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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., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Competing interests. The authors declare no competing interests.

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., Competing Interests: Competing Interests: The authors declare no competing financial interests.

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.

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., (© 2024. The Author(s).)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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.

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., (© 2022. The Author(s).)

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., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

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.

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., (Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

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., (© 2018 John Wiley & Sons Ltd.)

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., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

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., (Copyright © 2017, American Association for the Advancement of Science.)

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.

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.

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.

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., (Copyright © 2015, American Association for the Advancement of Science.)

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.

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We report genomic analysis of 300 meningiomas, the most common primary brain tumors, leading to the discovery of mutations in TRAF7, a proapoptotic E3 ubiquitin ligase, in nearly one-fourth of all meningiomas. Mutations in TRAF7 commonly occurred with a recurrent mutation (K409Q) in KLF4, a transcription factor known for its role in inducing pluripotency, or with AKT1(E17K), a mutation known to activate the PI3K pathway. SMO mutations, which activate Hedgehog signaling, were identified in ~5% of non-NF2 mutant meningiomas. These non-NF2 meningiomas were clinically distinctive-nearly always benign, with chromosomal stability, and originating from the medial skull base. In contrast, meningiomas with mutant NF2 and/or chromosome 22 loss were more likely to be atypical, showing genomic instability, and localizing to the cerebral and cerebellar hemispheres. Collectively, these findings identify distinct meningioma subtypes, suggesting avenues for targeted therapeutics.

Mechanisms involved with degeneration of motor neurons in amyotrophic lateral sclerosis (ALS; Lou Gehrig's Disease) are poorly understood, but genetically inherited forms, comprising ~10% of the cases, are potentially informative. Recent observations that several inherited forms of ALS involve the RNA binding proteins TDP43 and FUS raise the question as to whether RNA metabolism is generally disturbed in ALS. Here we conduct whole transcriptome profiling of motor neurons from a mouse strain, transgenic for a mutant human SOD1 (G85R SOD1-YFP), that develops symptoms of ALS and paralyzes at 5-6 months of age. Motor neuron cell bodies were laser microdissected from spinal cords at 3 months of age, a time when animals were presymptomatic but showed aggregation of the mutant protein in many lower motor neuron cell bodies and manifested extensive neuromuscular junction morphologic disturbance in their lower extremities. We observed only a small number of transcripts with altered expression levels or splicing in the G85R transgenic compared to age-matched animals of a wild-type SOD1 transgenic strain. Our results indicate that a major disturbance of polyadenylated RNA metabolism does not occur in motor neurons of mutant SOD1 mice, suggesting that the toxicity of the mutant protein lies at the level of translational or post-translational effects.

Divergence of serially homologous elements of organisms is a common evolutionary pattern contributing to increased phenotypic complexity. Here, we study the genomic intervals affecting the variational independence of fore- and hind limb traits within an experimental mouse population. We use an advanced intercross of inbred mouse strains to map the loci associated with the degree of autonomy between fore- and hind limb long bone lengths (loci affecting the relationship between traits, relationship quantitative trait loci [rQTL]). These loci have been proposed to interact locally with the products of pleiotropic genes, thereby freeing the local trait from the variational constraint due to pleiotropic mutations. Using the known polymorphisms (single nucleotide polymorphisms [SNPs]) between the parental strains, we characterized and compared the genomic regions in which the rQTL, as well as their interaction partners (intQTL), reside. We find that these two classes of QTL intervals harbor different kinds of molecular variation. SNPs in rQTL intervals more frequently reside in limb-specific cis-regulatory regions than SNPs in intQTL intervals. The intQTL loci modified by the rQTL, in contrast, show the signature of protein-coding variation. This result is consistent with the widely accepted view that protein-coding mutations have broader pleiotropic effects than cis-regulatory polymorphisms. For both types of QTL intervals, the underlying candidate genes are enriched for genes involved in protein binding. This finding suggests that rQTL effects are caused by local interactions among the products of the causal genes harbored in rQTL and intQTL intervals. This is the first study to systematically document the population-level molecular variation underlying the evolution of character individuation.

RNA-seq is becoming the de facto standard approach for transcriptome analysis with ever-reducing cost. It has considerable advantages over conventional technologies (microarrays) because it allows for direct identification and quantification of transcripts. Many time series RNA-seq datasets have been collected to study the dynamic regulations of transcripts. However, statistically rigorous and computationally efficient methods are needed to explore the time-dependent changes of gene expression in biological systems. These methods should explicitly account for the dependencies of expression patterns across time points. Here, we discuss several methods that can be applied to model timecourse RNA-seq data, including statistical evolutionary trajectory index (SETI), autoregressive time-lagged regression (AR(1)), and hidden Markov model (HMM) approaches. We use three real datasets and simulation studies to demonstrate the utility of these dynamic methods in temporal analysis.

