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.

CpG island turnover events predict evolutionary changes in enhancer activity.

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. Here we show that turnover of CpG islands (CGIs), which contribute to enhancer activation, is broadly associated with changes in enhancer activity across mammals, including humans. We integrated maps of CGIs and enhancer activity-associated histone modifications obtained from multiple tissues in nine mammalian species and found that CGI content in enhancers was strongly associated with increased histone modification levels. CGIs showed widespread turnover across species and species-specific CGIs were strongly enriched for enhancers exhibiting species-specific activity across all tissues and species we examined. Genes associated with enhancers with species-specific CGIs showed 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. Collectively, our results point to CGI turnover as a mechanism driving gene regulatory changes potentially underlying trait evolution in mammals.

The functional and evolutionary impacts of human-specific deletions in conserved elements.

Conserved genomic sequences disrupted in humans may underlie uniquely human phenotypic traits. We identified and characterized 10,032 human-specific conserved deletions (hCONDELs). These short (average 2.56 base pairs) deletions are enriched for human brain functions across genetic, epigenomic, and transcriptomic datasets. Using massively parallel reporter assays in six cell types, we discovered 800 hCONDELs conferring significant differences in regulatory activity, half of which enhance rather than disrupt regulatory function. We highlight several hCONDELs with putative human-specific effects on brain development, including HDAC5, CPEB4, and PPP2CA. Reverting an hCONDEL to the ancestral sequence alters the expression of LOXL2 and developmental genes involved in myelination and synaptic function. Our data provide a rich resource to investigate the evolutionary mechanisms driving new traits in humans and other species.

A systems biology approach identifies the role of dysregulated PRDM6 in the development of hypertension.

Genetic variants in the third intron of the PRDM6 gene have been associated with BP traits in multiple GWAS. By combining fine mapping, massively parallel reporter assays, and gene editing, we identified super enhancers that drive the expression of PRDM6 and are partly regulated by STAT1 as the causal variants for hypertension. The heterozygous disruption of Prdm6 in mice expressing Cre recombinase under the control of mouse smooth muscle cell protein 22-α promoter (Prdm6fl/+ SM22-Cre) exhibited a markedly higher number of renin-producing cells in the kidneys at E18.5 compared with WT littermates and developed salt-induced systemic hypertension that was completely responsive to the renin inhibitor aliskiren. Strikingly, RNA-Seq analysis of the mouse aortas identified a network of PRDM6-regulated genes that are located in GWAS-associated loci for blood pressure, most notably Sox6, which modulates renin expression in the kidney. Accordingly, the smooth muscle cell-specific disruption of Sox6 in Prdm6fl/+ SM22-Cre mice resulted in a dramatic reduction of renin. Fate mapping and histological studies also showed increased numbers of neural crest-derived cells accompanied by increased collagen deposition in the kidneys of Prdm6fl/+ Wnt1Cre-ZsGreen1Cre mice compared with WT mice. These findings establish the role of PRDM6 as a regulator of renin-producing cell differentiation into smooth muscle cells and as an attractive target for the development of antihypertensive drugs.

Modeling uniquely human gene regulatory function via targeted humanization of the mouse genome.

The evolution of uniquely human traits likely entailed changes in developmental gene regulation. Human Accelerated Regions (HARs), which include transcriptional enhancers harboring a significant excess of human-specific sequence changes, are leading candidates for driving gene regulatory modifications in human development. However, insight into whether HARs alter the level, distribution, and timing of endogenous gene expression remains limited. We examined the role of the HAR HACNS1 (HAR2) in human evolution by interrogating its molecular functions in a genetically humanized mouse model. We find that HACNS1 maintains its human-specific enhancer activity in the mouse embryo and modifies expression of Gbx2, which encodes a transcription factor, during limb development. Using single-cell RNA-sequencing, we demonstrate that Gbx2 is upregulated in the limb chondrogenic mesenchyme of HACNS1 homozygous embryos, supporting that HACNS1 alters gene expression in cell types involved in skeletal patterning. Our findings illustrate that humanized mouse models provide mechanistic insight into how HARs modified gene expression in human evolution.

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.

Evidence against tetrapod-wide digit identities and for a limited frame shift in bird wings.

In crown group tetrapods, individual digits are homologized in relation to a pentadactyl ground plan. However, testing hypotheses of digit homology is challenging because it is unclear whether digits represent distinct and conserved gene regulatory states. Here we show dramatic evolutionary dynamism in the gene expression profiles of digits, challenging the notion that five digits have conserved developmental identities across amniotes. Transcriptomics shows diversity in the patterns of gene expression differentiation of digits, although the anterior-most digit of the pentadactyl limb has a unique, conserved expression profile. Further, we identify a core set of transcription factors that are differentially expressed among the digits of amniote limbs; their spatial expression domains, however, vary between species. In light of these results, we reevaluate the frame shift hypothesis of avian wing evolution and conclude only the identity of the anterior-most digit has shifted position, suggesting a 1,3,4 digit identity in the bird wing.

High-Resolution Epigenomic Atlas of Human Embryonic Craniofacial Development.

Defects in patterning during human embryonic development frequently result in craniofacial abnormalities. The gene regulatory programs that build the craniofacial complex are likely controlled by information located between genes and within intronic sequences. However, systematic identification of regulatory sequences important for forming the human face has not been performed. Here, we describe comprehensive epigenomic annotations from human embryonic craniofacial tissues and systematic comparisons with multiple tissues and cell types. We identified thousands of tissue-specific craniofacial regulatory sequences and likely causal regions for rare craniofacial abnormalities. We demonstrate significant enrichment of common variants associated with orofacial clefting in enhancers active early in embryonic development, while those associated with normal facial variation are enriched near the end of the embryonic period. These data are provided in easily accessible formats for both craniofacial researchers and clinicians to aid future experimental design and interpretation of noncoding variation in those affected by craniofacial abnormalities.

