Identifying Gene Regulatory Activity Divergence in Cis and Trans with ATAC-STARR-seq

Abstract

Gene regulatory divergence between species can result from cis-acting local changes to regulatory element DNA sequences or global trans-acting changes to the regulatory environment. Understanding how these mechanisms drive regulatory evolution has been limited by a lack of methodology to identify cis and trans divergence on a global scale. In this dissertation, I present ATAC-STARR-seq, a new method I developed that allows direct identification of cis- and trans-divergent regulatory elements between human and rhesus macaque lymphoblastoid cells. In addition to thousands of cis changes, I discover an unexpected number (~10,000) of trans changes and show that cis and trans elements exhibit distinct patterns of sequence divergence and function. Overall, I find that 67% of divergent elements experienced changes in both cis and trans, revealing a substantial role for trans divergence—alone and together with cis changes—to regulatory differences between species. In addition to this evolutionary study, I show how ATAC-STARR-seq can be used to identify five distinct layers of gene regulation: 1) chromatin accessibility, 2) TF binding, 3) active regulatory activity, 4) silent regulatory activity, and 5) DNA regulatory element networks including active and silent regulatory elements. I additionally characterize key parameters of the assay and provide the field with computational and technical support to increase accessibility of the method to others. Overall, ATAC-STARR-seq will allow researchers to investigate new and important questions in gene regulation for years to come.