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Find video protocols related to scientific articles indexed in Pubmed.
Violation of the 12/23 rule of genomic V(D)J recombination is common in lymphocytes.
Genome Res.
PUBLISHED: 11-03-2014
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V(D)J genomic recombination joins single gene segments to encode an extensive repertoire of antigen receptor specificities in T and B lymphocytes. This process initiates with double-stranded breaks adjacent to conserved recombination signal sequences that contain either 12 or 23 nucleotide spacer regions. Only recombination between signal sequences with unequal spacers result in productive coding genes, a phenomenon known as the '12/23 rule'. Here we present two novel genomic tools that allow the capture and analysis of immune locus rearrangements from whole thymic and splenic tissues using second generation sequencing. Further, we provide strong evidence that the 12/23 rule of genomic recombination is frequently violated under physiological conditions resulting in unanticipated hybrid recombinations in ~10% of Tcra excision circles. Hence, we demonstrate that strict adherence to the 12/23 rule is intrinsic neither to recombination signal sequences nor to the catalytic process of recombination and propose that non-classical excision circles are liberated during the formation of antigen receptor diversity.
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Population and single-cell genomics reveal the Aire dependency, relief from Polycomb silencing, and distribution of self-antigen expression in thymic epithelia.
Genome Res.
PUBLISHED: 09-15-2014
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Promiscuous gene expression (PGE) by thymic epithelial cells (TEC) is essential for generating a diverse T cell antigen receptor repertoire tolerant to self-antigens, and thus for avoiding autoimmunity. Nevertheless, the extent and nature of this unusual expression program within TEC populations and single cells are unknown. Using deep transcriptome sequencing of carefully identified mouse TEC subpopulations, we discovered a program of PGE that is common between medullary (m) and cortical TEC, further elaborated in mTEC, and completed in mature mTEC expressing the autoimmune regulator gene (Aire). TEC populations are capable of expressing up to 19,293 protein-coding genes, the highest number of genes known to be expressed in any cell type. Remarkably, in mouse mTEC, Aire expression alone positively regulates 3980 tissue-restricted genes. Notably, the tissue specificities of these genes include known targets of autoimmunity in human AIRE deficiency. Led by the observation that genes induced by Aire expression are generally characterized by a repressive chromatin state in somatic tissues, we found these genes to be strongly associated with H3K27me3 marks in mTEC. Our findings are consistent with AIRE targeting and inducing the promiscuous expression of genes previously epigenetically silenced by Polycomb group proteins. Comparison of the transcriptomes of 174 single mTEC indicates that genes induced by Aire expression are transcribed stochastically at low cell frequency. Furthermore, when present, Aire expression-dependent transcript levels were 16-fold higher, on average, in individual TEC than in the mTEC population.
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Disruption of Visc-2, a Brain-Expressed Conserved Long Noncoding RNA, Does Not Elicit an Overt Anatomical or Behavioral Phenotype.
Cereb. Cortex
PUBLISHED: 09-12-2014
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Although long noncoding RNAs (lncRNAs) are proposed to play essential roles in mammalian neurodevelopment, we know little of their functions from their disruption in vivo. Combining evidence for evolutionary constraint and conserved expression data, we previously identified candidate lncRNAs that might play important and conserved roles in brain function. Here, we demonstrate that the sequence and neuronal transcription of lncRNAs transcribed from the previously uncharacterized Visc locus are conserved across diverse mammals. Consequently, one of these lncRNAs, Visc-2, was selected for targeted deletion in the mouse, and knockout animals were subjected to an extremely detailed anatomical and behavioral characterization. Despite a neurodevelopmental expression pattern of Visc-2 that is highly localized to the cortex and sites of neurogenesis, anomalies in neither cytoarchitecture nor neuroproliferation were identified in knockout mice. In addition, no abnormal motor, sensory, anxiety, or cognitive behavioral phenotypes were observed. These results are important because they contribute to a growing body of evidence that lncRNA loci contribute on average far less to brain and biological functions than protein-coding loci. A high-throughput knockout program focussing on lncRNAs, similar to that currently underway for protein-coding genes, will be required to establish the distribution of their organismal functions.
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The genomic substrate for adaptive radiation in African cichlid fish.
Nature
PUBLISHED: 09-03-2014
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Cichlid fishes are famous for large, diverse and replicated adaptive radiations in the Great Lakes of East Africa. To understand the molecular mechanisms underlying cichlid phenotypic diversity, we sequenced the genomes and transcriptomes of five lineages of African cichlids: the Nile tilapia (Oreochromis niloticus), an ancestral lineage with low diversity; and four members of the East African lineage: Neolamprologus brichardi/pulcher (older radiation, Lake Tanganyika), Metriaclima zebra (recent radiation, Lake Malawi), Pundamilia nyererei (very recent radiation, Lake Victoria), and Astatotilapia burtoni (riverine species around Lake Tanganyika). We found an excess of gene duplications in the East African lineage compared to tilapia and other teleosts, an abundance of non-coding element divergence, accelerated coding sequence evolution, expression divergence associated with transposable element insertions, and regulation by novel microRNAs. In addition, we analysed sequence data from sixty individuals representing six closely related species from Lake Victoria, and show genome-wide diversifying selection on coding and regulatory variants, some of which were recruited from ancient polymorphisms. We conclude that a number of molecular mechanisms shaped East African cichlid genomes, and that amassing of standing variation during periods of relaxed purifying selection may have been important in facilitating subsequent evolutionary diversification.
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Understanding functional miRNA-target interactions in vivo by site-specific genome engineering.
Nat Commun
PUBLISHED: 08-19-2014
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MicroRNA (miRNA) target recognition is largely dictated by short 'seed' sequences, and single miRNAs therefore have the potential to regulate a large number of genes. Understanding the contribution of specific miRNA-target interactions to the regulation of biological processes in vivo remains challenging. Here we use transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technologies to interrogate the functional relevance of predicted miRNA response elements (MREs) to post-transcriptional silencing in zebrafish and Drosophila. We also demonstrate an effective strategy that uses CRISPR-mediated homology-directed repair with short oligonucleotide donors for the assessment of MRE activity in human cells. These methods facilitate analysis of the direct phenotypic consequences resulting from blocking specific miRNA-MRE interactions at any point during development.
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Considerations when investigating lncRNA function in vivo.
Elife
PUBLISHED: 08-14-2014
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Although a small number of the vast array of animal long non-coding RNAs (lncRNAs) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell processes throughout development, differentiation and disease for the most part remains less clear. Phenotypes arising from deletion of an entire genomic locus cannot be unequivocally attributed either to the loss of the lncRNA per se or to the associated loss of other overlapping DNA regulatory elements. The distinction between cis- or trans-effects is also often problematic. We discuss the advantages and challenges associated with the current techniques for studying the in vivo function of lncRNAs in the light of different models of lncRNA molecular mechanism, and reflect on the design of experiments to mutate lncRNA loci. These considerations should assist in the further investigation of these transcriptional products of the genome.
