PROJECT SUMMARY (Abstract) In addition to genetics and environment, interindividual variation in epigenetic regulation may determine risk of obesity. Exceptional genomic loci called metastable epialleles offer unprecedented opportunities to test this. Metastable epialleles – essentially epigenetic polymorphisms – exhibit interindividual variation in DNA methylation that is neither tissue-specific nor genetically mediated. Establishment of DNA methylation at these loci is influenced by periconceptional environment, providing a potential explanation for how environmental influences during development increase risk of obesity later in life (developmental programming). We have shown that DNA methylation profiling across multiple tissues from multiple individuals is an effective approach to discover metastable epialleles, and have already identified several implicated in obesity. To test the overall hypothesis that individual differences in DNA methylation at human metastable epialleles predict risk of adult weight gain, we will partner with the NIH Genotype-Tissue Expression (GTEx) program and the Starr County Health Studies to pursue the following Specific Aims: Aim 1 - Screen for candidate metastable epialleles in 3 tissues from each of 10 GTEx donors. Aim 2 - Validate bona-fide metastable epialleles by studying 8 tissues from each of 100 GTEx donors. Aim 3 - Test whether DNA methylation at metastable epialleles affects risk of subsequent weight gain. This translational project will both discover new human MEs associated with obesity, and prospectively test whether these epigenetic variants are stable through adulthood, and affect risk of subsequent weight gain. Hence, the proposed research will provide crucial insights into how interindividual epigenetic variation affects risk of obesity.