Identifying functional enhancer mutations in the evolution of primate brain development

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Genetic variants in gene regulatory sequences are thought to contribute both to human disease and the evolution of uniquely human phenotypes. However, we lack the means to predict which variants in the genome will affect regulatory function, or to determine the extent of their effects. Current models are based on descriptive data of the function of gene regulatory elements such as evolutionary conservation, and thus suffer from low explanatory power and thus potentially high false positive rates due to a lack of direct experimental evidence.Here I propose a study to quantify the effects of mutations within enhancer sequences and to develop a predictive model of such mutations. Using a massively parallel reporter assay (MPRA), I will quantify the effects of human-specific substitutions in enhancers we previously identified as showing evolutionary gains in activity in the developing human neocortex. In a parallel approach, I will derive neural stem cells, which serve as a model for neurodevelopment, from induced pluripotent stem cells of humans and chimpanzees. I will conduct genome-wide comparisons of gene regulatory biochemical activity by analysing H3K27ac histone marks and chromatin accessibility in these cell lines and connect observed differences with human-specific enhancer substitutions. I will then use these two lines of empirical results to develop a predictive model to globally identify variants that alter enhancer activity. I will further use a large range of genome-wide parameters such as sequence content, transcription factor binding site predictions, evolutionary conservation, and biochemical markers to predict functional effects of enhancer substitutions. This two-pronged approach using data from a regulatory assay and from biochemical comparisons will enable me to identify substitutions that alter regulatory activity both through sequence change and through differential chromatin activity. Finally, I will validate the model by investigating variants with large effects as predicted by the model in human and chimpanzee neural stem cell lines by cross-comparing MPRA and biochemical comparisons and by analysing gene expression differences between species.This project will provide both specific insights into the evolution of human brain development and general insights into the evolution of regulatory function through enhancer sequence change. The direct experimental evidence my analyses will provide will enable us to better predict genetic variants that influence regulatory function. A powerful way to predict the effects of substitutions in gene regulatory elements may easily transform the way we understand how genetic variation affects regulatory function and thus biology.