1. Early Nutrition and the Establishment of Epigenotype at Metastable Epialleles Rob Waterland Houston, Texas

2. The Waterland Lab Funding: NIH-NIDDK, March of Dimes, USDA

4. Metabolic Imprinting Adaptive responses to early nutrition Persistent effect Susceptibility limited to critical period of development Waterland & Garza Am J Clin Nutr 1999;69

5. 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

6. Mammalian One Carbon Metabolism

7. 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

8. 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

9. The Viable Yellow Agouti (A vy ) Mouse

10. Maternal Methyl Donor Supplementation Affects Coat Color of A vy /a Offspring Waterland & Jirtle, Mol Cell Biol 2003

11. Supplementation Changes Coat Color by Increasing A vy Methylation Waterland & Jirtle, Mol Cell Biol 2003

12. Interpretation of A vy Experiment Specific transposable elements induce epigenetic instability, allowing early diet to influence epigenotype Transposable elements (SINEs, LINEs, etc.) comprise >40% of the human genome

13. 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 agoutiAxin fused

14. The Axin Fused (Axin Fu ) Mouse Axin regulates embryonic axis formation –Inhibitor of Wnt signaling pathway Axin Fu caused by IAP insertion into Axin intron 6 –Tail kink phenotype associated with expression of truncated transcript originating downstream of IAP –Axin Fu methylation silences the mutant transcript IAP 67 Vasicek et al Genetics 1997

15. Methods C57 (+/+) dams assigned to diets 2 weeks before mating with Axin Fu /+ males –Control: NIH-31 –Supplemented: NIH-31 with extra folic acid, B 12, betaine and choline Offspring rated for tail phenotype at age 21 d Axin Fu CpG methylation measured by bisulfite sequencing

16. Classification of Tail Phenotype None Slightly Kinky Kinky

17. Classification of Tail Phenotype Very Kinky

18. Methods C57 (+/+) dams assigned to diets 2 weeks before mating with Axin Fu /+ males –Control: NIH-31 –Supplemented: NIH-31 with extra folic acid, B 12, betaine and choline Offspring rated for tail phenotype at age 21 d Axin Fu CpG methylation measured by bisulfite sequencing with phosphor-imager quantitation

19. Characteristics of Offspring at Weaning ControlSupplemented Number of litters2422 Wean weight (g) † 8.2 ± 0.17.8 ± 0.1 * Litter size † 6.0 ± 0.35.7 ± 0.4 Proportion Axin Fu /+ pups per litter † 0.40 ± 0.040.56 ± 0.04 ** Total number of pups144125 Number of Axin Fu /+ pups5668 † mean ± sem * P < 0.05 ** P < 0.01

20. Axin Fu Methylation and Tail Phenotype Waterland et al, submitted

21. Supplementation Reduces Incidence of Tail Kinks in Axin Fu /+ Offspring P=0.002 Waterland et al, submitted P=0.002

22. Supplementation Does NOT Increase Axin Fu Methylation in Liver P=0.05

23. Axin Fu is Hypomethylated in Tail

24. Supplementation Prevents Tail Kinks by Reducing Tail-Specific Loss of Methylation P=0.009 Waterland et al, submitted P=0.05

25. Significance Similar to A vy, epigenetic metastability at Axin Fu confers lability to early nutrition Nutritional effects on DNA methylation during development may –Be tissue-specific –Occur at diverse ontogenic periods

26. Transgenerational Perpetuation of Obesity… By Epigenetic Mechanisms? A vy mouse is an ideal model –Spontaneously hyperphagic –Compare offspring of lean a/a dams and obese A vy /a dams

27. A vy Transgenerational Obesity Study Approach: –Maintain two separate populations of A vy /a mice on control (NIH-31) or methyl-supplemented diet (folic acid, vitamin B 12, betaine, choline) –Pass the A vy allele through the female germline for several generations –Assess cumulative effects on body weight of A vy /a and a/a offspring

28. Maternal Obesity Increases Body Weight at Weaning in F1 Offspring 56 121 A vy /a a/a P<0.001 Dams Offspring Body Weight

29. Transgenerational Effect of Maternal Obesity: Wean Weight by Generation F3 vs. F1 P=0.004 131 121 62 Average for offspring of a/a dams

30. Control Diet a/a Offspring Transgenerational Effects of Maternal Obesity Depend on Offspring Genotype and Maternal Diet

31. Control Diet a/a OffspringA vy /a Offspring Transgenerational Effects of Maternal Obesity Depend on Offspring Genotype and Maternal Diet

32. Control Diet Supplemented Diet a/a OffspringA vy /a Offspring Transgenerational Effects of Maternal Obesity Depend on Offspring Genotype and Maternal Diet

33. Control Diet Supplemented Diet a/a OffspringA vy /a Offspring Effect P value Generation <0.0001 Genotype <0.0001 Supplementation 0.002 N=498 total Transgenerational Effects of Maternal Obesity Depend on Offspring Genotype and Maternal Diet

34. Genotype-Epigenotype-Diet Interaction: Can too many vitamins make us fat? Maternal Obesity Methyl Donor Supplementation A vy Genotype Epigenetic Alterations Affecting Weight Gain?

35. Same Genotype, Different Epigenotype

36. Bisulfite Sequencing at Axin Fu T C 1 2 3 4 Site Unaffected Axin Fu Hypermethylated Affected Axin Fu less methylated

37. 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