Project Summary When genes move to new chromosomal locations they are often expressed at drastically different levels. We will use these chromosome position effects to investigate how different chromosomal locations affect the activity of cis-regulatory sequences (CRS). We propose to measure how the activities of a large and diverse collection of CRS change when they are moved to different chromosomal locations (Aim 1). To collect this data at scale we will introduce a new massively parallel reporter gene assay (MPRA). We aim to address several questions. 1. What accounts for the differences in regulatory potential between chromosome positions? When the activity of a CRS changes because it moves to a new location, are the changes due to the new chromatin state, the activity of nearby enhancers, or both? 2. When are the regulatory effects of CRS and genome position independent and when do they interact in complex ways? Do interactions depend on the properties of CRS and the specific chromatin landscape? We also propose to complement our investigation of gene expression levels with a study of how genome position influences gene expression dynamics (Aim 2). By measuring the cell- to-cell patterns of mRNA generated by different CRS at different chromosome locations, we will uncover how chromosome environments alter the dynamics of expression. The successful completion of the aims of this proposal will result in a quantitative and molecular understanding of the combinatorial regulation of gene expression by CRS and chromatin environment. A clear understanding of how CRS function in the genome will help identify and interpret disease- causing genetic variants that affect cis-regulation.