Early Nutrition and the Establishment of Epigenotype at Metastable Epialleles

Rob WaterlandHouston, Texas

The Waterland Lab

Funding: NIH-NIDDK, March of Dimes, USDA

Metabolic Imprinting

Adaptive responses to early nutrition

Persistent effect

Susceptibility limited to critical period of development

Waterland & Garza Am J Clin Nutr 1999;69

Early Nutrition and DNA Methylation

• Most cytosines within CpG dinucleotides are methylated

• Tissue-specific patterns of CpG methylation are established during development

• Methylation requires dietary methyl donors and cofactors

• Mitotically heritable

NH2

O

H

H

N

N

NH2

O

CH3

H

N

N

DNMT

SAM SAHR R

Cytosine 5-Methylcytosine

Mammalian One Carbon Metabolism

THF

5CH3THF

B12

1 2

6B6

3

4

5

Methionine

SAM

SAH

Homocysteine

B6

Cystathionine

Cysteine

Glutathione

CH3-X

Adenosine

X*DMG

Betaine

Choline

1 Methionine Synthase

2 Betaine-homocysteineMethyltransferase

3 Methionine Adenosyltransferase

4 Various Methyltransferases

5 SAH Hydrolase

6 Cystathionine β-Synthase

* X: Various Targets of Methylation

THF

5CH3THF

B12

1 2

6B6

3

4

5

Methionine

SAM

SAH

Homocysteine

B6

Cystathionine

Cysteine

Glutathione

CH3-X

Adenosine

X*DMG

Betaine

Choline

1 Methionine Synthase

2 Betaine-homocysteineMethyltransferase

3 Methionine Adenosyltransferase

4 Various Methyltransferases

5 SAH Hydrolase

6 Cystathionine β-Synthase

* X: Various Targets of Methylation

1 Methionine Synthase

2 Betaine-homocysteineMethyltransferase

3 Methionine Adenosyltransferase

4 Various Methyltransferases

5 SAH Hydrolase

6 Cystathionine β-Synthase

* X: Various Targets of Methylation

Early Nutrition and DNA Methylation

• Most cytosines within CpG dinucleotides are methylated

• Tissue-specific patterns of CpG methylation are established during development

• Methylation requires dietary methyl donors and cofactors

• Mitotically heritable

NH2

O

H

H

N

N

NH2

O

CH3

H

N

N

DNMT

SAM SAHR R

Cytosine 5-Methylcytosine

Overall Hypothesis

• Specific subsets of genes are especially sensitive to early nutritional influences on epigenetic regulation

– Genomically imprinted genes

– Metastable epialleles

Waterland and Jirtle, Nutrition 2004

The Viable Yellow Agouti (Avy) Mouse

Maternal Methyl Donor Supplementation Affects Coat Color of Avy/a Offspring

Waterland & Jirtle, Mol Cell Biol 2003

Supplementation Changes Coat Color by Increasing Avy Methylation

Waterland & Jirtle, Mol Cell Biol 2003

Interpretation of Avy Experiment

• Specific transposable elements induce epigenetic instability, allowing early diet to influence epigenotype

• Transposable elements (SINEs, LINEs, etc.) comprise >40% of the human genome

Metastable Epiallele

“An allele at which the epigenetic state can switch and establishment is a probabilistic event. Once established, the state is mitotically inherited.”

Rakyan et al Trends in Genetics 2002

Viable yellow agouti Axin fused

The Axin Fused (AxinFu) Mouse

• Axin regulates embryonic axis formation– Inhibitor of Wnt signaling pathway

• AxinFu caused by IAP insertion into Axin intron 6– Tail kink phenotype associated with expression of

truncated transcript originating downstream of IAP– AxinFu methylation silences the mutant transcript

IAP

6 7

Vasicek et al Genetics 1997

Methods

• C57 (+/+) dams assigned to diets 2 weeks before mating with AxinFu/+ males– Control: NIH-31– Supplemented: NIH-31 with extra

• folic acid, B12, betaine and choline

• Offspring rated for tail phenotype at age 21 d

• AxinFu CpG methylation measured by bisulfite sequencing

Classification of Tail Phenotype

None

Slightly Kinky

Kinky

Classification of Tail Phenotype

Very Kinky

Methods

• C57 (+/+) dams assigned to diets 2 weeks before mating with AxinFu/+ males– Control: NIH-31– Supplemented: NIH-31 with extra

