Human gene regulatory evolution is driven by the divergence of regulatory element function in both cis and trans

SUMMARYGene regulatory divergence between species can result fromcis-acting local changes to regulatory element DNA sequences or globaltrans-acting changes to the regulatory environment. Understanding how these mechanisms drive regulatory evolution has been limited by challenges in identifyingtrans-acting changes. We present a comprehensive approach to directly identifycis-andtrans-divergent regulatory elements between human and rhesus macaque lymphoblastoid cells using ATAC-STARR-seq. In addition to thousands ofcischanges, we discover an unexpected number (~10,000) oftranschanges and show thatcisandtranselements exhibit distinct patterns of sequence divergence and function. We further identify differentially expressed transcription factors that underlie >50% oftransdifferences and trace howcischanges can produce cascades oftranschanges. Overall, we find that most divergent elements (67%) experienced changes in bothcisandtrans, revealing a substantial role fortransdivergence—alone and together withcischanges—to regulatory differences between species.