The evolution of lineage-specific regulatory activities in the human embryonic limb.

The evolution of human anatomical features likely involved changes in gene regulation during development. However, the nature and extent of human-specific developmental regulatory functions remain unknown. We obtained a genome-wide view of cis-regulatory evolution in human embryonic tissues by comparing the histone modification H3K27ac, which provides a quantitative readout of promoter and enhancer activity, during human, rhesus, and mouse limb development. Based on increased H3K27ac, we find that 13% of promoters and 11% of enhancers have gained activity on the human lineage since the human-rhesus divergence. These gains largely arose by modification of ancestral regulatory activities in the limb or potential co-option from other tissues and are likely to have heterogeneous genetic causes. Most enhancers that exhibit gain of activity in humans originated in mammals. Gains at promoters and enhancers in the human limb are associated with increased gene expression, suggesting they include molecular drivers of human morphological evolution.

Coexpression networks implicate human midfetal deep cortical projection neurons in the pathogenesis of autism.

Autism spectrum disorder (ASD) is a complex developmental syndrome of unknown etiology. Recent studies employing exome- and genome-wide sequencing have identified nine high-confidence ASD (hcASD) genes. Working from the hypothesis that ASD-associated mutations in these biologically pleiotropic genes will disrupt intersecting developmental processes to contribute to a common phenotype, we have attempted to identify time periods, brain regions, and cell types in which these genes converge. We have constructed coexpression networks based on the hcASD "seed" genes, leveraging a rich expression data set encompassing multiple human brain regions across human development and into adulthood. By assessing enrichment of an independent set of probable ASD (pASD) genes, derived from the same sequencing studies, we demonstrate a key point of convergence in midfetal layer 5/6 cortical projection neurons. This approach informs when, where, and in what cell types mutations in these specific genes may be productively studied to clarify ASD pathophysiology.

Evolutionary innovations in conserved regulatory elements associate with developmental genes in mammals.

Transcriptional enhancers orchestrate cell type- and time point-specific gene expression programs. Genetic variation within enhancer sequences is an important contributor to phenotypic variation including evolutionary adaptations and human disease. Certain genes and pathways may be more prone to regulatory evolution than others, with different patterns across diverse organisms, but whether such patterns exist has not been investigated at a sufficient scale. To address this question, we identified signatures of accelerated sequence evolution in conserved enhancer elements throughout the mammalian phylogeny at an unprecedented scale. While different genes and pathways were enriched for regulatory evolution in different parts of the tree, we found a striking overall pattern of pleiotropic genes involved in gene regulatory and developmental processes being enriched for accelerated enhancer evolution. These genes were connected to more enhancers than other genes, which was the basis for having an increased amount of sequence acceleration over all their enhancers combined. We provide evidence that sequence acceleration is associated with turnover of regulatory function. Detailed study of one acceleration event in an enhancer of HES1 revealed that sequence evolution led to a new activity domain in the developing limb that emerged concurrently with the evolution of digit reduction in hoofed mammals. Our results provide evidence that enhancer evolution has been a frequent contributor to regulatory innovation at conserved developmental signaling genes in mammals.

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

Genetic changes that altered the function of gene regulatory elements have been implicated in the evolution of human traits such as the expansion of the cerebral cortex. However, identifying the particular changes that modified regulatory activity during human evolution remain challenging. Here we used massively parallel enhancer assays in neural stem cells to quantify the functional impact of >32,000 human-specific substitutions in >4,300 human accelerated regions (HARs) and human gain enhancers (HGEs), which include enhancers with novel activities in humans. We found that >30% of active HARs and HGEs exhibited differential activity between human and chimpanzee. We isolated the effects of human-specific substitutions from background genetic variation to identify the effects of genetic changes most relevant to human evolution. We found that substitutions interacted in both additive and nonadditive ways to modify enhancer function. Substitutions within HARs, which are highly constrained compared to HGEs, showed smaller effects on enhancer activity, suggesting that the impact of human-specific substitutions is buffered in enhancers with constrained ancestral functions. Our findings yield insight into how human-specific genetic changes altered enhancer function and provide a rich set of candidates for studies of regulatory evolution in humans.

A Statistical Framework for Mapping Risk Genes from De Novo Mutations in Whole-Genome-Sequencing Studies.

