We propose to develop a high-throughput method to test the functional activity of cis-regulatory elements in vivo, in specific cell types. Powered by Next Generation Sequencing, a range of high-throughput methods can generate data that allow investigators to predict the locations of potential cis-regulatory elements in the genome. Collectively these methods have generated hundreds of thousands of predictions. A major problem is that there are no corresponding technologies to validate the cis-regulatory activity of these predictions in vivo. To address this problem we propose to develop a massively parallel reporter gene assay to test the activity of cis-regulatory elements and their allelic variants in vivo, in specific cell types. Our plan is to develop methods to create large libraries of cis regulatory elements in lentiviral-based reporters, and to develop methods to assay the activity of these libraries from specific populations of cells in vivo. We propose to develop this technology first to study cis-regulation in specific cell types of the brain, but the approach will generalizeto a large range of cell types from different tissues in different organisms. Successful completion of our aims will result in a high-throughput method for testing the effects of cis-regulatory polymorphism in vivo, in specific cell types. Since a large fraction of disease causing variants are thought to reside in non-coding DNA, a scalable method to test the effects of allelic variation in cis-regulatory elements would have a large impact on the field.
The human genome stores information that codes for the structure of genes, as well as information that specifies when, where, and to what levels genes are active. Biologists have sophisticated methods for reading the information that codes for genes. Methods for reading and interpreting non-coding DNA that specifies when and where genes synthesize their products are far less developed. Since a large number of disease-causing DNA variations occur in non-coding DNA we propose to develop better methods for identifying non-coding DNA sequences that control where, when, and to what extent genes are active.
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