Disentangling cell-intrinsic and extrinsic factors underlying gene expression evolution.

Chimeras have played a foundational role in biology, for example by enabling the classification of developmental processes into those driven intrinsically by individual cells versus those driven extrinsically by their extracellular environment. Here, we extend this framework to decompose evolutionary divergence in gene expression and other quantitative traits into cell-intrinsic, extrinsic, and intrinsic-extrinsic interaction components. Applying this framework to reciprocal rat-mouse chimeras, we found that the majority of gene expression divergence is attributable to cell-intrinsic factors, though extrinsic factors also play an integral role. For example, a rat-like extracellular environment extrinsically up-regulates the expression of a key transcriptional regulator of the endoplasmic reticulum (ER) stress response in some but not all cell types, which in turn strongly predicts extrinsic up-regulation of its target genes and of the ER stress response pathway as a whole. This effect is also seen at the protein level, suggesting propagation through multiple regulatory levels. We also demonstrate that our framework is applicable to a cellular trait, neuronal differentiation, and estimated the intrinsic and extrinsic contributions to its divergence. Finally, we show that imprinted genes are dramatically mis-expressed in species-mismatched environments, suggesting that mismatch between rapidly evolving intrinsic and extrinsic mechanisms controlling gene imprinting may contribute to barriers to interspecies chimerism. Overall, our conceptual framework opens new avenues to investigate the mechanistic basis of evolutionary divergence in gene expression and other quantitative traits in any multicellular organism.

Cell-type-specific cis-regulatory divergence in gene expression and chromatin accessibility revealed by human-chimpanzee hybrid cells.

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 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.

Chromatin activity identifies differential gene regulation across human ancestries.

BACKGROUND: Current evidence suggests that cis-regulatory elements controlling gene expression may be the predominant target of natural selection in humans and other species. Detecting selection acting on these elements is critical to understanding evolution but remains challenging because we do not know which mutations will affect gene regulation. RESULTS: To address this, we devise an approach to search for lineage-specific selection on three critical steps in transcriptional regulation: chromatin activity, transcription factor binding, and chromosomal looping. Applying this approach to lymphoblastoid cells from 831 individuals of either European or African descent, we find strong signals of differential chromatin activity linked to gene expression differences between ancestries in numerous contexts, but no evidence of functional differences in chromosomal looping. Moreover, we show that enhancers rather than promoters display the strongest signs of selection associated with sites of differential transcription factor binding. CONCLUSIONS: Overall, our study indicates that some cis-regulatory adaptation may be more easily detected at the level of chromatin than DNA sequence. This work provides a vast resource of genomic interaction data from diverse human populations and establishes a novel selection test that will benefit future study of regulatory evolution in humans and other species.

Allele-specific expression reveals genetic drivers of tissue regeneration in mice.

In adult mammals, skin wounds typically heal by scarring rather than through regeneration. In contrast, "super-healer" Murphy Roths Large (MRL) mice have the unusual ability to regenerate ear punch wounds; however, the molecular basis for this regeneration remains elusive. Here, in hybrid crosses between MRL and non-regenerating mice, we used allele-specific gene expression to identify cis-regulatory variation associated with ear regeneration. Analyzing three major cell populations (immune, fibroblast, and endothelial), we found that genes with cis-regulatory differences specifically in fibroblasts were associated with wound-healing pathways and also co-localized with quantitative trait loci for ear wound-healing. Ectopic treatment with one of these proteins, complement factor H (CFH), accelerated wound repair and induced regeneration in typically fibrotic wounds. Through single-cell RNA sequencing (RNA-seq), we observed that CFH treatment dramatically reduced immune cell recruitment to wounds, suggesting a potential mechanism for CFH's effect. Overall, our results provide insights into the molecular drivers of regeneration with potential clinical implications.

Cell type-specific cis-regulatory divergence in gene expression and chromatin accessibility revealed by human-chimpanzee hybrid cells.

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 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.

Widespread epistasis among beneficial genetic variants revealed by high-throughput genome editing.

