generated using area

ratios (unlabelled vs. IS) for C, 5mC and 5hmC, and areas only for 5fC and

5caC. Excellent linearity was obtained for all analytes.

C

-1 0 1 2 3 40

1

2

3

4

5

Log10([C]) (nM)

Log 10

(Are

a ra

tio)

r2 = 0.9930

5mC

-2 -1 0 1 2 3-1

0

1

2

3

4

Log10([5mC]) (nM)

r2 = 0.9994

5hmC

-3 -2 -1 0 1 2-2

-1

0

1

2

3

Log10([5hmC]) (nM)

r2 = 0.9982

5fC

-3 -2 -1 0 1 24

5

6

7

8

9

Log10([5fC]) (nM)

Log 10

(Are

a) r2 = 0.9947

5caC

-3 -2 -1 0 1 24

5

6

7

8

9

Log10([5caC]) (nM)

r2 = 0.9924


6

Supplementary Figure 4. Example of extracted ion chromatograms for each

analyte and their corresponding internal standards obtained from a low-5fC-

containing mouse genomic DNA sample (adult spleen). Areas under the curve

were used for quantification. The numbers show retention times achieved on

a capillary column packed with 3 µm Hypercarb beads and a 20-min gradient.

C

5 10 15 200

1×1009

2×1009

3×1009

RT (min)

Inte

nsity

(AU)

9.57 min

C_IS

5 10 15 200

1×1006

2×1006

RT (min)

Inte

nsity

(AU)

9.55 min

5mC

5 10 15 200

1×1008

2×1008

3×1008

4×1008

RT (min)

10.20 min

5mC_IS

5 10 15 200

2×1007

4×1007

6×1007

RT (min)

10.18 min

5hmC

5 10 15 200

1×1006

2×1006

3×1006

4×1006

RT (min)

10.05 min

5hmC_IS

5 10 15 200

2×1006

4×1006

6×1006

RT (min)

10.02 min

5fC

5 10 15 200

2×1003

4×1003

6×1003

8×1003

1×1004

RT (min)

14.72 min

5fC zoom

14.0 14.5 15.0 15.50

2×1003

4×1003

6×1003

8×1003

1×1004

RT (min)


7

Supplementary Figure 5. Reproducibility of concentration measurements

(left) and global levels of 5mC, 5hmC, 5fC and 5caC (right). Each dot

represents the coefficient of variation (% CV) of 2 technical replicates for

genomic DNA samples. Bars represent mean ± SD of 47 random samples.

Reproducibility

[C][5m

C]

[5hmC]

[5fC]

[5caC

]

0

20

40

60

% C

V

Reproducibility

% 5mC

% 5hmC

ppm 5f

C

ppm 5c

aC

0

20

40

60


8

Supplementary Figure 6. Global levels of 5mC, 5hmC and 5fC in the

genomic DNA of all studied unlabelled C57BL/6 mouse tissues and mES

cells. Shown are mean ± SEM of 3 embryos (E11.5), 3 newborns (1 d old)

and mean and range for 2 adolescent (21 d old) and 2 adult (15 w old) mice.

Each biological sample was analysed in 2 technical replicates, and the mean

value was used.

Embryo (11.5 d)

Carcas

s

Forebra

in

Hindbra

inHea

rtAort

aLiv

er

mES (WT)

mES (TET-TKO)

0

1

2

3

4

5

Carcas

s

Forebra

in

Hindbra

inHea

rtAort

aLiv

er

mES (WT)

mES (TET-TKO)

0.0

0.2

0.4

0.6

0.8

Newborn (1 d)

Brain

TailHea

rt

Kidney Skin Gut

Liver

LungSple

en0

1

2

3

4

5

Brain

TailHea

rt

Kidney Skin Gut

Liver

LungSple

en0.0

0.2

0.4

0.6

0.8

Adolescent (21 d)

Brain

Cerebe

llum Tail

Muscle

SkinKidn

ey Lung

Tongu

eHea

rt

Dist gu

t

Mid gutColo

nLiv

erSple

en

Prox gu

t

Thymus

0

1

2

3

4

5

Brain

Cerebe

llum Tail

Muscle

SkinKidn

ey Lung

Tongu

eHea

rt

Dist gu

t

Mid gutColo

nLiv

erSple

en

Prox gu

t

Thymus

0.0

0.2

0.4

0.6

0.8

Adult (15 w)

