Genetic studies of human-chimpanzee divergence using stem cell fusions

Abstract

Complete genome sequencing has identified millions of DNA changes that differ between humans and chimpanzees. Although a subset of these changes likely underlies important phenotypic differences between humans and chimpanzees, it is currently difficult to distinguish causal from incidental changes and to map specific phenotypes to particular genome locations. To facilitate further genetic study of human-chimpanzee divergence, we have generated human and chimpanzee auto-tetraploids and allo-tetraploids by fusing induced pluripotent stem cells (iPSCs) of each species. The resulting tetraploid iPSCs can be stably maintained and retain the ability to differentiate along ectoderm, mesoderm, and endoderm lineages. RNA sequencing identifies thousands of genes whose expression differs between humans and chimpanzees when assessed in single-species diploid or auto-tetraploid iPSCs. Analysis of gene expression patterns in inter-specific allo-tetraploid iPSCs shows that human-chimpanzee expression differences arise from substantial contributions of both cis-acting changes linked to the genes themselves, and trans-acting changes elsewhere in the genome. To enable further genetic mapping of species differences, we tested chemical treatments for stimulating genome-wide mitotic recombination between human and chimpanzee chromosomes, and CRISPR methods for inducing species-specific changes on particular chromosomes in allo-tetraploid cells. We successfully generated derivative cells with nested deletions or inter-specific recombination on the X chromosome. These studies identify a long distance cis-regulatory domain of the Fragile X-associated gene (FMR1), confirm an important role for the X chromosome in trans-regulation of other expression differences, and illustrate the potential of this system for more detailed mapping of the molecular basis of human and chimpanzee evolution.

Human-specific genetics: new tools to explore the molecular and cellular basis of human evolution

“…Recent studies have used allotetraploid cells to identify candidate cis-regulatory changes in iPSCs, neural crest cells and neural lineage cells, revealing candidate cell types, such as astrocytes with an enrichment of cis-regulatory changes, and candidate genes, such as EVC2, that may influence craniofacial development 216,255,256 . Importantly, isolating trans-regulatory changes will still require consistent patterning and differentiation of human and chimpanzee contributor lines, including human-human and chimpanzee-chimpanzee autotetraploid cells, to fates similar to those of fused autotetraploid cells.…”

Accounting for cis-regulatory constraint prioritizes genes likely to affect species-specific traits

“…This approach has been successfully applied in many species and advanced our understanding of the evolution of gene regulation and its role in establishing phenotypic variation [10][11][12][13]. Furthermore, the recent development of human-chimpanzee allotetraploid "hybrid" cells and organoids enables detailed, accurate quantification of differences in gene expression between humans and our closest living relatives [8,9,14].…”