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8.2% of the Human genome is constrained: variation in rates of turnover across functional element classes in the human lineage.
PLoS Genet.
PUBLISHED: 07-01-2014
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Ten years on from the finishing of the human reference genome sequence, it remains unclear what fraction of the human genome confers function, where this sequence resides, and how much is shared with other mammalian species. When addressing these questions, functional sequence has often been equated with pan-mammalian conserved sequence. However, functional elements that are short-lived, including those contributing to species-specific biology, will not leave a footprint of long-lasting negative selection. Here, we address these issues by identifying and characterising sequence that has been constrained with respect to insertions and deletions for pairs of eutherian genomes over a range of divergences. Within noncoding sequence, we find increasing amounts of mutually constrained sequence as species pairs become more closely related, indicating that noncoding constrained sequence turns over rapidly. We estimate that half of present-day noncoding constrained sequence has been gained or lost in approximately the last 130 million years (half-life in units of divergence time, d1/2?=?0.25-0.31). While enriched with ENCODE biochemical annotations, much of the short-lived constrained sequences we identify are not detected by models optimized for wider pan-mammalian conservation. Constrained DNase 1 hypersensitivity sites, promoters and untranslated regions have been more evolutionarily stable than long noncoding RNA loci which have turned over especially rapidly. By contrast, protein coding sequence has been highly stable, with an estimated half-life of over a billion years (d1/2?=?2.1-5.0). From extrapolations we estimate that 8.2% (7.1-9.2%) of the human genome is presently subject to negative selection and thus is likely to be functional, while only 2.2% has maintained constraint in both human and mouse since these species diverged. These results reveal that the evolutionary history of the human genome has been highly dynamic, particularly for its noncoding yet biologically functional fraction.
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Diagnostically relevant facial gestalt information from ordinary photos.
Elife
PUBLISHED: 06-26-2014
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Craniofacial characteristics are highly informative for clinical geneticists when diagnosing genetic diseases. As a first step towards the high-throughput diagnosis of ultra-rare developmental diseases we introduce an automatic approach that implements recent developments in computer vision. This algorithm extracts phenotypic information from ordinary non-clinical photographs and, using machine learning, models human facial dysmorphisms in a multidimensional 'Clinical Face Phenotype Space'. The space locates patients in the context of known syndromes and thereby facilitates the generation of diagnostic hypotheses. Consequently, the approach will aid clinicians by greatly narrowing (by 27.6-fold) the search space of potential diagnoses for patients with suspected developmental disorders. Furthermore, this Clinical Face Phenotype Space allows the clustering of patients by phenotype even when no known syndrome diagnosis exists, thereby aiding disease identification. We demonstrate that this approach provides a novel method for inferring causative genetic variants from clinical sequencing data through functional genetic pathway comparisons.DOI: http://dx.doi.org/10.7554/eLife.02020.001.
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Ki-67 is a PP1-interacting protein that organises the mitotic chromosome periphery.
Elife
PUBLISHED: 05-29-2014
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When the nucleolus disassembles during open mitosis, many nucleolar proteins and RNAs associate with chromosomes, establishing a perichromosomal compartment coating the chromosome periphery. At present nothing is known about the function of this poorly characterised compartment. In this study, we report that the nucleolar protein Ki-67 is required for the assembly of the perichromosomal compartment in human cells. Ki-67 is a cell-cycle regulated protein phosphatase 1-binding protein that is involved in phospho-regulation of the nucleolar protein B23/nucleophosmin. Following siRNA depletion of Ki-67, NIFK, B23, nucleolin, and four novel chromosome periphery proteins all fail to associate with the periphery of human chromosomes. Correlative light and electron microscopy (CLEM) images suggest a near-complete loss of the entire perichromosomal compartment. Mitotic chromosome condensation and intrinsic structure appear normal in the absence of the perichromosomal compartment but significant differences in nucleolar reassembly and nuclear organisation are observed in post-mitotic cells.DOI: http://dx.doi.org/10.7554/eLife.01641.001.
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Transcriptional regulatory functions of nuclear long noncoding RNAs.
Trends Genet.
PUBLISHED: 05-14-2014
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Several nuclear localised intergenic long noncoding RNAs (lncRNAs) have been ascribed regulatory roles in transcriptional control and their number is growing rapidly. Initially, these transcripts were shown to function locally, near their sites of synthesis, by regulating the expression of neighbouring genes. More recently, lncRNAs have been demonstrated to interact with chromatin at several thousand different locations across multiple chromosomes and to modulate large-scale gene expression programs. Although the molecular mechanisms involved in targeting lncRNAs to distal binding sites remain poorly understood, the spatial organisation of the genome may have a role in specifying lncRNA function. Recent advances indicate that intergenic lncRNAs may exert more widespread effects on gene regulation than previously anticipated.
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No gene in the genome makes sense except in the light of evolution.
Annu Rev Genomics Hum Genet
PUBLISHED: 04-24-2014
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Evolutionary conservation has been an accurate predictor of functional elements across the first decade of metazoan genomics. More recently, there has been a move to define functional elements instead from biochemical annotations. Evolutionary methods are, however, more comprehensive than biochemical approaches can be and can assess quantitatively, especially for subtle effects, how biologically important--how injurious after mutation--different types of elements are. Evolutionary methods are thus critical for understanding the large fraction (up to 10%) of the human genome that does not encode proteins and yet might convey function. These methods can also capture the ephemeral nature of much noncoding functional sequence, with large numbers of functional elements having been gained and lost rapidly along each mammalian lineage. Here, we review how different strengths of purifying selection have impacted on protein-coding and non-protein-coding loci and on transcription factor binding sites in mammalian and fruit fly genomes.
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Monoallelic and biallelic mutations in MAB21L2 cause a spectrum of major eye malformations.
Am. J. Hum. Genet.
PUBLISHED: 03-24-2014
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We identified four different missense mutations in the single-exon gene MAB21L2 in eight individuals with bilateral eye malformations from five unrelated families via three independent exome sequencing projects. Three mutational events altered the same amino acid (Arg51), and two were identical de novo mutations (c.151C>T [p.Arg51Cys]) in unrelated children with bilateral anophthalmia, intellectual disability, and rhizomelic skeletal dysplasia. c.152G>A (p.Arg51His) segregated with autosomal-dominant bilateral colobomatous microphthalmia in a large multiplex family. The fourth heterozygous mutation (c.145G>A [p.Glu49Lys]) affected an amino acid within two residues of Arg51 in an adult male with bilateral colobomata. In a fifth family, a homozygous mutation (c.740G>A [p.Arg247Gln]) altering a different region of the protein was identified in two male siblings with bilateral retinal colobomata. In mouse embryos, Mab21l2 showed strong expression in the developing eye, pharyngeal arches, and limb bud. As predicted by structural homology, wild-type MAB21L2 bound single-stranded RNA, whereas this activity was lost in all altered forms of the protein. MAB21L2 had no detectable nucleotidyltransferase activity in vitro, and its function remains unknown. Induced expression of wild-type MAB21L2 in human embryonic kidney 293 cells increased phospho-ERK (pERK1/2) signaling. Compared to the wild-type and p.Arg247Gln proteins, the proteins with the Glu49 and Arg51 variants had increased stability. Abnormal persistence of pERK1/2 signaling in MAB21L2-expressing cells during development is a plausible pathogenic mechanism for the heterozygous mutations. The phenotype associated with the homozygous mutation might be a consequence of complete loss of MAB21L2 RNA binding, although the cellular function of this interaction remains unknown.