The phenotypic effect of any genetic variant can be altered by variation at other genomic loci. Known as epistasis, these genetic interactions shape the genotype-phenotype map of every species, yet their origins remain poorly understood. To investigate this, we employed high-throughput genome editing to measure the fitness effects of 1,826 naturally polymorphic variants in four strains of Saccharomyces cerevisiae. About 31% of variants affect fitness, of which 24% have strain-specific fitness effects indicative of epistasis. We found that beneficial variants are more likely to exhibit genetic interactions and that these interactions can be mediated by specific traits such as flocculation ability. This work suggests that adaptive evolution will often involve trade-offs where a variant is only beneficial in some genetic backgrounds, potentially explaining why many beneficial variants remain polymorphic. In sum, we provide a framework to understand the factors influencing epistasis with single-nucleotide resolution, revealing widespread epistasis among beneficial variants.

Gene-by-environment interactions are pervasive among natural genetic variants.

Gene-by-environment (GxE) interactions, in which a genetic variant's phenotypic effect is condition specific, are fundamental for understanding fitness landscapes and evolution but have been difficult to identify at the single-nucleotide level. Although many condition-specific quantitative trait loci (QTLs) have been mapped, these typically contain numerous inconsequential variants in linkage, precluding understanding of the causal GxE variants. Here, we introduce BARcoded Cas9 retron precise parallel editing via homology (CRISPEY-BAR), a high-throughput precision genome editing strategy, and use it to map GxE interactions of naturally occurring genetic polymorphisms impacting yeast growth. We identified hundreds of GxE variants within condition-specific QTLs, revealing unexpected genetic complexity. Moreover, we found that 93.7% of non-neutral natural variants within ergosterol biosynthesis pathway genes showed GxE interactions, including many impacting antifungal drug resistance through diverse molecular mechanisms. In sum, our results suggest an extremely complex, context-dependent fitness landscape characterized by pervasive GxE interactions while also demonstrating massively parallel genome editing as an effective means for investigating this complexity.

Evolution of Spatial and Temporal cis-Regulatory Divergence in Sticklebacks.

Cis-regulatory changes are thought to play a major role in adaptation. Threespine sticklebacks have repeatedly colonized freshwater habitats in the Northern Hemisphere, where they have evolved a suite of phenotypes that distinguish them from marine populations, including changes in physiology, behavior, and morphology. To understand the role of gene regulatory evolution in adaptive divergence, here we investigate cis-regulatory changes in gene expression between marine and freshwater ecotypes through allele-specific expression (ASE) in F1 hybrids. Surveying seven ecologically relevant tissues, including three sampled across two developmental stages, we identified cis-regulatory divergence affecting a third of genes, nearly half of which were tissue-specific. Next, we compared allele-specific expression in dental tissues at two timepoints to characterize cis-regulatory changes during development between marine and freshwater fish. Applying a genome-wide test for selection on cis-regulatory changes, we find evidence for lineage-specific selection on several processes between ecotypes, including the Wnt signaling pathway in dental tissues. Finally, we show that genes with ASE, particularly those that are tissue-specific, are strongly enriched in genomic regions of repeated marine-freshwater divergence, supporting an important role for these cis-regulatory differences in parallel adaptive evolution of sticklebacks to freshwater habitats. Altogether, our results provide insight into the cis-regulatory landscape of divergence between stickleback ecotypes across tissues and during development, and support a fundamental role for tissue-specific cis-regulatory changes in rapid adaptation to new environments.

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

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.

Transcriptome diversity is a systematic source of variation in RNA-sequencing data.

RNA sequencing has been widely used as an essential tool to probe gene expression. While standard practices have been established to analyze RNA-seq data, it is still challenging to interpret and remove artifactual signals. Several biological and technical factors such as sex, age, batches, and sequencing technology have been found to bias these estimates. Probabilistic estimation of expression residuals (PEER), which infers broad variance components in gene expression measurements, has been used to account for some systematic effects, but it has remained challenging to interpret these PEER factors. Here we show that transcriptome diversity-a simple metric based on Shannon entropy-explains a large portion of variability in gene expression and is the strongest known factor encoded in PEER factors. We then show that transcriptome diversity has significant associations with multiple technical and biological variables across diverse organisms and datasets. In sum, transcriptome diversity provides a simple explanation for a major source of variation in both gene expression estimates and PEER covariates.

cis-Regulatory changes in locomotor genes are associated with the evolution of burrowing behavior.

