James P. Noonan

Sequencing and Analysis of Neanderthal Genomic DNA

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 ∼706,000 years ago, and that the human and Neanderthal ancestral populations split ∼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.

Genomic Analysis of Non-NF2 Meningiomas Reveals Mutations in TRAF7, KLF4, AKT1, and SMO

Genetic Clues to Meningioma Meningiomas are the most common primary brain tumors in adults. Located within the layer of tissue covering the brain, these tumors are usually slow-growing and benign but can cause serious neurological complications. About half of these tumors have mutations in the neurofibromin 2 gene (NF2). To identify other genes that contribute to meningioma pathogenesis, Clark et al. (p. 1077, published online 24 January) performed genome sequence analysis on 300 tumors. Meningiomas fell into two general classes: benign tumors located at the skull base—which tend to harbor mutations in the TRAF7, KLF4, AKT1, and SMO genes—and higher-grade tumors located in the cerebral and cerebellar hemispheres harbor mutations in NF2. The mutational profiles of meningiomas, a common type of brain tumor, correlate with their anatomical location and clinical status. 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 AKT1E17K, 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.

Human-specific gain of function in a developmental enhancer.

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.

Genomics of Long-Range Regulatory Elements

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.

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.

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.

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.

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.

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.

Evolutionary changes in promoter and enhancer activity during human corticogenesis

Of mice, men, and macaque brains The human brain represents a unique evolutionary trajectory. To better understand how the human brain came to be, Reilly et al. sought to identify changes in gene expression between mice, macaques, and humans. They compared epigenetic marks in the embryonic cortex, which revealed changes in gene regulation in biological pathways associated with cortical development. Science, this issue p. 1155 Epigenetic marks associated with evolution of the human cortex gained activity relative to those in mice and monkeys. 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.