Analysis of de novo mutations (DNMs) from sequencing data of nuclear families has identified risk genes for many complex diseases, including multiple neurodevelopmental and psychiatric disorders. Most of these efforts have focused on mutations in protein-coding sequences. Evidence from genome-wide association studies (GWASs) strongly suggests that variants important to human diseases often lie in non-coding regions. Extending DNM-based approaches to non-coding sequences is challenging, however, because the functional significance of non-coding mutations is difficult to predict. We propose a statistical framework for analyzing DNMs from whole-genome sequencing (WGS) data. This method, TADA-Annotations (TADA-A), is a major advance of the TADA method we developed earlier for DNM analysis in coding regions. TADA-A is able to incorporate many functional annotations such as conservation and enhancer marks, to learn from data which annotations are informative of pathogenic mutations, and to combine both coding and non-coding mutations at the gene level to detect risk genes. It also supports meta-analysis of multiple DNM studies, while adjusting for study-specific technical effects. We applied TADA-A to WGS data of ∼300 autism-affected family trios across five studies and discovered several autism risk genes. The software is freely available for all research uses.

Disrupting the three-dimensional regulatory topology of the Pitx1 locus results in overtly normal development.

Developmental gene expression patterns are orchestrated by thousands of distant-acting transcriptional enhancers. However, identifying enhancers essential for the expression of their target genes has proven challenging. Maps of long-range regulatory interactions may provide the means to identify enhancers crucial for developmental gene expression. To investigate this hypothesis, we used circular chromosome conformation capture coupled with interaction maps in the mouse limb to characterize the regulatory topology of Pitx1, which is essential for hindlimb development. We identified a robust hindlimb-specific interaction between Pitx1 and a putative hindlimb-specific enhancer. To interrogate the role of this interaction in Pitx1 regulation, we used genome editing to delete this enhancer in mouse. Although deletion of the enhancer completely disrupts the interaction, Pitx1 expression in the hindlimb is only mildly affected, without any detectable compensatory interactions between the Pitx1 promoter and potentially redundant enhancers. Pitx1 enhancer null mice did not exhibit any of the characteristic morphological defects of the Pitx1-/- mutant. Our results suggest that robust, tissue-specific physical interactions at essential developmental genes have limited predictive value for identifying enhancer mutations with strong loss-of-function phenotypes.

Genome-wide analysis of the regulation of Cu metabolism in Cryptococcus neoformans.

The ability of the human fungal pathogen Cryptococcus neoformans to adapt to variable copper (Cu) environments within the host is key for successful dissemination and colonization. During pulmonary infection, host alveolar macrophages compartmentalize Cu into the phagosome and C. neoformans Cu-detoxifying metallothioneins, MT1 and MT2, are required for survival of the pathogen. In contrast, during brain colonization the C. neoformans Cu+ importers Ctr1 and Ctr4 are required for virulence. Central for the regulation and expression of both the Cu detoxifying MT1/2 and the Cu acquisition Ctr1/4 proteins is the Cu-metalloregulatory transcription factor Cuf1, an established C. neoformans virulence factor. Due to the importance of the distinct C. neoformans Cu homeostasis mechanisms during host colonization and virulence, and to the central role of Cuf1 in regulating Cu homeostasis, we performed a combination of RNA-Seq and ChIP-Seq experiments to identify differentially transcribed genes between conditions of high and low Cu. We demonstrate that the transcriptional regulation exerted by Cuf1 is intrinsically complex and that Cuf1 also functions as a transcriptional repressor. The Cu- and Cuf1-dependent regulon in C. neoformans reveals new adaptive mechanisms for Cu homeostasis in this pathogenic fungus and identifies potential new pathogen-specific targets for therapeutic intervention in fungal infections.

Evolution of Gene Regulation in Humans.

As a species, we possess unique biological features that distinguish us from other primates. Here, we review recent efforts to identify changes in gene regulation that drove the evolution of novel human phenotypes. We discuss genotype-directed comparisons of human and nonhuman primate genomes to identify human-specific genetic changes that may encode new regulatory functions. We also review phenotype-directed approaches, which use comparisons of gene expression or regulatory function in homologous human and nonhuman primate cells and tissues to identify changes in expression levels or regulatory activity that may be due to genetic changes in humans. Together, these studies are beginning to reveal the landscape of regulatory innovation in human evolution and point to specific regulatory changes for further study. Finally, we highlight two novel strategies to model human-specific regulatory functions in vivo: primate induced pluripotent stem cells and the generation of humanized mice by genome editing.

Origin and evolution of developmental enhancers in the mammalian neocortex.