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Cross-talking noncoding RNAs contribute to cell-specific neurodegeneration in SCA7.
Nat. Struct. Mol. Biol.
PUBLISHED: 02-08-2014
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What causes the tissue-specific pathology of diseases resulting from mutations in housekeeping genes? Specifically, in spinocerebellar ataxia type 7 (SCA7), a neurodegenerative disorder caused by a CAG-repeat expansion in ATXN7 (which encodes an essential component of the mammalian transcription coactivation complex, STAGA), the factors underlying the characteristic progressive cerebellar and retinal degeneration in patients were unknown. We found that STAGA is required for the transcription initiation of miR-124, which in turn mediates the post-transcriptional cross-talk between lnc-SCA7, a conserved long noncoding RNA, and ATXN7 mRNA. In SCA7, mutations in ATXN7 disrupt these regulatory interactions and result in a neuron-specific increase in ATXN7 expression. Strikingly, in mice this increase is most prominent in the SCA7 disease-relevant tissues, namely the retina and cerebellum. Our results illustrate how noncoding RNA-mediated feedback regulation of a ubiquitously expressed housekeeping gene may contribute to specific neurodegeneration.
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The long non-coding RNA Paupar regulates the expression of both local and distal genes.
EMBO J.
PUBLISHED: 02-01-2014
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Although some long noncoding RNAs (lncRNAs) have been shown to regulate gene expression in cis, it remains unclear whether lncRNAs can directly regulate transcription in trans by interacting with chromatin genome-wide independently of their sites of synthesis. Here, we describe the genomically local and more distal functions of Paupar, a vertebrate-conserved and central nervous system-expressed lncRNA transcribed from a locus upstream of the gene encoding the PAX6 transcription factor. Knockdown of Paupar disrupts the normal cell cycle profile of neuroblastoma cells and induces neural differentiation. Paupar acts in a transcript-dependent manner both locally, to regulate Pax6, as well as distally by binding and regulating genes on multiple chromosomes, in part through physical association with PAX6 protein. Paupar binding sites are enriched near promoters and can function as transcriptional regulatory elements whose activity is modulated by Paupar transcript levels. Our findings demonstrate that a lncRNA can function in trans at transcriptional regulatory elements distinct from its site of synthesis to control large-scale transcriptional programmes.
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Intergenic lncRNAs and the evolution of gene expression.
Curr. Opin. Genet. Dev.
PUBLISHED: 01-30-2014
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Eukaryote genomes encode a surprisingly large number of noncoding transcripts. Around two-thirds of human transcribed loci do not encode protein, and many are intergenic and produce long (>200 nucleotides) noncoding RNAs (lncRNAs). Extensive analyses using comparative genomics and transcriptomics approaches have established that lncRNA sequence and transcription tend to turn over rapidly during evolution. Our appreciation of the biological roles of lncRNAs, based only on a handful of transcripts with well-characterized functions, is that lncRNAs have diverse roles in regulating gene expression. These proposed roles together with their rapid rates of evolution suggest that lncRNAs could contribute to the divergent expression patterns observed among species and potentially to the origin of new traits.
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A code for RanGDP binding in ankyrin repeats defines a nuclear import pathway.
Cell
PUBLISHED: 01-22-2014
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Regulation of nuclear import is fundamental to eukaryotic biology. The majority of nuclear import pathways are mediated by importin-cargo interactions. Yet not all nuclear proteins interact with importins, necessitating the identification of a general importin-independent nuclear import pathway. Here, we identify a code that determines importin-independent nuclear import of ankyrin repeats (ARs), a structural motif found in over 250 human proteins with diverse functions. AR-containing proteins (ARPs) with a hydrophobic residue at the 13th position of two consecutive ARs bind RanGDP efficiently, and consequently enter the nucleus. This code, experimentally tested in 17 ARPs, predicts the nuclear-cytoplasmic localization of over 150 annotated human ARPs with high accuracy and is acquired by the most common familial melanoma-associated CDKN2A mutation, leading to nuclear accumulation of mutant p16ink4a. The RaDAR (RanGDP/AR) pathway represents a general importin-independent nuclear import pathway and is frequently used by AR-containing transcriptional regulators, especially those regulating NF-?B/p53.
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Sequencing depth and coverage: key considerations in genomic analyses.
Nat. Rev. Genet.
PUBLISHED: 01-18-2014
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Sequencing technologies have placed a wide range of genomic analyses within the capabilities of many laboratories. However, sequencing costs often set limits to the amount of sequences that can be generated and, consequently, the biological outcomes that can be achieved from an experimental design. In this Review, we discuss the issue of sequencing depth in the design of next-generation sequencing experiments. We review current guidelines and precedents on the issue of coverage, as well as their underlying considerations, for four major study designs, which include de novo genome sequencing, genome resequencing, transcriptome sequencing and genomic location analyses (for example, chromatin immunoprecipitation followed by sequencing (ChIP-seq) and chromosome conformation capture (3C)).
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CGAT: computational genomics analysis toolkit.
Bioinformatics
PUBLISHED: 01-05-2014
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Computational genomics seeks to draw biological inferences from genomic datasets, often by integrating and contextualizing next-generation sequencing data. CGAT provides an extensive suite of tools designed to assist in the analysis of genome scale data from a range of standard file formats. The toolkit enables filtering, comparison, conversion, summarization and annotation of genomic intervals, gene sets and sequences. The tools can both be run from the Unix command line and installed into visual workflow builders, such as Galaxy.
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The RFTS domain of Raf2 is required for Cul4 interaction and heterochromatin integrity in fission yeast.
PLoS ONE
PUBLISHED: 01-01-2014
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Centromeric heterochromatin assembly in fission yeast is critical for faithful chromosome segregation at mitosis. Its assembly requires a concerted pathway of events whereby the RNA interference (RNAi) pathway guides H3K9 methylation to target sequences. H3K9 methylation, a hallmark of heterochromatin structure, is mediated by the single histone methyltransferase Clr4 (equivalent to metazoan Suv3-9), a component of the CLRC complex. Loss of or defects in CLRC components disrupts heterochromatin formation due to loss of H3K9 methylation, thus an intact, fully functional CLRC complex is required for heterochromatin integrity. Despite its importance, little is known about the contribution of the CLRC component Raf2 to H3K9 methylation and heterochromatin assembly. We demonstrate that Raf2 is concentrated at centromeres and contrary to other analyses, we find that loss of Raf2 does not affect CENP-ACnp1 localisation or recruitment to centromeres. Our sequence alignments show that Raf2 contains a Replication Foci Targeting Sequence (RFTS) domain homologous to the RFTS domain of the human DNA methyltransferase DNMT1. We show that the Raf2 RFTS domain is required for centromeric heterochromatin formation as its mutation disrupts H3K9 methylation but not the processing of centromeric transcripts into small interfering RNAs (siRNAs) by the RNAi pathway. Analysis of biochemical interactions demonstrates that the RFTS domain mediates an interaction between Raf2 and the CLRC component Cul4. We conclude that the RFTS domain of Raf2 is a protein interaction module that plays an important role in heterochromatin formation at centromeres.
