Developmental Relaxation of InsulinLike Growth Factor 2 Imprinting in Kidney is Determined by Weanli

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Early Nutrition and the Establishment of
Epigenotype at Metastable Epialleles
Rob Waterland Houston, Texas
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The Waterland Lab
Funding NIH-NIDDK, March of Dimes, USDA
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(No Transcript)
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Metabolic Imprinting
Adaptive responses to early nutrition Persistent
effect Susceptibility limited to critical period
of development Waterland Garza Am J Clin
Nutr 199969
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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

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Mammalian One Carbon Metabolism
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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

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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
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The Viable Yellow Agouti (Avy) Mouse
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Maternal Methyl Donor Supplementation Affects
Coat Color of Avy/a Offspring
Waterland Jirtle, Mol Cell Biol 2003
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Supplementation Changes Coat Color by Increasing
Avy Methylation
Waterland Jirtle, Mol Cell Biol 2003
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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

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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
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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

Vasicek et al Genetics 1997
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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

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Classification of Tail Phenotype
None
Slightly Kinky
Kinky
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Classification of Tail PhenotypeVery Kinky
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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

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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 20
AxinFu Methylation and Tail Phenotype
Waterland et al, submitted
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Supplementation Reduces Incidence of Tail Kinks
in AxinFu/ Offspring
P0.002
P0.002
Waterland et al, submitted
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Supplementation Does NOT Increase AxinFu
Methylation in Liver
P0.05
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AxinFu is Hypomethylated in Tail
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Supplementation Prevents Tail Kinks by Reducing
Tail-Specific Loss of Methylation
P0.009
P0.05
Waterland et al, submitted
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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

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Transgenerational Perpetuation of ObesityBy
Epigenetic Mechanisms?

• Avy mouse is an ideal model
• Spontaneously hyperphagic
• Compare offspring of lean
• a/a dams and obese Avy/a dams

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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

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Maternal Obesity Increases Body Weight at Weaning
in F1 Offspring
Offspring Body Weight
Dams
PAvy/a
a/a
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Transgenerational Effect of Maternal
ObesityWean Weight by Generation
F3 vs. F1 P0.004
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131
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Average for offspring of a/a dams
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Transgenerational Effects of Maternal Obesity
Depend on Offspring Genotype and Maternal Diet
a/a Offspring
Control Diet
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Transgenerational Effects of Maternal Obesity
Depend on Offspring Genotype and Maternal Diet
a/a Offspring
Avy/a Offspring
Control Diet
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Transgenerational Effects of Maternal Obesity
Depend on Offspring Genotype and Maternal Diet
a/a Offspring
Avy/a Offspring
Control Diet
Supplemented Diet
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Transgenerational Effects of Maternal Obesity
Depend on Offspring Genotype and Maternal Diet
a/a Offspring
Avy/a Offspring
Effect P value Generation
Control Diet
Supplemented Diet
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Genotype-Epigenotype-Diet InteractionCan too
many vitamins make us fat?
Maternal Obesity
Methyl Donor Supplementation
Avy Genotype
Epigenetic Alterations Affecting Weight Gain?
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Same Genotype, Different Epigenotype
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Bisulfite Sequencing at AxinFu
Unaffected AxinFu Hypermethylated
Affected AxinFu less methylated
T C
T C
Site
1
2
3
4
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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