Morphological innovations such as the mammalian neocortex may involve the evolution of novel regulatory sequences. However, de novo birth of regulatory elements active during morphogenesis has not been extensively studied in mammals. Here, we use H3K27ac-defined regulatory elements active during human and mouse corticogenesis to identify enhancers that were likely active in the ancient mammalian forebrain. We infer the phylogenetic origins of these enhancers and find that ∼20% arose in the mammalian stem lineage, coincident with the emergence of the neocortex. Implementing a permutation strategy that controls for the nonrandom variation in the ages of background genomic sequences, we find that mammal-specific enhancers are overrepresented near genes involved in cell migration, cell signaling, and axon guidance. Mammal-specific enhancers are also overrepresented in modules of coexpressed genes in the cortex that are associated with these pathways, notably ephrin and semaphorin signaling. Our results also provide insight into the mechanisms of regulatory innovation in mammals. We find that most neocortical enhancers did not originate by en bloc exaptation of transposons. Young neocortical enhancers exhibit smaller H3K27ac footprints and weaker evolutionary constraint in eutherian mammals than older neocortical enhancers. Based on these observations, we present a model of the enhancer life cycle in which neocortical enhancers initially emerge from genomic background as short, weakly constrained "proto-enhancers." Many proto-enhancers are likely lost, but some may serve as nucleation points for complex enhancers to evolve.

The genomic landscape of cohesin-associated chromatin interactions.

Cohesin is implicated in establishing tissue-specific DNA loops that target enhancers to promoters, and also localizes to sites bound by the insulator protein CTCF, which blocks enhancer-promoter communication. However, cohesin-associated interactions have not been characterized on a genome-wide scale. Here we performed chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) of the cohesin subunit SMC1A in developing mouse limb. We identified 2264 SMC1A interactions, of which 1491 (65%) involved sites co-occupied by CTCF. SMC1A participates in tissue-specific enhancer-promoter interactions and interactions that demarcate regions of correlated regulatory output. In contrast to previous studies, we also identified interactions between promoters and distal sites that are maintained in multiple tissues but are poised in embryonic stem cells and resolve to tissue-specific activated or repressed chromatin states in the mouse embryo. Our results reveal the diversity of cohesin-associated interactions in the genome and highlight their role in establishing the regulatory architecture of development.

Chromatin state signatures associated with tissue-specific gene expression and enhancer activity in the embryonic limb.

The regulatory elements that direct tissue-specific gene expression in the developing mammalian embryo remain largely unknown. Although chromatin profiling has proven to be a powerful method for mapping regulatory sequences in cultured cells, chromatin states characteristic of active developmental enhancers have not been directly identified in embryonic tissues. Here we use whole-transcriptome analysis coupled with genome-wide profiling of H3K27ac and H3K27me3 to map chromatin states and enhancers in mouse embryonic forelimb and hindlimb. We show that gene-expression differences between forelimb and hindlimb, and between limb and other embryonic cell types, are correlated with tissue-specific H3K27ac signatures at promoters and distal sites. Using H3K27ac profiles, we identified 28,377 putative enhancers, many of which are likely to be limb specific based on strong enrichment near genes highly expressed in the limb and comparisons with tissue-specific EP300 sites and known enhancers. We describe a chromatin state signature associated with active developmental enhancers, defined by high levels of H3K27ac marking, nucleosome displacement, hypersensitivity to sonication, and strong depletion of H3K27me3. We also find that some developmental enhancers exhibit components of this signature, including hypersensitivity, H3K27ac enrichment, and H3K27me3 depletion, at lower levels in tissues in which they are not active. Our results establish histone modification profiling as a tool for developmental enhancer discovery, and suggest that enhancers maintain an open chromatin state in multiple embryonic tissues independent of their activity level.

Transcriptional programs in transient embryonic zones of the cerebral cortex defined by high-resolution mRNA sequencing.

Characterizing the genetic programs that specify development and evolution of the cerebral cortex is a central challenge in neuroscience. Stem cells in the transient embryonic ventricular and subventricular zones generate neurons that migrate across the intermediate zone to the overlying cortical plate, where they differentiate and form the neocortex. It is clear that not one but a multitude of molecular pathways are necessary to progress through each cellular milestone, yet the underlying transcriptional programs remain unknown. Here, we apply differential transcriptome analysis on microscopically isolated cell populations, to define five transcriptional programs that represent each transient embryonic zone and the progression between these zones. The five transcriptional programs contain largely uncharacterized genes in addition to transcripts necessary for stem cell maintenance, neurogenesis, migration, and differentiation. Additionally, we found intergenic transcriptionally active regions that possibly encode unique zone-specific transcripts. Finally, we present a high-resolution transcriptome map of transient zones in the embryonic mouse forebrain.

Reconstructing human-specific regulatory functions in model systems.