Brain

Cerebe

llum

Prox gu

t

Tongu

eTail

Liver Skin

Mid gut

Spleen

Thymus

Heart

MuscleKidn

ey

Dist gu

tColo

nLun

g0

1

2

3

4

5

% m

C (o

ver t

otal

C)

Brain

Cerebe

llum

Prox gu

t

Tongu

eTail

Liver Skin

Mid gut

Spleen

Thymus

Heart

MuscleKidn

ey

Dist gu

tColo

nLun

g0.0

0.2

0.4

0.6

0.8

% h

mC

(ove

r tot

al C

)

Brain

TailHea

rt

Kidney Skin Gut

Liver

LungSple

en0

6

12

18

Brain

Cerebe

llum Tail

Muscle

SkinKidn

ey Lung

Tongu

eHea

rt

Dist gu

t

Mid gutColo

nLiv

erSple

en

Prox gu

t

Thymus

0

6

12

18

Brain

Cerebe

llum

Prox gu

t

Tongu

eTail

Liver Skin

Mid gut

Spleen

Thymus

Heart

MuscleKidn

ey

Dist gu

tColo

nLun

g0

6

12

18

ppm

5fC

(ove

r tot

al C

)


9

Supplementary Figure 7. Global levels of 5mC, 5hmC, 5fC and 5caC in adult

12-week old female CD1 mice. Shown are mean ± SEM of 3 animals. The

high 5fC levels in adult spleen are consistent with values reported by Ito et al.

(strain not given)1.

CD1 mice (12 w)

BrainKidn

eyHea

rt

Muscle Lun

gLiv

er TailColo

n

Tong

ue Skin

Dist gu

t

Mid gut

Prox gu

t

Spleen

Thymus

0

1

2

3

4

5

% 5

mC

(ove

r tot

al C

)

CD1 mice (12 w)

BrainKidn

eyHea

rt

Muscle Lun

gLiv

er TailColo

n

Tong

ue Skin

Dist gu

t

Mid gut

Prox gu

t

Spleen

Thymus

0.0

0.2

0.4

0.6

% 5

hmC

(ove

r tot

al C

)Brai

nKidn

eyHea

rt

Muscle Lun

gLiv

er TailColo

n

Tong

ue Skin

Dist gu

t

Mid gut

Prox gu

t

Spleen

Thymus

0

5

10

15

ppm

5fC

(ove

r tot

al C

)

BrainKidn

eyHea

rt

Muscle Lun

gLiv

er TailColo

n

Tong

ue Skin

Dist gu

t

Mid gut

Prox gu

t

Spleen

Thymus

0.0

0.5

1.0

1.5

2.0

ppm

5ca

C (o

ver t

otal

C)


10

Supplementary Figure 8. Correlations between global levels of 5fC, 5hmC

and 5mC in DNA from the same tissues and individuals. Shown are mean

values from individual biological replicates presented in Fig. 2 and brain

samples are indicated with an asterisk.

5fC vs. 5mC - E11.5

2.5 3.0 3.5 4.0 4.5 5.00

5

10

15

20

% 5mC (over total C)

ppm

fC (o

ver t

otal

C)

r2 = 0.30

5fC vs. 5hmC - E11.5

0.00 0.05 0.10 0.15 0.200

5

10

15

20

% 5hmC (over total C)

ppm

fC (o

ver t

otal

C)

r2 = 0.24

5fC vs. 5mC - newborn

2.6 3.0 3.4 3.8 4.20

2

4

6

8


r2 = 0.02 including brainr2 = 0.00 omitting brain

* **

5fC vs. 5mC - adolescent

2.4 3.0 3.6 4.2 4.80

5

10

15

20


r2 = 0.15 including brainr2 = 0.01 omitting brain *

*

5fC vs. 5mC - adult

2.8 3.2 3.6 4.0 4.4 4.80

5

10

15

% 5mC (over total C)

