Nucleic Acids as Direct Effectors in Gene Regulation: From Argonaute Proteins to Scalp Epigenomics

摘要

Much of the complexity of biology lies in the problem of how cells sharing the same, relatively static genetic code produce the vast diversity of cellular states and functions necessary for multicellular life. DNA and RNA are the storage and message molecules, respectively, of genetic information transfer, but these macromolecules have also been co-opted as direct functional players in the control of specific, contextdependent gene regulation. The vastness of sequence space and the challenge of generating quantitative, genome-scale datasets make understanding, predicting, and intervening in these modes of gene regulation daunting.The first part of this work addresses Argonaute family proteins, which load short nucleic acid guides to program specific binding to nucleic acid targets to regulate gene expression, host defense, and other biological functions. We deploy multiple highthroughput sequencing-based assays to measure the association rates, binding affinities, and single turnover cleavage rates for mouse Ago2 loaded with specific RNA guides against >40,000 unique RNA targets. We map sequence to structure to function relationships for Ago2 binding and cleavage, and show that our in vitro measurements can be used to predict gene repression in an engineered cellular system. We next use similar methodologic approaches to study an Argonaute protein derived from the bacterium Thermus thermophilus, TtAgo, that uses DNA guides to bind and cleave DNA targets at extreme temperatures. By measuring the binding of multiple DNA guides against thousands of targets each, we were able to construct general, quantitative models of association kinetics and binding affinity. We also show that guide sequence composition has dramatic effects on cleavage activity, suggesting that only a subset of guides are capable of cleaving targets at physiologically relevant temperatures.In the second part of this work, we examine a different form of gene regulation– the use of enhancers and other cis-regulatory elements to control gene expression in the many distinct cell types comprising human scalp. We generated paired single cell RNAand ATAC-sequencing datasets of primary human scalp. We use these integrated datasets to identify 'highly-regulated genes' linked to a disproportionately large number of enhancers and show that for a given highly-regulated gene expressed in multiple cell types, a greater number of linked enhancers is associated with higher levels of transcription. We demonstrate that genetic variation associated with skin and hair disease is specifically enriched in open chromatin regions of implicated cell types, including a strong association between dermal papilla cells and androgenetic alopecia. Using machine learning approaches, we further prioritize specific genetic variants that putatively disrupt transcription factor binding sites, leading to altered expression at disease-relevant genes.