Uniquely human physical traits, such as an expanded cerebral cortex and changes in limb morphology that allow us to use tools and walk upright, are in part due to human-specific genetic changes that altered when, where, and how genes are expressed during development. Over 20 000 putative regulatory elements with potential human-specific functions have been discovered. Understanding how these elements contributed to human evolution requires identifying candidates most likely to have shaped human traits, then studying them in genetically modified animal models. Here, we review the progress and challenges in generating and studying such models and propose a pathway for advancing the field. Finally, we highlight that large-scale collaborations across multiple research domains are essential to decipher what makes us human.

CpG island turnover events predict evolutionary changes in enhancer activity.

BACKGROUND: Genetic changes that modify the function of transcriptional enhancers have been linked to the evolution of biological diversity across species. Multiple studies have focused on the role of nucleotide substitutions, transposition, and insertions and deletions in altering enhancer function. CpG islands (CGIs) have recently been shown to influence enhancer activity, and here we test how their turnover across species contributes to enhancer evolution. RESULTS: We integrate maps of CGIs and enhancer activity-associated histone modifications obtained from multiple tissues in nine mammalian species and find that CGI content in enhancers is strongly associated with increased histone modification levels. CGIs show widespread turnover across species and species-specific CGIs are strongly enriched for enhancers exhibiting species-specific activity across all tissues and species. Genes associated with enhancers with species-specific CGIs show concordant biases in their expression, supporting that CGI turnover contributes to gene regulatory innovation. Our results also implicate CGI turnover in the evolution of Human Gain Enhancers (HGEs), which show increased activity in human embryonic development and may have contributed to the evolution of uniquely human traits. Using a humanized mouse model, we show that a highly conserved HGE with a large CGI absent from the mouse ortholog shows increased activity at the human CGI in the humanized mouse diencephalon. CONCLUSIONS: Collectively, our results point to CGI turnover as a mechanism driving gene regulatory changes potentially underlying trait evolution in mammals.

Human Accelerated Regions regulate gene networks implicated in apical-to-basal neural progenitor fate transitions.

The evolution of the human cerebral cortex involved modifications in the composition and proliferative potential of the neural stem cell (NSC) niche during brain development. Human Accelerated Regions (HARs) exhibit a significant excess of human-specific sequence changes and have been implicated in human brain evolution. Multiple studies support that HARs include neurodevelopmental enhancers with novel activities in humans, but their biological functions in NSCs have not been empirically assessed at scale. Here we conducted a direct-capture Perturb-seq screen repressing 180 neurodevelopmentally active HARs in human iPSC-derived NSCs with single-cell transcriptional readout. After profiling >188,000 NSCs, we identified a set of HAR perturbations with convergent transcriptional effects on gene networks involved in NSC apicobasal polarity, a cellular process whose precise regulation is critical to the developmental emergence of basal radial glia (bRG), a progenitor population that is expanded in humans. Across multiple HAR perturbations, we found convergent dysregulation of specific apicobasal polarity and adherens junction regulators, including PARD3, ABI2, SETD2 , and PCM1 . We found that the repression of one candidate from the screen, HAR181, as well as its target gene CADM1 , disrupted apical PARD3 localization and NSC rosette formation. Our findings reveal interconnected roles for HARs in NSC biology and cortical development and link specific HARs to processes implicated in human cortical expansion.

Cell type-specific dysregulation of gene expression due to Chd8 haploinsufficiency during mouse cortical development.

Disruptive variants in the chromodomain helicase CHD8, which acts as a transcriptional regulator during neurodevelopment, are strongly associated with risk for autism spectrum disorder (ASD). Loss of CHD8 function is hypothesized to perturb gene regulatory networks in the developing brain, thereby contributing to ASD etiology. However, insight into the cell type-specific transcriptional effects of CHD8 loss of function remains limited. We used single-cell and single-nucleus RNA-sequencing to globally profile gene expression and identify dysregulated genes in the embryonic and juvenile wild type and Chd8 +/- mouse cortex, respectively. Chd8 and other ASD risk-associated genes showed a convergent expression trajectory that was largely conserved between the mouse and human developing cortex, increasing from the progenitor zones to the cortical plate. Genes associated with risk for neurodevelopmental disorders and genes involved in neuron projection development, chromatin remodeling, signaling, and migration were dysregulated in Chd8 +/- embryonic day (E) 12.5 radial glia. Genes implicated in synaptic organization and activity were dysregulated in Chd8 +/- postnatal day (P) 25 deep- and upper-layer excitatory cortical neurons, suggesting a delay in synaptic maturation or impaired synaptogenesis due to CHD8 loss of function. Our findings reveal a complex pattern of transcriptional dysregulation in Chd8 +/- developing cortex, potentially with distinct biological impacts on progenitors and maturing neurons in the excitatory neuronal lineage.