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.

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.

Genetic selection from libraries expressing proteins with randomized amino acid segments is a powerful approach to identify proteins with novel biological activities. Here, we assessed the utility of deep DNA sequencing to characterize the composition, diversity, size and stability of such randomized libraries. We used 454 pyrosequencing to sequence a retroviral library expressing small proteins with randomized transmembrane domains. Despite the potential for unintended random mutagenesis during its construction, the overall hydrophobic composition and diversity of the proteins encoded by the sequenced library conformed well to its design. In addition, our sequencing results allowed us to calculate a more accurate estimate of the number of different proteins encoded by the library and suggested that the traditional methods for estimating the size of randomized libraries may overestimate their true size. Our results further demonstrated that no significant genetic bottlenecks exist in the methods used to express complex retrovirus libraries in mammalian cells and recover library sequences from these cells. These findings suggest that deep sequencing can be used to determine the quality and content of other libraries with randomized segments and to follow individual sequences during selection.

Humans possess unique physical and cognitive characteristics relative to other primates. Comparative analyses of the human and chimpanzee genomes are beginning to reveal sequence changes on the human lineage that may have contributed to the evolution of human traits. However, these studies cannot identify the genetic differences that distinguish modern humans from archaic human species. Here, I will discuss efforts to obtain genomic sequence from Neanderthal, the closest known relative of modern humans. Recent studies in this nascent field have focused on developing methods to recover nuclear DNA from Neanderthal remains. The success of these early studies has inspired a Neanderthal genome project, which promises to produce a reference Neanderthal genome sequence in the near future. Technical issues, such as the level of Neanderthal sequence coverage that can realistically be obtained from a single specimen and the presence of modern human contaminating sequences, reduce the detection of authentic human-Neanderthal sequence differences but may be remedied by methodological improvements. More critical for the utility of a Neanderthal genome sequence is the evolutionary relationship of humans and Neanderthals. Current evidence suggests that the modern human and Neanderthal lineages diverged before the emergence of contemporary humans. A fraction of biologically relevant human-chimpanzee sequence differences are thus likely to have arisen and become fixed exclusively on the modern human lineage. A reconstructed Neanderthal genome sequence could be integrated into human-primate genome comparisons to help reveal the evolutionary genetic events that produced modern humans.

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., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Transcriptional regulation of gene expression plays a significant role in establishing the diversity of human cell types and biological functions from a common set of genes. The components of regulatory control in the human genome include cis-acting elements that act across immense genomic distances to influence the spatial and temporal distribution of gene expression. Here we review the established categories of distant-acting regulatory elements, discussing the classical and contemporary evidence of their regulatory potential and clinical importance. Current efforts to identify regulatory sequences throughout the genome and elucidate their biological significance depend heavily on advances in sequence conservation-based analyses and on increasingly large-scale efforts applying transgenic technologies in model organisms. We discuss the advantages and limitations of sequence conservation as a predictor of regulatory function and present complementary emerging technologies now being applied to annotate regulatory elements in vertebrate genomes.

Changes in gene regulation have long been thought to underlie biological differences between humans and other primates. Recent advances have facilitated the study of human-specific sequence changes in regulatory DNAs in the context of human development. Comparative genomic analyses coupled with genome-wide in vivo developmental enhancer screens have identified thousands of known and likely regulatory elements in the genome. These have provided the substrate for statistical and experimental identification of regulatory sequences with human-specific developmental activities. On the basis of these early results, the outlines of an integrated strategy have emerged that combines gene expression atlases of human development, in vivo reverse genetic studies of regulatory DNAs, and maps of human-specific sequence change to reveal the genetic basis of unique human biology.

Changes in gene regulation are thought to have contributed to the evolution of human development. However, in vivo evidence for uniquely human developmental regulatory function has remained elusive. In transgenic mice, a conserved noncoding sequence (HACNS1) that evolved extremely rapidly in humans acted as an enhancer of gene expression that has gained a strong limb expression domain relative to the orthologous elements from chimpanzee and rhesus macaque. This gain of function was consistent across two developmental stages in the mouse and included the presumptive anterior wrist and proximal thumb. In vivo analyses with synthetic enhancers, in which human-specific substitutions were introduced into the chimpanzee enhancer sequence or reverted in the human enhancer to the ancestral state, indicated that 13 substitutions clustered in an 81-base pair module otherwise highly constrained among terrestrial vertebrates were sufficient to confer the human-specific limb expression domain.