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Brain-expressed 3'UTR extensions strengthen miRNA cross-talk between ion channel/transporter encoding mRNAs.
Front Genet
PUBLISHED: 01-01-2014
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Why protein-coding genes express transcripts with longer 3'untranslated regions (3'UTRs) in the brain rather than in other tissues remains poorly understood. Given the established role of 3'UTRs in post-transcriptional regulation of transcript abundance and their recently highlighted contributions to miRNA-mediated cross-talk between mRNAs, we hypothesized that 3'UTR lengthening enhances coordinated expression between functionally-related genes in the brain. To test this hypothesis, we annotated 3'UTRs of human brain-expressed genes and found that transcripts encoding ion channels or transporters are specifically enriched among those genes expressing their longest 3'UTR extension in this tissue. These 3'UTR extensions have high density of response elements predicted for those miRNAs that are specifically expressed in the human frontal cortex (FC). Importantly, these miRNA response elements are more frequently shared among ion channel/transporter-encoding mRNAs than expected by chance. This indicates that miRNA-mediated cross-talk accounts, at least in part, for the observed coordinated expression of ion channel/transporter genes in the adult human brain. We conclude that extension of these genes' 3'UTRs enhances the miRNA-mediated cross-talk among their transcripts which post-transcriptionally regulates their mRNAs' relative levels.
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Adult pallium transcriptomes surprise in not reflecting predicted homologies across diverse chicken and mouse pallial sectors.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 07-22-2013
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The thorniest problem in comparative neurobiology is the identification of the particular brain region of birds and reptiles that corresponds to the mammalian neocortex [Butler AB, Reiner A, Karten HJ (2011) Ann N Y Acad Sci 1225:14-27; Wang Y, Brzozowska-Prechtl A, Karten HJ (2010) Proc Natl Acad Sci USA 107(28):12676-12681]. We explored which genes are actively transcribed in the regions of controversial ancestry in a representative bird (chicken) and mammal (mouse) at adult stages. We conducted four analyses comparing the expression patterns of their 5,130 most highly expressed one-to-one orthologous genes that considered global patterns of expression specificity, strong gene markers, and coexpression networks. Our study demonstrates transcriptomic divergence, plausible convergence, and, in two exceptional cases, conservation between specialized avian and mammalian telencephalic regions. This large-scale study potentially resolves the complex relationship between developmental homology and functional characteristics on the molecular level and settles long-standing evolutionary debates.
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Tiki, at the head of a new superfamily of enzymes.
Bioinformatics
PUBLISHED: 07-18-2013
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Tiki proteins appear to antagonize Wnt signalling pathway by acting as Wnt proteases, thereby affecting Wnt solubility by its amino-terminal cleavage. Tiki1 protease activity was shown to be metal ion-dependent and was inhibited by chelating agents and thus was tentatively proposed to be a metalloprotease. Nevertheless, Tiki proteins exhibit no detectable sequence similarity to previously described metalloproteases, but instead have been reported as being homologues of TraB proteins (Pfam ID: PF01963), a widely distributed family of unknown function and structure. Here, we show that Tiki proteins are members of a new superfamily of domains contained not just in TraB proteins, but also in erythromycin esterase (Pfam ID: PF05139), DUF399 (domain of unknown function 399; Pfam ID: PF04187) and MARTX toxins that contribute to host invasion and pathogenesis by bacteria. We establish the core fold of this enzymatic domain and its catalytic residues.
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Chromatin signatures at transcriptional start sites separate two equally populated yet distinct classes of intergenic long noncoding RNAs.
Genome Biol.
PUBLISHED: 07-17-2013
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Mammalian transcriptomes contain thousands of long noncoding RNAs (lncRNAs). Some lncRNAs originate from intragenic enhancers which, when active, behave as alternative promoters producing transcripts that are processed using the canonical signals of their host gene. We have followed up this observation by analyzing intergenic lncRNAs to determine the extent to which they might also originate from intergenic enhancers.
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Identification and function of long non-coding RNAs.
Essays Biochem.
PUBLISHED: 07-09-2013
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It is now clear that eukaryotic cells produce many thousands of non-coding RNAs. The least well-studied of these are longer than 200 nt and are known as lncRNAs (long non-coding RNAs). These loci are of particular interest as their biological relevance remains uncertain. Sequencing projects have identified thousands of these loci in a variety of species, from flies to humans. Genome-wide scans for functionality, such as evolutionary and expression analyses, suggest that many of these molecules have functional roles to play in the cell. Nevertheless, only a handful of lncRNAs have been experimentally investigated, and most of these appear to possess roles in regulating gene expression at a variety of different levels. Several lncRNAs have also been implicated in cancer. This evidence suggests that lncRNAs represent a new class of non-coding gene whose importance should become clearer upon further experimental investigation.
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GAT: a simulation framework for testing the association of genomic intervals.
Bioinformatics
PUBLISHED: 06-18-2013
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A common question in genomic analysis is whether two sets of genomic intervals overlap significantly. This question arises, for example, when interpreting ChIP-Seq or RNA-Seq data in functional terms. Because genome organization is complex, answering this question is non-trivial.
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Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system.
Cell Rep
PUBLISHED: 06-05-2013
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Here, we present a simple and highly efficient method for generating and detecting mutations of any gene in Drosophila melanogaster through the use of the CRISPR/Cas9 system (clustered regularly interspaced palindromic repeats/CRISPR-associated). We show that injection of RNA into the Drosophila embryo can induce highly efficient mutagenesis of desired target genes in up to 88% of injected flies. These mutations can be transmitted through the germline to make stable lines. Our system provides at least a 10-fold improvement in efficiency over previously published reports, enabling wider application of this technique. We also describe a simple and highly sensitive method of detecting mutations in the target gene by high-resolution melt analysis and discuss how the new technology enables the study of gene function.
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Network topologies and convergent aetiologies arising from deletions and duplications observed in individuals with autism.
PLoS Genet.
