Geller, E. | Search bioRxiv

bioRxiv2019

Massively parallel discovery of human-specific substitutions that alter neurodevelopmental enhancer activity

Genetic changes that altered the function of gene regulatory elements have been implicated in the evolution of the human brain. However, identifying the particular changes that modified regulatory activity during neurodevelopment remains challenging. Here we used massively parallel enhancer assays in human neural stem cells to measure the impact of 32,776 human-specific substitutions on enhancer activity in 1,363 Human Accelerated Regions (HARs) and 3,027 Human Gain Enhancers (HGEs), which include enhancers with novel activities in humans. We found that 31.9% of active HARs and 36.4% of active HGEs exhibited differential activity between human and chimpanzee. This enabled us to isolate the effects of 401 human-specific substitutions from other types of genetic variation in HARs and HGEs. Substitutions acted in both an additive and non-additive manner to alter enhancer activity. Human-specific substitutions altered predicted binding sites for a specific set of human transcription factors (TFs) that were a subset of TF binding sites associated with enhancer activity in our assay. Substitutions within HARs, which are overall highly constrained compared to HGEs, showed smaller effects on enhancer activity, suggesting that the impact of human-specific substitutions may be buffered in enhancers with constrained ancestral functions. Our findings yield insight into the mechanisms by which human-specific genetic changes impact enhancer function and provide a rich set of candidates for experimental studies of regulatory evolution in humans.

genomics↗

bioRxiv2019

Massively parallel disruption of enhancers active during human corticogenesis

Changes in gene regulation have been linked to the expansion of the human cerebral cortex and to neurodevelopmental disorders. However, the biological effects of genetic variation within developmental regulatory elements on human corticogenesis are not well understood. We used sgRNA-Cas9 genetic screens in human neural stem cells (hNSCs) to disrupt 10,674 expressed genes and 2,227 enhancers active in the developing human cortex and determine the resulting effects on cellular proliferation. Gene disruptions affecting proliferation were enriched for genes associated with risk for human neurodevelopmental phenotypes including primary microcephaly and autism spectrum disorder. Although disruptions in enhancers had overall weaker effects on proliferation than gene disruptions, we identified enhancer disruptions that severely perturbed hNSC self-renewal. Disruptions in Human Accelerated Regions and Human Gain Enhancers, regulatory elements implicated in the evolution of the human brain, also perturbed hNSC proliferation, establishing a role for these elements in human neurodevelopment. Integrating proliferation phenotypes with chromatin interaction maps revealed regulatory relationships between enhancers and target genes that contribute to neurogenesis and potentially to human cortical evolution.

genomics↗