The Genotype-Tissue Expression project (GTEx) is expanding the availability of primary tissue samples for studying the impact of genetic variation on gene expression across individuals and tissues. However, inter-individual variation in gene expression level can often result from the combined effects of multiple variants, each of which may affect the activity of different regulatory regions in complex ways, and also integrate complex post-transcriptional events. Thus, to directly assess the effect of sequence variants on regulatory elements, we propose to systematic profile epigenetic modifications on stored GTEx biospecimens to enhance the analysis of gene expression variation already available and planned by the GTEx consortium. Aim 1. We will use whole-genome bisulfite sequencing (WGBS) to profile DNA methylation levels for all 28M CpGs in the genome of 20 individuals in 8 GTEx tissues. We will use these to identify variable methylated regions (VMRs) across cell types and across individuals, to evaluate the effects of eQTL SNPs on DNA methylation levels of neighboring CpGs, and to recognize allele-specific methylation (ASM) regions and their tissue specificity. Aim 2. We will systematically profile DNA methylation levels across 250 individuals in the same 8 tissues using hybrid-selection bisulfite sequencing (HSBS) for a subset of 2M regions selected within enhancer-associated, eQTL-associated, and inter-individual variable regions. We will use these to predict methylation quantitative trait loci (meQTLs) in each tissue, relate these to GTEx expression QTLs (eQTLs) for the same tissues to provide mechanistic hypotheses for inter- individual gene expression variation, and to discover meQTLs for genes where no single eQTL is significant. Aim 3. We will use chromatin immunoprecipitation sequencing (ChIP-Seq) to profile enhancer- and promoter-associated histone modification H3K27ac in 6 GTEx brain samples across 100 individuals, to recognize genotype-associated enhancer regions and enhQTLs. We will relate these to eQTLs in the same tissues and meQTLs discovered using HSBS on enhancer-proximal and eQTL proximal CpGs to generate mechanistic models for regulatory changes associated with genotype variation. We will work closely with the GTEx Analysis Consortium to integrate GTEx eQTLs with our predicted meQTLs, ASM regions, and enhQTLs to relate regulatory region variation to gene expression variation and to genome-wide association studies to understand the role of sequence variation in tissue-specific gene regulation, gene expression, and human disease.