PUBLISHED: 06-01-2013
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Autism Spectrum Disorders (ASD) are highly heritable and characterised by impairments in social interaction and communication, and restricted and repetitive behaviours. Considering four sets of de novo copy number variants (CNVs) identified in 181 individuals with autism and exploiting mouse functional genomics and known protein-protein interactions, we identified a large and significantly interconnected interaction network. This network contains 187 genes affected by CNVs drawn from 45% of the patients we considered and 22 genes previously implicated in ASD, of which 192 form a single interconnected cluster. On average, those patients with copy number changed genes from this network possess changes in 3 network genes, suggesting that epistasis mediated through the network is extensive. Correspondingly, genes that are highly connected within the network, and thus whose copy number change is predicted by the network to be more phenotypically consequential, are significantly enriched among patients that possess only a single ASD-associated network copy number changed gene (p?=?0.002). Strikingly, deleted or disrupted genes from the network are significantly enriched in GO-annotated positive regulators (2.3-fold enrichment, corrected p?=?2×10(-5)), whereas duplicated genes are significantly enriched in GO-annotated negative regulators (2.2-fold enrichment, corrected p?=?0.005). The direction of copy change is highly informative in the context of the network, providing the means through which perturbations arising from distinct deletions or duplications can yield a common outcome. These findings reveal an extensive ASD-associated molecular network, whose topology indicates ASD-relevant mutational deleteriousness and that mechanistically details how convergent aetiologies can result extensively from CNVs affecting pathways causally implicated in ASD.
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Homozygous mutations in a predicted endonuclease are a novel cause of congenital dyserythropoietic anemia type I.
Haematologica
PUBLISHED: 05-28-2013
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The congenital dyserythropoietic anemias are a heterogeneous group of rare disorders primarily affecting erythropoiesis with characteristic morphological abnormalities and a block in erythroid maturation. Mutations in the CDAN1 gene, which encodes Codanin-1, underlie the majority of congenital dyserythropoietic anemia type I cases. However, no likely pathogenic CDAN1 mutation has been detected in approximately 20% of cases, suggesting the presence of at least one other locus. We used whole genome sequencing and segregation analysis to identify a homozygous T to A transversion (c.533T>A), predicted to lead to a p.L178Q missense substitution in C15ORF41, a gene of unknown function, in a consanguineous pedigree of Middle-Eastern origin. Sequencing C15ORF41 in other CDAN1 mutation-negative congenital dyserythropoietic anemia type I pedigrees identified a homozygous transition (c.281A>G), predicted to lead to a p.Y94C substitution, in two further pedigrees of SouthEast Asian origin. The haplotype surrounding the c.281A>G change suggests a founder effect for this mutation in Pakistan. Detailed sequence similarity searches indicate that C15ORF41 encodes a novel restriction endonuclease that is a member of the Holliday junction resolvase family of proteins.
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Next-generation Sequencing of Advanced Prostate Cancer Treated with Androgen-deprivation Therapy.
Eur. Urol.
PUBLISHED: 05-27-2013
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Androgen-deprivation therapy (ADT) is standard treatment for locally advanced or metastatic prostate cancer (PCa). Many patients develop castration resistance (castration-resistant PCa [CRPC]) after approximately 2-3 yr, with a poor prognosis. The molecular mechanisms underlying CRPC progression are unclear.
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Sixty years of genome biology.
Genome Biol.
PUBLISHED: 04-25-2013
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Sixty years after Watson and Crick published the double helix model of DNAs structure, thirteen members of Genome Biologys Editorial Board select key advances in the field of genome biology subsequent to that discovery.
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Rapid evolution of Beta-keratin genes contribute to phenotypic differences that distinguish turtles and birds from other reptiles.
Genome Biol Evol
PUBLISHED: 04-12-2013
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Sequencing of vertebrate genomes permits changes in distinct protein families, including gene gains and losses, to be ascribed to lineage-specific phenotypes. A prominent example of this is the large-scale duplication of beta-keratin genes in the ancestors of birds, which was crucial to the subsequent evolution of their beaks, claws, and feathers. Evidence suggests that the shell of Pseudomys nelsoni contains at least 16 beta-keratins proteins, but it is unknown whether this is a complete set and whether their corresponding genes are orthologous to avian beak, claw, or feather beta-keratin genes. To address these issues and to better understand the evolution of the turtle shell at a molecular level, we surveyed the diversity of beta-keratin genes from the genome assemblies of three turtles, Chrysemys picta, Pelodiscus sinensis, and Chelonia mydas, which together represent over 160 Myr of chelonian evolution. For these three turtles, we found 200 beta-keratins, which indicate that, as for birds, a large expansion of beta-keratin genes in turtles occurred concomitantly with the evolution of a unique phenotype, namely, their plastron and carapace. Phylogenetic reconstruction of beta-keratin gene evolution suggests that separate waves of gene duplication within a single genomic location gave rise to scales, claws, and feathers in birds, and independently the scutes of the shell in turtles.
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The western painted turtle genome, a model for the evolution of extreme physiological adaptations in a slowly evolving lineage.
Genome Biol.
PUBLISHED: 03-28-2013
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BACKGROUND: We describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species physiological capacities to withstand extreme anoxia and tissue freezing. RESULTS: Our phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented. CONCLUSIONS: Our comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtles extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders.
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MISP is a novel Plk1 substrate required for proper spindle orientation and mitotic progression.
J. Cell Biol.
PUBLISHED: 03-20-2013
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Precise positioning of the mitotic spindle determines the correct cell division axis and is crucial for organism development. Spindle positioning is mediated through a cortical machinery by capturing astral microtubules, thereby generating pushing/pulling forces at the cell cortex. However, the molecular link between these two structures remains elusive. Here we describe a previously uncharacterized protein, MISP (C19orf21), as a substrate of Plk1 that is required for correct mitotic spindle positioning. MISP is an actin-associated protein throughout the cell cycle. MISP depletion led to an impaired metaphase-to-anaphase transition, which depended on phosphorylation by Plk1. Loss of MISP induced mitotic defects including spindle misorientation accompanied by shortened astral microtubules. Furthermore, we find that MISP formed a complex with and regulated the cortical distribution of the +TIP binding protein p150(glued), a subunit of the dynein-dynactin complex. We propose that Plk1 phosphorylates MISP, thus stabilizing cortical and astral microtubule attachments required for proper mitotic spindle positioning.
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Mutations within lncRNAs are effectively selected against in fruitfly but not in human.
Genome Biol.
PUBLISHED: 02-25-2013
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Previous studies in Drosophila and mammals have revealed levels of long non-coding RNAs (lncRNAs) sequence conservation that are intermediate between neutrally evolving and protein-coding sequence. These analyses compared conservation between species that diverged up to 75 million years ago. However, analysis of sequence polymorphisms within a species population can provide an understanding of essentially contemporaneous selective constraints that are acting on lncRNAs and can quantify the deleterious effect of mutations occurring within these loci.
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The African coelacanth genome provides insights into tetrapod evolution.