How evolution modifies complex, innate behaviors is largely unknown. Divergence in many morphological traits, and some behaviors, is linked to cis-regulatory changes in gene expression. Given this, we compare brain gene expression of two interfertile sister species of Peromyscus mice that show large and heritable differences in burrowing behavior. Species-level differential expression and allele-specific expression in F1 hybrids indicate a preponderance of cis-regulatory divergence, including many genes whose cis-regulation is affected by burrowing behavior. Genes related to locomotor coordination show the strongest signals of lineage-specific selection on burrowing-induced cis-regulatory changes. Furthermore, genetic markers closest to these candidate genes associate with variation in burrow shape in a genetic cross, suggesting an enrichment for loci affecting burrowing behavior near these candidate locomotor genes. Our results provide insight into how cis-regulated gene expression can depend on behavioral context and how this dynamic regulatory divergence between species may contribute to behavioral evolution.

Bacterial Retrons Enable Precise Gene Editing in Human Cells.

Retrons are bacterial genetic elements involved in anti-phage defense. They have the unique ability to reverse transcribe RNA into multicopy single-stranded DNA (msDNA) that remains covalently linked to their template RNA. Retrons coupled with CRISPR-Cas9 in yeast have been shown to improve the efficiency of precise genome editing via homology-directed repair (HDR). In human cells, HDR editing efficiency has been limited by challenges associated with delivering extracellular donor DNA encoding the desired mutation. In this study, we tested the ability of retrons to produce msDNA as donor DNA and facilitate HDR by tethering msDNA to guide RNA in HEK293T and K562 cells. Through heterologous reconstitution of retrons from multiple bacterial species with the CRISPR-Cas9 system, we demonstrated HDR rates of up to 11.4%. Overall, our findings represent the first step in extending retron-based precise gene editing to human cells.

Divergent patterns of selection on metabolite levels and gene expression.

BACKGROUND: Natural selection can act on multiple genes in the same pathway, leading to polygenic adaptation. For example, adaptive changes were found to down-regulate six genes involved in ergosterol biosynthesis-an essential pathway targeted by many antifungal drugs-in some strains of the yeast Saccharomyces cerevisiae. However, the impact of this polygenic adaptation on metabolite levels was unknown. Here, we performed targeted mass spectrometry to measure the levels of eight metabolites in this pathway in 74 yeast strains from a genetic cross. RESULTS: Through quantitative trait locus (QTL) mapping we identified 19 loci affecting ergosterol pathway metabolite levels, many of which overlap loci that also impact gene expression within the pathway. We then used the recently developed v-test, which identified selection acting upon three metabolite levels within the pathway, none of which were predictable from the gene expression adaptation. CONCLUSIONS: These data showed that effects of selection on metabolite levels were complex and not predictable from gene expression data. This suggests that a deeper understanding of metabolism is necessary before we can understand the impacts of even relatively straightforward gene expression adaptations on metabolic pathways.

GRINS: Genetic elements that recode assembly-line polyketide synthases and accelerate their diversification.

Assembly-line polyketide synthases (PKSs) are large and complex enzymatic machineries with a multimodular architecture, typically encoded in bacterial genomes by biosynthetic gene clusters. Their modularity has led to an astounding diversity of biosynthesized molecules, many with medical relevance. Thus, understanding the mechanisms that drive PKS evolution is fundamental for both functional prediction of natural PKSs as well as for the engineering of novel PKSs. Here, we describe a repetitive genetic element in assembly-line PKS genes which appears to play a role in accelerating the diversification of closely related biosynthetic clusters. We named this element GRINS: genetic repeats of intense nucleotide skews. GRINS appear to recode PKS protein regions with a biased nucleotide composition and to promote gene conversion. GRINS are present in a large number of assembly-line PKS gene clusters and are particularly widespread in the actinobacterial genus Streptomyces While the molecular mechanisms associated with GRINS appearance, dissemination, and maintenance are unknown, the presence of GRINS in a broad range of bacterial phyla and gene families indicates that these genetic elements could play a fundamental role in protein evolution.

The cis-regulatory effects of modern human-specific variants.