Our knowledge of Neanderthals is based on a limited number of remains and artifacts from which we must make inferences about their biology, behavior, and relationship to ourselves. Here, we describe the characterization of these extinct hominids from a new perspective, based on the development of a Neanderthal metagenomic library and its high-throughput sequencing and analysis. Several lines of evidence indicate that the 65,250 base pairs of hominid sequence so far identified in the library are of Neanderthal origin, the strongest being the ascertainment of sequence identities between Neanderthal and chimpanzee at sites where the human genomic sequence is different. These results enabled us to calculate the human-Neanderthal divergence time based on multiple randomly distributed autosomal loci. Our analyses suggest that on average the Neanderthal genomic sequence we obtained and the reference human genome sequence share a most recent common ancestor approximately 706,000 years ago, and that the human and Neanderthal ancestral populations split approximately 370,000 years ago, before the emergence of anatomically modern humans. Our finding that the Neanderthal and human genomes are at least 99.5% identical led us to develop and successfully implement a targeted method for recovering specific ancient DNA sequences from metagenomic libraries. This initial analysis of the Neanderthal genome advances our understanding of the evolutionary relationship of Homo sapiens and Homo neanderthalensis and signifies the dawn of Neanderthal genomics.

Changes in gene regulation likely influenced the profound phenotypic divergence of humans from other mammals, but the extent of adaptive substitution in human regulatory sequences remains unknown. We identified 992 conserved noncoding sequences (CNSs) with a significant excess of human-specific substitutions. These accelerated elements were disproportionately found near genes involved in neuronal cell adhesion. To assess the uniqueness of human noncoding evolution, we examined CNSs accelerated in chimpanzee and mouse. Although we observed a similar enrichment near neuronal adhesion genes in chimpanzee, the accelerated CNSs themselves exhibited almost no overlap with those in human, suggesting independent evolution toward different neuronal phenotypes in each species. CNSs accelerated in mouse showed no bias toward neuronal cell adhesion. Our results indicate that widespread cis-regulatory changes in human evolution may have contributed to uniquely human features of brain development and function.

Despite the greater information content of genomic DNA, ancient DNA studies have largely been limited to the amplification of mitochondrial sequences. Here we describe metagenomic libraries constructed with unamplified DNA extracted from skeletal remains of two 40,000-year-old extinct cave bears. Analysis of approximately 1 megabase of sequence from each library showed that despite significant microbial contamination, 5.8 and 1.1% of clones contained cave bear inserts, yielding 26,861 base pairs of cave bear genome sequence. Comparison of cave bear and modern bear sequences revealed the evolutionary relationship of these lineages. The metagenomic approach used here establishes the feasibility of ancient DNA genome sequencing programs.

The coelacanth is one of the nearest living relatives of tetrapods. However, a teleost species such as zebrafish or Fugu is typically used as the outgroup in current tetrapod comparative sequence analyses. Such studies are complicated by the fact that teleost genomes have undergone a whole-genome duplication event, as well as individual gene-duplication events. Here, we demonstrate the value of coelacanth genome sequence by complete sequencing and analysis of the protocadherin gene cluster of the Indonesian coelacanth, Latimeria menadoensis. We found that coelacanth has 49 protocadherin cluster genes organized in the same three ordered subclusters, alpha, beta, and gamma, as the 54 protocadherin cluster genes in human. In contrast, whole-genome and tandem duplications have generated two zebrafish protocadherin clusters comprised of at least 97 genes. Additionally, zebrafish protocadherins are far more prone to homogenizing gene conversion events than coelacanth protocadherins, suggesting that recombination- and duplication-driven plasticity may be a feature of teleost genomes. Our results indicate that coelacanth provides the ideal outgroup sequence against which tetrapod genomes can be measured. We therefore present L. menadoensis as a candidate for whole-genome sequencing.

Chromosome 5 is one of the largest human chromosomes and contains numerous intrachromosomal duplications, yet it has one of the lowest gene densities. This is partially explained by numerous gene-poor regions that display a remarkable degree of noncoding conservation with non-mammalian vertebrates, suggesting that they are functionally constrained. In total, we compiled 177.7 million base pairs of highly accurate finished sequence containing 923 manually curated protein-coding genes including the protocadherin and interleukin gene families. We also completely sequenced versions of the large chromosome-5-specific internal duplications. These duplications are very recent evolutionary events and probably have a mechanistic role in human physiological variation, as deletions in these regions are the cause of debilitating disorders including spinal muscular atrophy.