• folic acid, B12, betaine and choline

• Offspring rated for tail phenotype at age 21 d

• AxinFu CpG methylation measured by bisulfite sequencing with phosphor-imager quantitation

Characteristics of Offspring at Weaning

Control Supplemented

Number of litters 24 22

Wean weight (g)† 8.2 ± 0.1 7.8 ± 0.1 *

Litter size† 6.0 ± 0.3 5.7 ± 0.4

Proportion AxinFu/+ pups per litter† 0.40 ± 0.04 0.56 ± 0.04 **

Total number of pups 144 125

Number of AxinFu/+ pups 56 68

† mean ± sem

* P < 0.05

** P < 0.01

AxinFu Methylation and Tail Phenotype

65’ 7 3’

5’3’IAP 1

6

200 bp

None Slightly kinky Kinky Very kinky

A

B

C

None Slightly kinky Kinky Very kinky

Tail Phenotype

% M

eth

ylat

ion

0

20

40

60

80

100

5721

29

17

65’ 7 3’

5’3’IAP 1

6

200 bp

65’ 7 3’

5’3’IAP 1

6

200 bp

None Slightly kinky Kinky Very kinky

A

B

C

None Slightly kinky Kinky Very kinky

Tail Phenotype

% M

eth

ylat

ion

0

20

40

60

80

100

5721

29

17

None Slightly kinky Kinky Very kinky

Tail Phenotype

% M

eth

ylat

ion

0

20

40

60

80

100

5721

29

17

None Slightly kinky Kinky Very kinky

Tail Phenotype

% M

eth

ylat

ion

0

20

40

60

80

100

5721

29

17

Waterland et al, submitted

Supplementation Reduces Incidence of Tail Kinks in AxinFu/+ Offspring

P=0.002

Waterland et al, submitted

None Slightly kinky Kinky Very kinky

Tail Phenotype

0

10

20

30

40

50

60 Control

Supplemented

% o

f all

Axi

nF

u /+

Off

sprin

g

None Slightly kinky Kinky Very kinky

Tail Phenotype

0

10

20

30

40

50

60 Control

Supplemented

% o

f all

Axi

nF

u /+

Off

sprin

g

P=0.002

Supplementation Does NOT Increase AxinFu Methylation in Liver

1 2 3 4 5 60

20

40

60

80

100

1 2 3 4 5 6

CpG Site

% M

ethy

latio

n

Control Supplemented

CpG Site

Liver

1 2 3 4 5 60

20

40

60

80

100

1 2 3 4 5 6

CpG Site

% M

ethy

latio

n

Control Supplemented

CpG Site

Liver

1 2 3 4 5 60

20

40

60

80

100

1 2 3 4 5 61 2 3 4 5 60

20

40

60

80

100

1 2 3 4 5 6

CpG Site

% M

ethy

latio

n

Control Supplemented

CpG Site

Liver

P=0.05

AxinFu is Hypomethylated in Tail

0

20

40

60

80

100Brain

Kidney

Liver

Tail

% M

eth

yla

tion

AxinFu/+ Mouse1 2 3 4 5 6 7 8

0

20

40

60

80

100Brain

Kidney

Liver

Tail

% M

eth

yla

tion

AxinFu/+ Mouse1 2 3 4 5 6 7 8

Supplementation Prevents Tail Kinks by Reducing Tail-Specific Loss of Methylation

1 2 3 4 5 60

20

40

60

80

100

1 2 3 4 5 6

CpG Site

% M

eth

yla

tion

Control Supplemented

CpG Site

Liver

1 2 3 4 5 60

20

40

60

80

100

1 2 3 4 5 6

CpG Site

% M

eth

yla

tion

Control Supplemented

CpG Site

Liver

1 2 3 4 5 60

20

40

60

80

100

1 2 3 4 5 61 2 3 4 5 60

20

40

60

80

100

1 2 3 4 5 6

CpG Site

% M

eth

yla

tion

Control Supplemented

CpG Site

Liver

1 2 3 4 5 60

20

40

60

80

100

1 2 3 4 5 6

CpG Site%

Me

thyl

atio

n

Control Supplemented

CpG Site

Tail

1 2 3 4 5 60

20

40

60

80

100

1 2 3 4 5 6

CpG Site%

Me

thyl

atio

n

Control Supplemented

CpG Site

Tail

1 2 3 4 5 60

20

40

60

80

100

1 2 3 4 5 61 2 3 4 5 60

20

40

60

80

100

1 2 3 4 5 6

CpG Site%

Me

thyl

atio

n

Control Supplemented

CpG Site

Tail

P=0.009

Waterland et al, submitted

P=0.05

Significance

• Similar to Avy, epigenetic metastability at AxinFu confers lability to early nutrition

• Nutritional effects on DNA methylation during development may – Be tissue-specific – Occur at diverse ontogenic periods

Transgenerational Perpetuation of Obesity…By Epigenetic Mechanisms?