Chris T Amemiya, Jessica Alföldi, Alison P Lee, Shaohua Fan, Hervé Philippe, Iain MacCallum, Ingo Braasch, Tereza Manousaki, Igor Schneider, Nicolas Rohner, Chris Organ, Domitille Chalopin, Jeramiah J Smith, Mark Robinson, Rosemary A Dorrington, Marco Gerdol, Bronwen Aken, Maria Assunta Biscotti, Marco Barucca, Denis Baurain, Aaron M Berlin, Gregory L Blatch, Francesco Buonocore, Thorsten Burmester, Michael S Campbell, Adriana Canapa, John P Cannon, Alan Christoffels, Gianluca De Moro, Adrienne L Edkins, Lin Fan, Anna Maria Fausto, Nathalie Feiner, Mariko Forconi, Junaid Gamieldien, Sante Gnerre, Andreas Gnirke, Jared V Goldstone, Wilfried Haerty, Mark E Hahn, Uljana Hesse, Steve Hoffmann, Jeremy Johnson, Sibel I Karchner, Shigehiro Kuraku, Marcia Lara, Joshua Z Levin, Gary W Litman, Evan Mauceli, Tsutomu Miyake, M Gail Mueller, David R Nelson, Anne Nitsche, Ettore Olmo, Tatsuya Ota, Alberto Pallavicini, Sumir Panji, Barbara Picone, Chris P Ponting, Sonja J Prohaska, Dariusz Przybylski, Nil Ratan Saha, Vydianathan Ravi, Filipe J Ribeiro, Tatjana Sauka-Spengler, Giuseppe Scapigliati, Stephen M J Searle, Ted Sharpe, Oleg Simakov, Peter F Stadler, John J Stegeman, Kenta Sumiyama, Diana Tabbaa, Hakim Tafer, Jason Turner-Maier, Peter Van Heusden, Simon White, Louise Williams, Mark Yandell, Henner Brinkmann, Jean-Nicolas Volff, Clifford J Tabin, Neil Shubin, Manfred Schartl, David B Jaffe, John H Postlethwait, Byrappa Venkatesh, Federica Di Palma, Eric S Lander, Axel Meyer, Kerstin Lindblad-Toh.
Nature
PUBLISHED: 02-20-2013
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The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.
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Predicting long non-coding RNAs using RNA sequencing.
Methods
PUBLISHED: 01-30-2013
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The advent of next-generation sequencing, and in particular RNA-sequencing (RNA-seq), technologies has expanded our knowledge of the transcriptional capacity of human and other animal, genomes. In particular, recent RNA-seq studies have revealed that transcription is widespread across the mammalian genome, resulting in a large increase in the number of putative transcripts from both within, and intervening between, known protein-coding genes. Long transcripts that appear to lack protein-coding potential (long non-coding RNAs, lncRNAs) have been the focus of much recent research, in part owing to observations of their cell-type and developmental time-point restricted expression patterns. A variety of sequencing protocols are currently available for identifying lncRNAs including RNA polymerase II occupancy, chromatin state maps and - the focus of this review - deep RNA sequencing. In addition, there are numerous analytical methods available for mapping reads and assembling transcript models that predict the presence and structure of lncRNAs from RNA-seq data. Here we review current methods for identifying lncRNAs using large-scale sequencing data from RNA-seq experiments and highlight analytical considerations that are required when undertaking such projects.
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Insights into the evolution of Darwins finches from comparative analysis of the Geospiza magnirostris genome sequence.
BMC Genomics
PUBLISHED: 01-23-2013
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A classical example of repeated speciation coupled with ecological diversification is the evolution of 14 closely related species of Darwins (Galápagos) finches (Thraupidae, Passeriformes). Their adaptive radiation in the Galápagos archipelago took place in the last 2-3 million years and some of the molecular mechanisms that led to their diversification are now being elucidated. Here we report evolutionary analyses of genome of the large ground finch, Geospiza magnirostris.
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Epigenetic conservation at gene regulatory elements revealed by non-methylated DNA profiling in seven vertebrates.
Elife
PUBLISHED: 01-15-2013
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Two-thirds of gene promoters in mammals are associated with regions of non-methylated DNA, called CpG islands (CGIs), which counteract the repressive effects of DNA methylation on chromatin. In cold-blooded vertebrates, computational CGI predictions often reside away from gene promoters, suggesting a major divergence in gene promoter architecture across vertebrates. By experimentally identifying non-methylated DNA in the genomes of seven diverse vertebrates, we instead reveal that non-methylated islands (NMIs) of DNA are a central feature of vertebrate gene promoters. Furthermore, NMIs are present at orthologous genes across vast evolutionary distances, revealing a surprising level of conservation in this epigenetic feature. By profiling NMIs in different tissues and developmental stages we uncover a unifying set of features that are central to the function of NMIs in vertebrates. Together these findings demonstrate an ancient logic for NMI usage at gene promoters and reveal an unprecedented level of epigenetic conservation across vertebrate evolution. DOI:http://dx.doi.org/10.7554/eLife.00348.001.
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Wrangling for microRNAs provokes much crosstalk.
Genome Biol.
PUBLISHED: 11-21-2011
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Levels of transcripts sharing microRNA response elements are co-regulated. These RNA-RNA interactions imply that combinations of microRNAs modulate cell-specific transcript networks.
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Preparation of high-quality next-generation sequencing libraries from picogram quantities of target DNA.
Genome Res.
PUBLISHED: 11-16-2011
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New sequencing technologies can address diverse biomedical questions but are limited by a minimum required DNA input of typically 1 ?g. We describe how sequencing libraries can be reproducibly created from 20 pg of input DNA using a modified transpososome-mediated fragmentation technique. Resulting libraries incorporate in-line bar-coding, which facilitates sample multiplexes that can be sequenced using Illumina platforms with the manufacturers sequencing primer. We demonstrate this technique by providing deep coverage sequence of the Escherichia coli K-12 genome that shows equivalent target coverage to a 1-?g input library prepared using standard Illumina methods. Reducing template quantity does, however, increase the proportion of duplicate reads and enriches coverage in low-GC regions. This finding was confirmed with exhaustive resequencing of a mouse library constructed from 20 pg of gDNA input (about seven haploid genomes) resulting in ?0.4-fold statistical coverage of uniquely mapped fragments. This implies that a near-complete coverage of the mouse genome is obtainable with this approach using 20 genomes as input. Application of this new method now allows genomic studies from low mass samples and routine preparation of sequencing libraries from enrichment procedures.
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Death of PRDM9 coincides with stabilization of the recombination landscape in the dog genome.
Genome Res.