The synaptic cell adhesion molecules encoded by the protocadherin gene cluster are hypothesized to provide a molecular code involved in the generation of synaptic complexity in the developing brain. Variation in copy number and sequence content of protocadherin cluster genes among vertebrate species could reflect adaptive differences in protocadherin function. We have completed an analysis of zebrafish protocadherin cluster genes. Zebrafish have two unlinked protocadherin clusters, DrPcdh1 and DrPcdh2. Like mammalian protocadherin clusters, DrPcdh1 has both alpha and gamma variable and constant region exons. A consensus protocadherin promoter motif sequence identified in mammals is also conserved in zebrafish. Few orthologous relationships, however, are apparent between zebrafish and mammalian protocadherin proteins. Here we show that protocadherin cluster genes in human, mouse, rat, and zebrafish are subject to striking gene conversion events. These events are restricted to regions of the coding sequence, particularly the coding sequences of ectodomain 6 and the cytoplasmic domain. Diversity among paralogs is restricted to particular ectodomains that are excluded from conversion events. Conversion events are also strongly correlated with an increase in third-position GC content. We propose that the combination of lineage-specific duplication, restricted gene conversion, and adaptive variation in diversified ectodomains drives vertebrate protocadherin cluster evolution.

Regions of extensive linkage disequilibrium (LD) appear to be a common feature of the human genome. However, the mechanisms that maintain these regions are unknown. In an effort to understand whether gene density contributes to LD, we determined the degree of promoter sequence variation in a large tandem-arrayed gene family, the human protocadherin alpha cluster, on chromosome 5. These genes are expressed at synaptic junctions in the developing brain and the adult brain and may be involved in the determination of synaptic complexity. We sequenced the promoters of all 13 alpha protocadherin genes in 96 European Americans and identified polymorphisms in the promoters alpha 1, alpha 3, alpha 4, alpha 5, alpha 7, alpha 9, alpha 11, and alpha 13. In these promoters, 11 common SNPs are in extensive LD, forming two 48-kb haplotypes of equal frequency, in this population, that extend from the alpha1 through alpha 7 genes. We sequenced these promoters in East Asians and African Americans, and we estimated haplotype frequencies and calculated LD statistics for all three populations. Our results indicate that, although extensive LD is an ancient feature of the alpha cluster, it has eroded over time. SNPs 3' of alpha 7 are involved in ancestral recombination events in all populations, and overall alpha-cluster LD is reduced in African Americans. We obtained significant positive values for Tajima's D test for all alpha promoter SNPs in Europeans (D=3.03) and East Asians (D=2.64), indicating an excess of intermediate-frequency variants, which is a signature of balancing selection. We also discovered a 16.7-kb deletion that truncates the alpha 8 gene and completely removes the alpha 9 and alpha 10 genes. This deletion appears in unaffected individuals from multiple populations, suggesting that a reduction in protocadherin gene number is not obviously deleterious.

The genomic organization of the human protocadherin alpha, beta, and gamma gene clusters (designated Pcdh alpha [gene symbol PCDHA], Pcdh beta [PCDHB], and Pcdh gamma [PCDHG]) is remarkably similar to that of immunoglobulin and T-cell receptor genes. The extracellular and transmembrane domains of each protocadherin protein are encoded by an unusually large "variable" region exon, while the intracellular domains are encoded by three small "constant" region exons located downstream from a tandem array of variable region exons. Here we report the results of a comparative DNA sequence analysis of the orthologous human (750 kb) and mouse (900 kb) protocadherin gene clusters. The organization of Pcdh alpha and Pcdh gamma gene clusters in the two species is virtually identical, whereas the mouse Pcdh beta gene cluster is larger and contains more genes than the human Pcdh beta gene cluster. We identified conserved DNA sequences upstream of the variable region exons, and found that these sequences are more conserved between orthologs than between paralogs. Within this region, there is a highly conserved DNA sequence motif located at about the same position upstream of the translation start codon of each variable region exon. In addition, the variable region of each gene cluster contains a rich array of CpG islands, whose location corresponds to the position of each variable region exon. These observations are consistent with the proposal that the expression of each variable region exon is regulated by a distinct promoter, which is highly conserved between orthologous variable region exons in mouse and human.