Principles of regulatory evolution inferred from early differentiation in primates

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Changes in regulatory sequences have undoubtedly played an important role during human evolution. However, we still know very little about them. This is in part because we do not understand very well how gene regulation is encoded in the human genome. Here, we suggest taking an evolutionary approach by generating and analysing single-cell expression and epigenomic data from four primate species during embryoid body formation to learn more about this "regulatory code".One central insight from recent studies of the evolution of regulatory elements is that they have a high turnover rate even though the regulated processes are very conserved across species. Hence, it has been speculated that compensatory evolution is common in regulatory evolution. With the proposed study design we can evaluate how common compensatory evolution is for transcriptional regulatory elements (TREs) during primate evolution: We will differentiate induced pluripotent stem cells from humans, gorillas, orangutans and cynomolgus macaques to embryoid bodies (EBs) and generate scRNA-seq and scATAC-seq data from a total of ~150,000 cells. With this novel data set we can reconstruct the developmental trajectories from all three germ layers and compare them among the four species. Using the scRNA-seq data, we will be able to estimate the conservation of gene regulation and regulatory networks during early primate differentiation, and using the scATAC-seq data we can infer and compare the active TREs for each gene, species and trajectory. We will then integrate the scRNA-seq and the scATAC-seq data to connect measures for the conservation of gene regulation, with the conservation of TRE accessibility and TRE sequence evolution during early primate differentiation. Now, we can identify cases of compensatory evolution as genes that have a conserved expression trajectory, but non-conserved TREs. In the easiest case, we will find functionally analogous, but non-orthologous TREs (faTREs). We will validate the functional equivalence of the faTREs using a massively parallel reporter assay (MPRA). Once we have established functional equivalence, we can identify sequence features such as the diversity or number of transcription factor binding sites that can predict compensatory evolution. In summary, our approach will reveal principles of regulatory evolution that will improve our understanding of human gene regulation.