Abstract: Understanding the genetic basis of gene regulation is key to understanding the evolutionary processes that have shaped specific traits in humans and non-human apes. In turn, identifying and characterizing the genetic variants that lead to inter-individual responses to different exposures is critical to gain insight into gene by environment interactions and associated phenotypic consequences. By elucidating the genetic variation that underlies regulatory differences between species, and the genetic variation that is associated with individual differences in response to environmental cues, we can gain a better understanding of the evolutionary processes that led to human-specific traits, as well as the genetic basis of complex traits and diseases. The challenge is that to carry out this work in humans and other apes, one must rely on in vitro systems. During the first term of the MIRA award, my lab focused on understanding the molecular mechanisms that underlie the evolution of gene expression, such as natural selection, the influence of epigenetic marks, and the effects of gene duplication. We developed and broadly shared a comparative panel of iPSCs from humans and chimpanzees, and we established a new approach for iPSC differentiation (we call it ‘guided differentiation’), which makes it feasible for us to study gene regulation across a broad range of cell types and contexts. We used these systems to study the impact of variation in gene regulatory networks on complex diseases and the role of gene regulation in the generation of phenotypic diversity. We explored the roles of gene expression in human diseases, such as cancer and neurological disorders, and developed methods for analyzing comparative single-cell gene expression data. In the next term of this award, we propose to continue to study similar broad areas. We will sharpen our focus on gene by environment interactions and develop a better understanding of the genetic variation that leads to regulatory variation in response to different exposures. We will employ a dynamic eQTL mapping approach to offer additional insight into the genetic basis for disease. We will also explore the role of epigenetic mechanisms, such as DNA methylation, histone modifications, and non-coding RNAs, in mediating gene by environment interactions. We will use the comparative iPSC panel to explore the relative impact and evolutionary consequences of changes in cis and trans regulatory mechanisms in dozens of different tissues and cell types and continue to share the panel and derivative cultures with the community. Finally, we will take advantage of improved single-cell technologies to characterize the dispersion and robustness associated with single-cell gene regulation and identify genetic mechanisms that underlie the regulation of gene regulatory noise.