Evolutionary innovation in conserved regulatory elements across the mammalian tree of life.

Transcriptional enhancers orchestrate cell type- and time point-specific gene expression programs. Evolution of enhancer sequences can alter target gene expression without causing detrimental misexpression in other contexts. It has long been thought that this modularity allows evolutionary changes in enhancers to escape pleiotropic constraints, which is especially important for evolutionary constrained developmental patterning genes. However, there is still little data supporting this hypothesis. Here we identified signatures of accelerated evolution in conserved enhancer elements across the mammalian phylogeny. We found that pleiotropic genes involved in gene regulatory and developmental processes were enriched for accelerated sequence evolution within their enhancer elements. These genes were associated with an excess number of enhancers compared to other genes, and due to this they exhibit a substantial degree of sequence acceleration over all their enhancers combined. We provide evidence that sequence acceleration is associated with turnover of regulatory function. We studied one acceleration event in depth and found that its sequence evolution led to the emergence of a new enhancer activity domain that may be involved in the evolution of digit reduction in hoofed mammals. Our results provide tangible evidence that enhancer evolution has been a frequent contributor to modifications involving constrained developmental signaling genes in mammals.

Massively parallel disruption of enhancers active in human neural stem cells.

Changes in gene regulation have been linked to the expansion of the human cerebral cortex and to neurodevelopmental disorders, potentially by altering neural progenitor proliferation. However, the effects of genetic variation within regulatory elements on neural progenitors remain obscure. We use sgRNA-Cas9 screens in human neural stem cells (hNSCs) to disrupt 10,674 genes and 26,385 conserved regions in 2,227 enhancers active in the developing human cortex and determine effects on proliferation. Genes with proliferation phenotypes are associated with neurodevelopmental disorders and show biased expression in specific fetal human brain neural progenitor populations. Although enhancer disruptions overall have weaker effects than gene disruptions, we identify enhancer disruptions that severely alter hNSC self-renewal. Disruptions in human accelerated regions, implicated in human brain evolution, also alter proliferation. Integrating proliferation phenotypes with chromatin interactions reveals regulatory relationships between enhancers and their target genes contributing to neurogenesis and potentially to human cortical evolution.

Resolving the three-dimensional interactome of Human Accelerated Regions during human and chimpanzee neurodevelopment.

Human Accelerated Regions (HARs) are highly conserved across species but exhibit a significant excess of human-specific sequence changes, suggesting they may have gained novel functions in human evolution. HARs include transcriptional enhancers with human-specific activity and have been implicated in the evolution of the human brain. However, our understanding of how HARs contributed to uniquely human features of the brain is hindered by a lack of insight into the genes and pathways that HARs regulate. It is unclear whether HARs acted by altering the expression of gene targets conserved between HARs and their chimpanzee orthologs or by gaining new gene targets in human, a mechanism termed enhancer hijacking. We generated a high-resolution map of chromatin interactions for 1,590 HARs and their orthologs in human and chimpanzee neural stem cells (NSCs) to comprehensively identify gene targets in both species. HARs and their chimpanzee orthologs targeted a conserved set of 2,963 genes enriched for neurodevelopmental processes including neurogenesis and synaptic transmission. Changes in HAR enhancer activity were correlated with changes in conserved gene target expression. Conserved targets were enriched among genes differentially expressed between human and chimpanzee NSCs or between human and non-human primate developing and adult brain. Species-specific HAR gene targets did not converge on known biological functions and were not significantly enriched among differentially expressed genes, suggesting that HARs did not alter gene expression via enhancer hijacking. HAR gene targets, including differentially expressed targets, also showed cell type-specific expression patterns in the developing human brain, including outer radial glia, which are hypothesized to contribute to human cortical expansion. Our findings support that HARs influenced human brain evolution by altering the expression of conserved gene targets and provide the means to functionally link HARs with novel human brain features.

Gene regulation and the origins of human biological uniqueness.

What makes us human? It is likely that changes in gene expression and regulation, in addition to those in protein-coding genes, drove the evolution of uniquely human biological traits. In this review, we discuss how efforts to annotate regulatory functions in the human genome are being combined with maps of human-specific sequence acceleration to identify cis-regulatory elements with human-specific activity. Although the evolutionary interpretation of these events is a subject of considerable debate, the technical and analytical means are now at hand to identify the set of evolutionary genetic events that shaped our species.