PUBLISHED: 10-17-2011
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Analysis of diverse eukaryotes has revealed that recombination events cluster in discrete genomic locations known as hotspots. In humans, a zinc-finger protein, PRDM9, is believed to initiate recombination in >40% of hotspots by binding to a specific DNA sequence motif. However, the PRDM9 coding sequence is disrupted in the dog genome assembly, raising questions regarding the nature and control of recombination in dogs. By analyzing the sequences of PRDM9 orthologs in a number of dog breeds and several carnivores, we show here that this gene was inactivated early in canid evolution. We next use patterns of linkage disequilibrium using more than 170,000 SNP markers typed in almost 500 dogs to estimate the recombination rates in the dog genome using a coalescent-based approach. Broad-scale recombination rates show good correspondence with an existing linkage-based map. Significant variation in recombination rate is observed on the fine scale, and we are able to detect over 4000 recombination hotspots with high confidence. In contrast to human hotspots, 40% of canine hotspots are characterized by a distinct peak in GC content. A comparative genomic analysis indicates that these peaks are present also as weaker peaks in the panda, suggesting that the hotspots have been continually reinforced by accelerated and strongly GC biased nucleotide substitutions, consistent with the long-term action of biased gene conversion on the dog lineage. These results are consistent with the loss of PRDM9 in canids, resulting in a greater evolutionary stability of recombination hotspots. The genetic determinants of recombination hotspots in the dog genome may thus reflect a fundamental process of relevance to diverse animal species.
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What fraction of the human genome is functional?
Genome Res.
PUBLISHED: 08-29-2011
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Many evolutionary studies over the past decade have estimated ?(sel), the proportion of all nucleotides in the human genome that are subject to purifying selection because of their biological function. Most of these studies have estimated the nucleotide substitution rates from genome sequence alignments across many diverse mammals. Some ?(sel) estimates will be affected by the heterogeneity of substitution rates in neutral sequence across the genome. Most will also be inaccurate if change in the functional sequence repertoire occurs rapidly relative to the separation of lineages that are being compared. Evidence gathered from both evolutionary and experimental analyses now indicate that rates of "turnover" of functional, predominantly noncoding, sequence are, indeed, high. They are sufficiently high that an estimated 50% of mouse constrained noncoding sequence is predicted not to be shared with rat, a closely related rodent. The rapidity of turnover results in, at least, a twofold underestimate of ?(sel) by analyses that measure constraint across the eutherian phylogeny. Approaches that take account of turnover estimate that the steady-state value of ?(sel) lies between 10% and 15%. Experimental studies corroborate the predicted rates of loss and gain of noncoding functional sites. These studies show the limitations inherent in the use of deep sequence conservation for identifying functional sequence. Experimental investigations focusing on lineage-specific, noncoding, and functional sequence are now essential if we are to appreciate the complete functional repertoire of the human genome.
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Transcriptomic analysis supports similar functional roles for the two thymuses of the tammar wallaby.
BMC Genomics
PUBLISHED: 08-19-2011
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The thymus plays a critical role in the development and maturation of T-cells. Humans have a single thoracic thymus and presence of a second thymus is considered an anomaly. However, many vertebrates have multiple thymuses. The tammar wallaby has two thymuses: a thoracic thymus (typically found in all mammals) and a dominant cervical thymus. Researchers have known about the presence of the two wallaby thymuses since the 1800s, but no genome-wide research has been carried out into possible functional differences between the two thymic tissues. Here, we used pyrosequencing to compare the transcriptomes of a cervical and thoracic thymus from a single 178 day old tammar wallaby.
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Structure and evolutionary history of DISC1.
Hum. Mol. Genet.
PUBLISHED: 08-18-2011
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Evolutionary and protein structural analyses can provide functional insights into genes implicated in human psychiatric diseases. Even eukaryotic organisms lacking nervous systems contain homologues of many key signalling molecules of animal neurons implying that human cognition derives, in part, from modifications of ancestral molecules and complexes. One protein whose evolutionary origin is obscure is DISC1 (disrupted in schizophrenia 1) whose gene locus has been associated with many psychiatric conditions including schizophrenia, clinical depression and bipolar disorder. This proteins rapid evolution and its unusual amino acid and ?-helix composition have hindered searches for DISC1 homologues in species other than vertebrates. Here, we review the evolution and structure of the DISC1 protein in the light of in-depth sequence analyses. These predict DISC1 orthologues in diverse eukaryotic organisms, including early-branching animals such as amphioxus, sea anemone, amoebas and Trichoplax, and in plants and algae. DISC1 thus is widespread among eukaryotes, although it remains absent from fungi, nematodes and Diptera, including fruit flies. These observations now permit studies of DISC1 function in simple non-vertebrate model organisms. Surprisingly, these analyses also identify between two and four sequence repeats in DISC1 orthologues. The first two of these repeats show significant sequence similarity to the UVR family of globular domains. These UVR-like repeats are predicted to contain, not coiled coil structures, but rather two closely associated antiparallel ?-helices. One common missense variant in DISC1 (L607F) lies within the second DISC1 UVR-like domain. These observations should assist in delineating the functional regions of the DISC1 protein.
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The Anolis lizard genome: an amniote genome without isochores.
Genome Biol Evol
PUBLISHED: 07-27-2011
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Isochores are large regions of relatively homogeneous nucleotide composition and are present in the genomes of all mammals and birds that have been sequenced to date. The newly sequenced genome of Anolis carolinensis provides the first opportunity to quantify isochore structure in a nonavian reptile. We find Anolis to have the most compositionally homogeneous genome of all amniotes sequenced thus far, a homogeneity exceeding that for the frog Xenopus. Based on a Bayesian algorithm, Anolis has smaller and less GC-rich isochores compared with human and chicken. Correlates generally associated with GC-rich isochores, including shorter introns and higher gene density, have all but disappeared from the Anolis genome. Using genic GC as a proxy for isochore structure so as to compare with other vertebrates, we found that GC content has substantially decreased in the lineage leading to Anolis since diverging from the common ancestor of Reptilia ?275 Ma, perhaps reflecting weakened or reversed GC-biased gene conversion, a nonadaptive substitution process that is thought to be important in the maintenance and trajectory of isochore evolution. Our results demonstrate that GC composition in Anolis is not associated with important features of genome structure, including gene density and intron size, in contrast to patterns seen in mammal and bird genomes.
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Mouse genomic variation and its effect on phenotypes and gene regulation.
Nature
PUBLISHED: 07-05-2011
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We report genome sequences of 17 inbred strains of laboratory mice and identify almost ten times more variants than previously known. We use these genomes to explore the phylogenetic history of the laboratory mouse and to examine the functional consequences of allele-specific variation on transcript abundance, revealing that at least 12% of transcripts show a significant tissue-specific expression bias. By identifying candidate functional variants at 718 quantitative trait loci we show that the molecular nature of functional variants and their position relative to genes vary according to the effect size of the locus. These sequences provide a starting point for a new era in the functional analysis of a key model organism.
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Rapid turnover of functional sequence in human and other genomes.
Annu Rev Genomics Hum Genet
PUBLISHED: 07-05-2011
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The amount of a genomes sequence that is functional has been surprisingly difficult to estimate accurately. This has severely hindered analyses asking whether the amount of functional genomic sequence correlates with organismal complexity. Most studies estimate these amounts by considering nucleotide substitution rates within aligned sequences. These approaches show reduced power to identify sequence that is aligned, functional, and constrained only within narrowly defined phyla. The neutral indel model exploits insertions or deletions (indels) rather than substitutions in predicting functional sequence. Surprisingly, this method indicates that half of all functional sequence is specific to individual eutherian lineages. This review considers the rates at which coding or noncoding and functional or nonfunctional sequence changes among mammalian genomes. In contrast to the slow rate at which protein-coding sequence changes, functional noncoding sequence appears to change or be turned over at rapid rates in mammals.
