Characterization of silencer element-associated chromatin

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Abstract Spatio-temporal control of gene expression is a fundamental process in biology. While the role of enhancers to positively regulate gene expression has been appreciated for decades, silencers are conspicuously absent from most models. A recent string of high-throughput screens in multiple systems has identified thousands of silencers. Strikingly, a large number of these silencers fall within bifunctional cis-regulatory elements that also act as enhancers in other cell types or developmental times. Silencers are also enriched for eQTLs and GWAS hits, suggesting they are essential for understanding the genetics of complex disease. Silencers are distinct from insulators, which prevent enhancer-promoter interactions. Silencers actively dampen expression of their target genes and, like enhancers, act independently of position and orientation. Even though a large number of silencers have been functionally identified in reporter assays, it is still difficult to predict new silencer elements. While enhancers, promoters, and insulators can be predicted by ChIP-seq for specific histone post- translational modifications, no chromatin modifications are known to be predictive of silencers. Additionally, the set of DNA binding proteins and their cofactors at silencers is largely unknown. Identification of ChIP-seq target that are highly predictive of silencers would vastly improve the ability of researchers to study these fundamental elements in new contexts and can give insights into silencing mechanisms. In order to address fundamental lingering questions regarding silencers, I will perform a proteomic survey to identify factors that distinguish silencer and enhancer states of bifunctional cis-regulatory elements in Drosophila mesoderm. This will give key insights into the mechanisms of silencer elements. I will also investigate the three-dimensional contacts formed between silencers and their targets. It has already been observed that silencers bound by Snail do not contact TSSs, but instead disrupt normal enhancer-promoter contacts, a process known as anti- looping. Completion of this project will fill address many of the outstanding questions regarding silencer elements, including the factors responsible for silencing and the effect they have on their target genes. This project will uncover specific markers of silencers that will allow future investigators to identify them via ChIP- seq similar to how other functional elements can be annotated. This research project will give valuable experience in experimental design and analysis that I hope to carry with me into a career as an academic investigator.