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

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 challenges in identifying trans-acting changes. We present a comprehensive approach to directly identify cis- and trans-divergent regulatory elements between human and rhesus macaque lymphoblastoid cells using ATAC-STARR-seq. In addition to thousands of cis changes, we discover an unexpected number (~10,000) of trans changes and show that cis and trans elements exhibit distinct patterns of sequence divergence and function. We further identify differentially expressed transcription factors that underlie >50% of trans differences and trace how cis changes can produce cascades of trans changes. Overall, we find that most divergent elements (67%) 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.