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.

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.

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.

Molecular and cellular reorganization of neural circuits in the human lineage.

To better understand the molecular and cellular differences in brain organization between human and nonhuman primates, we performed transcriptome sequencing of 16 regions of adult human, chimpanzee, and macaque brains. Integration with human single-cell transcriptomic data revealed global, regional, and cell-type-specific species expression differences in genes representing distinct functional categories. We validated and further characterized the human specificity of genes enriched in distinct cell types through histological and functional analyses, including rare subpallial-derived interneurons expressing dopamine biosynthesis genes enriched in the human striatum and absent in the nonhuman African ape neocortex. Our integrated analysis of the generated data revealed diverse molecular and cellular features of the phylogenetic reorganization of the human brain across multiple levels, with relevance for brain function and disease.

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 autism-associated chromatin modifier CHD8 regulates other autism risk genes during human neurodevelopment.

Recent studies implicate chromatin modifiers in autism spectrum disorder (ASD) through the identification of recurrent de novo loss of function mutations in affected individuals. ASD risk genes are co-expressed in human midfetal cortex, suggesting that ASD risk genes converge in specific regulatory networks during neurodevelopment. To elucidate such networks, we identify genes targeted by CHD8, a chromodomain helicase strongly associated with ASD, in human midfetal brain, human neural stem cells (hNSCs) and embryonic mouse cortex. CHD8 targets are strongly enriched for other ASD risk genes in both human and mouse neurodevelopment, and converge in ASD-associated co-expression networks in human midfetal cortex. CHD8 knockdown in hNSCs results in dysregulation of ASD risk genes directly targeted by CHD8. Integration of CHD8-binding data into ASD risk models improves detection of risk genes. These results suggest loss of CHD8 contributes to ASD by perturbing an ancient gene regulatory network during human brain development.

Evolutionary genomics. Evolutionary changes in promoter and enhancer activity during human corticogenesis.

Human higher cognition is attributed to the evolutionary expansion and elaboration of the human cerebral cortex. However, the genetic mechanisms contributing to these developmental changes are poorly understood. We used comparative epigenetic profiling of human, rhesus macaque, and mouse corticogenesis to identify promoters and enhancers that have gained activity in humans. These gains are significantly enriched in modules of coexpressed genes in the cortex that function in neuronal proliferation, migration, and cortical-map organization. Gain-enriched modules also showed correlated gene expression patterns and similar transcription factor binding site enrichments in promoters and enhancers, suggesting that they are connected by common regulatory mechanisms. Our results reveal coordinated patterns of potential regulatory changes associated with conserved developmental processes during corticogenesis, providing insight into human cortical evolution.

Chromatin immunoprecipitation with fixed animal tissues and preparation for high-throughput sequencing.

Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) is a powerful method used to identify genome-wide binding patterns of transcription factors and distribution of various histone modifications associated with different chromatin states. In most published studies, ChIP-Seq has been performed on cultured cells grown under controlled conditions, allowing generation of large amounts of material in a homogeneous biological state. Although such studies have provided great insight into the dynamic landscapes of animal genomes, they do not allow the examination of transcription factor binding and chromatin states in adult tissues, developing embryonic structures, or tumors. Such knowledge is critical to understanding the information required to create and maintain a complex biological tissue and to identify noncoding regions of the genome directly involved in tissues affected by complex diseases such as autism. Studying these tissue types with ChIP-Seq can be challenging due to the limited availability of tissues and the lack of complex biological states able to be achieved in culture. These inherent differences require alterations of standard cross-linking and chromatin extraction typically used in cell culture. Here we describe a general approach for using small amounts of animal tissue to perform ChIP-Seq directed at histone modifications and transcription factors. Tissue is homogenized before treatment with formaldehyde to ensure proper cross-linking, and a two-step nuclear isolation is performed to increase extraction of soluble chromatin. Small amounts of soluble chromatin are then used for immunoprecipitation (IP) and prepared for multiplexed high-throughput sequencing.

DAWN: a framework to identify autism genes and subnetworks using gene expression and genetics.

BACKGROUND: De novo loss-of-function (dnLoF) mutations are found twofold more often in autism spectrum disorder (ASD) probands than their unaffected siblings. Multiple independent dnLoF mutations in the same gene implicate the gene in risk and hence provide a systematic, albeit arduous, path forward for ASD genetics. It is likely that using additional non-genetic data will enhance the ability to identify ASD genes. METHODS: To accelerate the search for ASD genes, we developed a novel algorithm, DAWN, to model two kinds of data: rare variations from exome sequencing and gene co-expression in the mid-fetal prefrontal and motor-somatosensory neocortex, a critical nexus for risk. The algorithm casts the ensemble data as a hidden Markov random field in which the graph structure is determined by gene co-expression and it combines these interrelationships with node-specific observations, namely gene identity, expression, genetic data and the estimated effect on risk. RESULTS: Using currently available genetic data and a specific developmental time period for gene co-expression, DAWN identified 127 genes that plausibly affect risk, and a set of likely ASD subnetworks. Validation experiments making use of published targeted resequencing results demonstrate its efficacy in reliably predicting ASD genes. DAWN also successfully predicts known ASD genes, not included in the genetic data used to create the model. CONCLUSIONS: Validation studies demonstrate that DAWN is effective in predicting ASD genes and subnetworks by leveraging genetic and gene expression data. The findings reported here implicate neurite extension and neuronal arborization as risks for ASD. Using DAWN on emerging ASD sequence data and gene expression data from other brain regions and tissues would likely identify novel ASD genes. DAWN can also be used for other complex disorders to identify genes and subnetworks in those disorders.