The Neanderthal and Denisovan genomes enabled the discovery of sequences that differ between modern and archaic humans, the majority of which are noncoding. However, our understanding of the regulatory consequences of these differences remains limited, in part due to the decay of regulatory marks in ancient samples. Here, we used a massively parallel reporter assay in embryonic stem cells, neural progenitor cells, and bone osteoblasts to investigate the regulatory effects of the 14,042 single-nucleotide modern human-specific variants. Overall, 1791 (13%) of sequences containing these variants showed active regulatory activity, and 407 (23%) of these drove differential expression between human groups. Differentially active sequences were associated with divergent transcription factor binding motifs, and with genes enriched for vocal tract and brain anatomy and function. This work provides insight into the regulatory function of variants that emerged along the modern human lineage and the recent evolution of human gene expression.

Primate cell fusion disentangles gene regulatory divergence in neurodevelopment.

Among primates, humans display a unique trajectory of development that is responsible for the many traits specific to our species. However, the inaccessibility of primary human and chimpanzee tissues has limited our ability to study human evolution. Comparative in vitro approaches using primate-derived induced pluripotent stem cells have begun to reveal species differences on the cellular and molecular levels1,2. In particular, brain organoids have emerged as a promising platform to study primate neural development in vitro3-5, although cross-species comparisons of organoids are complicated by differences in developmental timing and variability of differentiation6,7. Here we develop a new platform to address these limitations by fusing human and chimpanzee induced pluripotent stem cells to generate a panel of tetraploid hybrid stem cells. We applied this approach to study species divergence in cerebral cortical development by differentiating these cells into neural organoids. We found that hybrid organoids provide a controlled system for disentangling cis- and trans-acting gene-expression divergence across cell types and developmental stages, revealing a signature of selection on astrocyte-related genes. In addition, we identified an upregulation of the human somatostatin receptor 2 gene (SSTR2), which regulates neuronal calcium signalling and is associated with neuropsychiatric disorders8,9. We reveal a human-specific response to modulation of SSTR2 function in cortical neurons, underscoring the potential of this platform for elucidating the molecular basis of human evolution.

Human-chimpanzee fused cells reveal cis-regulatory divergence underlying skeletal evolution.

Gene regulatory divergence is thought to play a central role in determining human-specific traits. However, our ability to link divergent regulation to divergent phenotypes is limited. Here, we utilized human-chimpanzee hybrid induced pluripotent stem cells to study gene expression separating these species. The tetraploid hybrid cells allowed us to separate cis- from trans-regulatory effects, and to control for nongenetic confounding factors. We differentiated these cells into cranial neural crest cells, the primary cell type giving rise to the face. We discovered evidence of lineage-specific selection on the hedgehog signaling pathway, including a human-specific sixfold down-regulation of EVC2 (LIMBIN), a key hedgehog gene. Inducing a similar down-regulation of EVC2 substantially reduced hedgehog signaling output. Mice and humans lacking functional EVC2 show striking phenotypic parallels to human-chimpanzee craniofacial differences, suggesting that the regulatory divergence of hedgehog signaling may have contributed to the unique craniofacial morphology of humans.

Lineage-specific selection and the evolution of virulence in the Candida clade.

Candida albicans is the most common cause of systemic fungal infections in humans and is considerably more virulent than its closest known relative, Candida dubliniensis. To investigate this difference, we constructed interspecies hybrids and quantified mRNA levels produced from each genome in the hybrid. This approach systematically identified expression differences in orthologous genes arising from cis-regulatory sequence changes that accumulated since the two species last shared a common ancestor, some 10 million y ago. We documented many orthologous gene-expression differences between the two species, and we pursued one striking observation: All 15 genes coding for the enzymes of glycolysis showed higher expression from the C. albicans genome than the C. dubliniensis genome in the interspecies hybrid. This pattern requires evolutionary changes to have occurred at each gene; the fact that they all act in the same direction strongly indicates lineage-specific natural selection as the underlying cause. To test whether these expression differences contribute to virulence, we created a C. dubliniensis strain in which all 15 glycolysis genes were produced at modestly elevated levels and found that this strain had significantly increased virulence in the standard mouse model of systemic infection. These results indicate that small expression differences across a deeply conserved set of metabolism enzymes can play a significant role in the evolution of virulence in fungal pathogens.