Proper organismal function requires fine-tuned regulation of gene expression, yet variation exists within and between species. This variation can take the form of multiple disease states in humans as well as phenotypic differences between species. Despite its importance, regulatory evolution is not yet well understood. Previous studies have shown that regulatory divergence is governed by changes in cis- and trans-regulatory elements that affect the level of expression of genes. cis-regulatory changes have effects on allele-specific expression, while trans-regulatory variation contributes to the expression of both alleles in a diploid cell. The effects of cis- and trans-regulatory elements can be disentangled using measures of allele-specific gene expression. This proposal aims to address questions such as: What are the relative contributions of cis- and trans-regulatory changes to divergent gene expression? Do the relative contributions of cis- and trans-regulatory changes vary with divergence time? Are different functional classes affected more often by changes in cis- or trans-regulatory elements? To address these questions, we propose a novel strategy for distinguishing cis- and trans-regulatory effects on variable gene expression genome-wide. Using high- throughput sequencing, we will measure allele-specific expression in pairs of inbred strains from the same and different species. Alelle-specific expression will also be determined in F1 hybrids produced by crossing pairs of these strains. New computational tools have been developed to analyze the enormous amount of data produced by the massively parallel sequencing, and these tools will be made available to the broader research community. Allele-specific measurements of gene expression will be used to compare the relative contribution of cis- and trans-regulatory variants among crosses. We will also make allele-specific transgenes and use them for functional tests of cis- regulatory divergence. Finally, we will test the hypothesis that cis-regulatory changes accumulate faster than trans-regulatory changes over time, using the whole transcriptome of strains/species with divergence times ranging from 10,000-7 million years ago. PUBLIC HEALTH RELEVANCE: Relevance: Proper gene regulation can mean the difference between healthy and disease states in humans. Determining the contributions that different regulatory variants make to differences in gene expression is an important step towards understanding the relationship between genotypes and phenotypes. The conclusions drawn with the proposed study of Drosophila regulatory variation can then be used as a model for human variation in gene expression regulation.