Development of massively parallel reporter assays that use cognate promoters

University Of California, San Francisco, San Francisco CA

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Abstract

Mutations in gene regulatory elements are a major cause of human disease. The ENCODE, Epigenome Roadmap and other projects have identified millions of putative regulatory elements across more than one hundred cell types and tissues. While these maps have significantly expanded our knowledge of regulatory sequences, they are only descriptive and further high-throughput functional assays are needed in order to understand the biology of these elements. In addition, with whole-genomes on the verge of being commonly available, there is a pressing need to develop high-throughput assays that can rapidly analyze the functional effect of the thousands of variants detected in these genomes. Massively parallel reporter assays (MPRAs) provide such a technique, enabling the testing of thousands of sequences and their variants for reporter activity. However, they currently have several caveats. These include amongst others the inability to test long DNA sequences and the majority of MPRAs testing sequences in an episomal manner and alongside a minimal promoter instead of the target gene promoter. Here, we will develop a novel MPRA technology that will address all of these caveats. Using capture Hi-C technology that allows for the hybridization of regulatory elements to their target promoter, we will generate an MPRA library that encompasses long regulatory sequences cloned in front of their target promoters. Using a lentivirus based MPRA (lentiMPRA) method that we developed, we will test these sequences in a genome integrated manner. To functionally validate that there are indeed differences between a minimal promoter versus a target promoter based MPRA library, we will compare similar sequences in both contexts. Finally, we will also dissect enhancer-promoter interactions to pinpoint important sequences driving these interactions. The technology that we will develop will enable the testing of thousands of regulatory elements and their variants alongside their target promoter. As such, it will increase our understanding of regulatory element function and how gene regulatory elements communicate with their target promoter.