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Abstract

Enhancers are regulatory DNA sequences that control gene spatial-temporal patterning based on their primary DNA sequence. Through the binding of proteins called Transcription Factors (TFs), enhancers turn genes “on” or “off” across fields of cells to express genes in complex patterns throughout development. To this day, we still cannot accurately and precisely synthesize an enhancer de-novo based on our best models. These findings suggest that we still have a limited understanding of how much regulatory information is encoded within the primary sequence of an enhancer. Furthermore, it is thought that enhancers are one of the primary drivers of evolution. Yet, we are far from predicting enhancer evolution due to limited technology and sparse experimental data. In this thesis, I review the field of enhancers, their evolution, and the regulation behind the shavenbaby locus. I next highlight the high-throughput technology developed to study enhancer mutants at a higher throughput with the help of a custom liquid-handling robot called Flyspresso and an adaptive-feedback confocal microscopy plugin. With this automated pipeline, I carry out a mutational scanning experiment on an enhancer at the shavenbaby locus called E3N to simulate possible paths and modes of evolution. I find that developmental enhancers are densely encoded and highly pleiotropic. I also identified new TF binding sites and examples of developmental biases that either constrain or drive evolution. I then discuss a mutational hotspot that evolves ectopic expression of shavenbaby in the developing wing and haltere, which I hypothesize is due to a transcriptional repressor. I additionally create a gene expression atlas for the late Drosophila embryo to map fragile and robust components of the E3N expression pattern and identify more TF binding sites. Finally, I summarize this thesis with an updated working model for E3N and an explanation to what extent we can predict E3N’s evolution.