r2 = 0.05 including brainr2 = 0.00 omitting brain

**

5fC vs. 5hmC - newborn

0.0 0.1 0.2 0.30

2

4

6

8



**

5fC vs. 5hmC - adolescent

0.0 0.2 0.4 0.60

5

10

15

20



*

5fC vs. 5hmC - adult

0.0 0.2 0.4 0.60

4

8

12


r2 = 0.64 including brainr2 = 0.10 omitting brain * *


11

Supplementary Figure 9. Mass spectra of all nucleoside base fragments

acquired at a resolution of 70,000 and used for quantification of labelling

ratios of 5mC, 5hmC and 5fC. Theoretical and found accurate masses are

listed in Supplementary Table 1. Theoretical masses ± 5 ppm were used to

generate extracted ion chromatograms and obtain areas under the curve for

quantification.

5mC

126.05 126.07 126.090

1×1006

2×1006

3×1006

m/z

5mC[+4]

130.07 130.09 130.110

5×1006

1×1007

2×1007

m/z

5hmC

142.04 142.06 142.080.0

5.0×1004

1.0×1005

1.5×1005

m/z

5hmC[+3]

145.06 145.08 145.100

2×1005

4×1005

6×1005

8×1005

m/z

5fC

140.03 140.05 140.070.0

5.0×1003

1.0×1004

1.5×1004

2.0×1004

2.5×1004

m/z

5fC[+2]

142.04 142.06 142.080

2×1003

4×1003

6×1003

8×1003

1×1004

m/z

NH

NH+

NH2

O

H

O

NH

NH+

NH2

O

D

DD

*

NH

NH+

NH2

O

H

OHH

NH

NH+

NH2

O

D

O

*

NH

NH+

NH2

O

H

HH

NH

NH+

NH2

O

D

OHD

*


12

Supplementary Table 1. A list of all nucleosides measured in this study. The

Q Exactive mass spectrometer was set to select given parent ions (± 0.2 Da)

in the quadrupole mass filter and perform fragmentation and acquisition of full

scans (50 - 300 Da) for each ion. Indicated accurate masses of fragment ions

(± 5 ppm) were used for quantification. IS = internal standard. [13CD3] L-met =

[methyl-13CD3] L-methionine.

Base Source Isotopes

Parent ion

formula

Parent ion

[M+H]+

Fragment

formula

Fragment

[M+H]+

C Natural - C9H14O4N3 228.1 C4H6ON3 112.05054

C IS 15N, D2 C9H12D2O4N215N 231.1 C4H4D2ON2

15N 115.06013

5mC Natural - C10H16O4N3 242.1 C5H8ON3 126.06619

5mC IS D3 C10H13D3O4N3 245.1 C5H5D3ON3 129.08502

5mC [13CD3] L-met 13C, D3 C913CH13D3O4N3 246.1 C4

13CH5D3ON3 130.08837

5hmC Natural - C10H16O5N3 258.1 C5H8O2N3 142.06110

5hmC IS D3 C10H13D3O5N3 261.1 C5H5D3O2N3 145.07993

5hmC [13CD3] L-met 13C, D2 C913CH14D2O5N3 261.1 C4

13CH6D2O2N3 145.07701

5fC Natural - C10H14O5N3 256.1 C5H6O2N3 140.04545

5fC [13CD3] L-met 13C, D C913CH13DO5N3 258.1 C4

13CH5DO2N3 142.05508

5caC Natural - C10H14O6N3 272.1 C5H6O3N3 156.04037

RNA 5mC Natural - C10H16O5N3 258.1 C5H8ON3 126.06619

RNA 5mC [13CD3] L-met 13C, D3 C913CH13D3O5N3 262.1 C4

13CH5D3ON3 130.08837


13

References

1. Ito, S. et al. Tet proteins can convert 5-methylcytosine to 5-

formylcytosine and 5-carboxylcytosine. Science 333, 1300-3 (2011).


Documents

UK - media.nature.com · 1 Supplementary Information for: 5-Formylcytosine can be a stable DNA modification in mammals Martin Bachman1,2, Santiago Uribe-Lewis2, Xiaoping Yang2, Heather