Measuring allele-specific expression in interspecies hybrids is a powerful way to detect cis-regulatory changes underlying adaptation. However, it remains difficult to identify genes most likely to explain species-specific traits. Here, we outline a simple strategy that leverages population-scale allele-specific RNA-seq data to identify genes that show constrained cis-regulation within species yet show divergence between species. Applying this strategy to data from human-chimpanzee hybrid cortical organoids, we identify signatures of lineage-specific selection on genes related to saccharide metabolism, neurodegeneration, and primary cilia. We also highlight cis-regulatory divergence in CUX1 and EDNRB that may shape the trajectory of human brain development.

Measuring differences in allele specific gene expression in hybrids between two different species is a powerful way to detect cis-regulatory changes underlying adaptation. However, these studies frequently identify thousands of differentially expressed genes. This makes it difficult to differentiate candidate genes most likely to explain interspecies phenotypic differences from genes whose expression levels diverge with little evolutionary constraint. Here we outline a simple strategy that leverages population-scale allele-specific RNA-seq data to identify genes that have constrained cis-regulation within species yet show divergence between species. By applying this strategy to data from human/chimpanzee hybrid cortical spheroids we identify signatures of lineage-specific selection on the expression of genes related to cellular proliferation, speech, glucose metabolism, and glycan degradation. In addition, we highlight human-derived differences in the expression of CUX1 and EDNRB that may contribute to differences in neural connectivity and brain size between humans and other primates.

Although gene expression divergence has long been postulated to be the primary driver of human evolution, identifying the genes and genetic variants underlying uniquely human traits has proven to be quite challenging. Theory suggests that cell type-specific cis-regulatory variants may fuel evolutionary adaptation due to the specificity of their effects. These variants can precisely tune the expression of a single gene in a single cell type, avoiding the potentially deleterious consequences of trans-acting changes and non-cell type-specific changes that can impact many genes and cell types, respectively. It has recently become possible to quantify human-specific cis-acting regulatory divergence by measuring allele-specific expression in human-chimpanzee hybrid cells—the product of fusing induced pluripotent stem (iPS) cells of each species in vitro. However, these cis-regulatory changes have only been explored in a limited number of tissues and cell types. Here, we quantify human-chimpanzee cis-regulatory divergence in gene expression and chromatin accessibility across six cell types, enabling the identification of highly cell type-specific cis-regulatory changes. We find that cell type-specific genes and regulatory elements evolve faster than those shared across cell types, suggesting an important role for genes with cell type-specific expression in human evolution. Furthermore, we identify several instances of lineage-specific natural selection that may have played key roles in specific cell types, such as coordinated changes in the cis-regulation of dozens of genes involved in neuronal firing in motor neurons. Finally, using novel metrics and a machine learning model, we identify genetic variants that likely alter chromatin accessibility and transcription factor binding, leading to neuron-specific changes in the expression of the neurodevelopmentally important genes FABP7 and GAD1. Overall, our results demonstrate that integrative analysis of cis-regulatory divergence in chromatin accessibility and gene expression across cell types is a promising approach to identify the specific genes and genetic variants that make us human.

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Although gene expression divergence has long been postulated to be the primary driver of human evolution, identifying the genes and genetic variants underlying uniquely human traits has proven to be quite challenging. Theory suggests that cell type-specificcis-regulatory variants may fuel evolutionary adaptation due to the specificity of their effects. These variants can precisely tune the expression of a single gene in a single cell type, avoiding the potentially deleterious consequences oftrans-acting changes and non-cell type-specific changes that can impact many genes and cell types, respectively. It has recently become possible to quantify human-specificcis-acting regulatory divergence by measuring allele-specific expression in human-chimpanzee hybrid cells—the product of fusing induced pluripotent stem (iPS) cells of each speciesin vitro. However, thesecis-regulatory changes have only been explored in a limited number of tissues and cell types. Here, we quantify human-chimpanzeecis-regulatory divergence in gene expression and chromatin accessibility across six cell types, enabling the identification of highly cell type-specificcis-regulatory changes. We find that cell type-specific genes and regulatory elements evolve faster than those shared across cell types, suggesting an important role for genes with cell type-specific expression in human evolution. Furthermore, we identify several instances of lineage-specific natural selection that may have played key roles in specific cell types, such as coordinated changes in thecis-regulation of dozens of genes involved in neuronal firing in motor neurons. Finally, using novel metrics and a machine learning model, we identify genetic variants that likely alter chromatin accessibility and transcription factor binding, leading to neuron-specific changes in the expression of the neurodevelopmentally important genesFABP7andGAD1. Overall, our results demonstrate that integrative analysis ofcis-regulatory divergence in chromatin accessibility and gene expression across cell types is a promising approach to identify the specific genes and genetic variants that make us human.