Enhancer screens to study enhancer regulation in development and evolution

Enhancers are sequences in the genome that act as switches to turn on gene expression in the right time and place during development. Enhancers are regulated by binding of transcription factors to recruit transcriptional machinery. However, exactly how the sequence of an enhancer encodes this function is poorly understood. Reporter assays test enhancers for activity, and mutational approaches to evaluate important sequences within an enhancer help us better understand enhancer regulation. However, testing of individual enhancers is slow and tedious process. Massively parallel reporter assays (MPRAs) can test thousands to millions of enhancers within a single experiment. In this dissertation, I discuss the use of two enhancer MPRAs to further our understanding of enhancer regulation in development and evolution. First, I performed an MPRA to study enhancer regulation in the developing notochord of Ciona robusta and discovered notochord logics and grammars, the interplay between transcription factor order, orientation, spacing, and binding affinity, important for driving notochord-specific expression. These enhancer logics and grammars show signatures of conservation across chordates. In Chapter 2, I developed enhancer MPRAs in the chicken limb bud, which is the first of its kind in developing vertebrate embryos. Using this method, I identified new enhancers active in the forelimb and hindlimb that could be further studied to understand how sequence changes impact enhancer activity. Overall, the methods I developed to test genomic regions in developing chordate and vertebrate embryos will enable unprecedented insight into how enhancers encode the instructions for development