• Avy mouse is an ideal model– Spontaneously hyperphagic– Compare offspring of lean

a/a dams and obese Avy/a dams

Avy Transgenerational Obesity Study• Approach:

– Maintain two separate populations of Avy/a mice on control (NIH-31) or methyl-supplemented diet (folic acid, vitamin B12, betaine, choline)

– Pass the Avy allele through the female germline for several generations

– Assess cumulative effects on body weight of Avy/a and a/a offspring

Maternal Obesity Increases Body Weight at Weaning in F1 Offspring

0

1

2

3

4

5

6

7

8

9

10

11

a/a dam Avy/a dam

d 21

bod

y w

eigh

t (g) 56

121

Avy/aa/a

P<0.001

Dams

Offspring Body Weight

Transgenerational Effect of Maternal Obesity:Wean Weight by Generation

8

8.5

9

9.5

10

10.5

11

11.5

12

F1 F2 F3

d 21

bod

y w

eigh

t (g)

F3 vs. F1 P=0.004

131121

62

Average for

offspring of

a/a dams

Control Diet

a/a Offspring

Transgenerational Effects of Maternal Obesity Depend on Offspring Genotype and Maternal Diet

8

8.5

9

9.5

10

10.5

11

11.5

12

12.5

F1 F2 F3

d 21

bod

y w

eigh

t (g

)

Control Diet

a/a Offspring Avy/a Offspring

Transgenerational Effects of Maternal Obesity Depend on Offspring Genotype and Maternal Diet

8

8.5

9

9.5

10

10.5

11

11.5

12

12.5

F1 F2 F3

d 21

bod

y w

eigh

t (g

)

8

8.5

9

9.5

10

10.5

11

11.5

12

12.5

F1 F2 F3

d 21

bod

y w

eigh

t (g

)

Control Diet

Supplemented Diet

a/a Offspring Avy/a Offspring

Transgenerational Effects of Maternal Obesity Depend on Offspring Genotype and Maternal Diet

8

8.5

9

9.5

10

10.5

11

11.5

12

12.5

F1 F2 F3

d 21

bod

y w

eigh

t (g

)

8

8.5

9

9.5

10

10.5

11

11.5

12

12.5

F1 F2 F3

d 21

bod

y w

eigh

t (g

)

8

8.5

9

9.5

10

10.5

11

11.5

12

12.5

F1 F2 F3

d 21

bod

y w

eigh

t (g

)

Control Diet

Supplemented Diet

a/a Offspring Avy/a Offspring

Effect P valueGeneration <0.0001 Genotype <0.0001Supplementation 0.002

N=498 total

Transgenerational Effects of Maternal Obesity Depend on Offspring Genotype and Maternal Diet

8

8.5

9

9.5

10

10.5

11

11.5

12

12.5

F1 F2 F3

d 21

bod

y w

eigh

t (g

)

8

8.5

9

9.5

10

10.5

11

11.5

12

12.5

F1 F2 F3

d 21

bod

y w

eigh

t (g

)

8

8.5

9

9.5

10

10.5

11

11.5

12

12.5

F1 F2 F3

d 21

bod

y w

eigh

t (g

)

8

8.5

9

9.5

10

10.5

11

11.5

12

12.5

F1 F2 F3

d 21

bod

y w

eigh

t (g

)

Genotype-Epigenotype-Diet Interaction:Can too many vitamins make us fat?

Maternal Obesity

Methyl DonorSupplementation

Avy Genotype

Epigenetic AlterationsAffecting Weight Gain?

Same Genotype, Different Epigenotype

Bisulfite Sequencing at AxinFu

T C T C

1

23

4

Site

Unaffected

AxinFu Hypermethylated

Affected

AxinFu less methylated

Validation of Quantitative Bisulfite Sequencing: Exploit H19 DMR in C57BL/6 x Cast/Ei F1 Mice

SacI digestion eliminates paternal allele

MfeI digestion eliminates maternal allele

Measure % methylation in known mixtures of SacI and MfeI digested DNA

Waterland et al Hum Mol Genet 2006