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A transcriptomic atlas of mouse neocortical layers.
Neuron
PUBLISHED: 06-20-2011
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In the mammalian cortex, neurons and glia form a patterned structure across six layers whose complex cytoarchitectonic arrangement is likely to contribute to cognition. We sequenced transcriptomes from layers 1-6b of different areas (primary and secondary) of the adult (postnatal day 56) mouse somatosensory cortex to understand the transcriptional levels and functional repertoires of coding and noncoding loci for cells constituting these layers. A total of 5,835 protein-coding genes and 66 noncoding RNA loci are differentially expressed ("patterned") across the layers, on the basis of a machine-learning model (naive Bayes) approach. Layers 2-6b are each associated with specific functional and disease annotations that provide insights into their biological roles. This new resource (http://genserv.anat.ox.ac.uk/layers) greatly extends currently available resources, such as the Allen Mouse Brain Atlas and microarray data sets, by providing quantitative expression levels, by being genome-wide, by including novel loci, and by identifying candidate alternatively spliced transcripts that are differentially expressed across layers.
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Cdc45: the missing RecJ ortholog in eukaryotes?
Bioinformatics
PUBLISHED: 06-08-2011
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DNA replication is one of the most ancient of cellular processes and functional similarities among its molecular machinery are apparent across all cellular life. Cdc45 is one of the essential components of the eukaryotic replication fork and is required for the initiation and elongation of DNA replication, but its molecular function is currently unknown. In order to trace its evolutionary history and to identify functional domains, we embarked on a computational sequence analysis of the Cdc45 protein family. Our findings reveal eukaryotic Cdc45 and prokaryotic RecJ to possess a common ancestry and Cdc45 to contain a catalytic site within a predicted exonuclease domain. The likely orthology between Cdc45 and RecJ reveals new lines of enquiry into DNA replication mechanisms in eukaryotes.
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Repo-Man coordinates chromosomal reorganization with nuclear envelope reassembly during mitotic exit.
Dev. Cell
PUBLISHED: 05-05-2011
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Repo-Man targets protein phosphatase 1 ? (PP1?) to chromatin at anaphase onset and regulates chromosome structure during mitotic exit. Here, we show that a Repo-Man:PP1 complex forms in anaphase following dephosphorylation of Repo-Man. Upon activation, the complex localizes to chromosomes and causes the dephosphorylation of histone H3 (Thr3, Ser10, and Ser28). In anaphase, Repo-Man has both catalytic and structural functions that are mediated by two separate domains. A C-terminal domain localizes Repo-Man to bulk chromatin in early anaphase. There, it targets PP1 for the dephosphorylation of histone H3 and possibly other chromosomal substrates. An N-terminal domain localizes Repo-Man to the chromosome periphery later in anaphase. There, it is responsible for the recruitment of nuclear components such as Importin ? and Nup153 in a PP1-independent manner. These observations identify Repo-Man as a key factor that coordinates chromatin remodeling and early events of nuclear envelope reformation during mitotic exit.
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Regulation of DNA replication through Sld3-Dpb11 interaction is conserved from yeast to humans.
Curr. Biol.
PUBLISHED: 04-27-2011
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Cyclin-dependent kinases (CDKs) play crucial roles in promoting DNA replication and preventing rereplication in eukaryotic cells [1-4]. In budding yeast, CDKs promote DNA replication by phosphorylating two proteins, Sld2 and Sld3, which generates binding sites for pairs of BRCT repeats (breast cancer gene 1 [BRCA1] C terminal repeats) in the Dpb11 protein [5, 6]. The Sld3-Dpb11-Sld2 complex generated by CDK phosphorylation is required for the assembly and activation of the Cdc45-Mcm2-7-GINS (CMG) replicative helicase. In response to DNA replication stress, the interaction between Sld3 and Dpb11 is blocked by the checkpoint kinase Rad53 [7], which prevents late origin firing [7, 8]. Here we show that the two key CDK sites in Sld3 are conserved in the human Sld3-related protein Treslin/ticrr and are essential for DNA replication. Moreover, phosphorylation of these two sites mediates interaction with the orthologous pair of BRCT repeats in the human Dpb11 ortholog, TopBP1. Finally, we show that DNA replication stress prevents the interaction between Treslin/ticrr and TopBP1 via the Chk1 checkpoint kinase. Our results indicate that Treslin/ticrr is a genuine ortholog of Sld3 and that the Sld3-Dpb11 interaction has remained a critical nexus of S phase regulation through eukaryotic evolution.
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The genome of the green anole lizard and a comparative analysis with birds and mammals.
Nature
PUBLISHED: 04-21-2011
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The evolution of the amniotic egg was one of the great evolutionary innovations in the history of life, freeing vertebrates from an obligatory connection to water and thus permitting the conquest of terrestrial environments. Among amniotes, genome sequences are available for mammals and birds, but not for non-avian reptiles. Here we report the genome sequence of the North American green anole lizard, Anolis carolinensis. We find that A. carolinensis microchromosomes are highly syntenic with chicken microchromosomes, yet do not exhibit the high GC and low repeat content that are characteristic of avian microchromosomes. Also, A. carolinensis mobile elements are very young and diverse-more so than in any other sequenced amniote genome. The GC content of this lizard genome is also unusual in its homogeneity, unlike the regionally variable GC content found in mammals and birds. We describe and assign sequence to the previously unknown A. carolinensis X chromosome. Comparative gene analysis shows that amniote egg proteins have evolved significantly more rapidly than other proteins. An anole phylogeny resolves basal branches to illuminate the history of their repeated adaptive radiations.
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Natural genetic variation caused by small insertions and deletions in the human genome.
Genome Res.
PUBLISHED: 04-01-2011
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Human genetic variation is expected to play a central role in personalized medicine. Yet only a fraction of the natural genetic variation that is harbored by humans has been discovered to date. Here we report almost 2 million small insertions and deletions (INDELs) that range from 1 bp to 10,000 bp in length in the genomes of 79 diverse humans. These variants include 819,363 small INDELs that map to human genes. Small INDELs frequently were found in the coding exons of these genes, and several lines of evidence indicate that such variation is a major determinant of human biological diversity. Microarray-based genotyping experiments revealed several interesting observations regarding the population genetics of small INDEL variation. For example, we found that many of our INDELs had high levels of linkage disequilibrium (LD) with both HapMap SNPs and with high-scoring SNPs from genome-wide association studies. Overall, our study indicates that small INDEL variation is likely to be a key factor underlying inherited traits and diseases in humans.
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Evc2 is a positive modulator of Hedgehog signalling that interacts with Evc at the cilia membrane and is also found in the nucleus.
BMC Biol.
PUBLISHED: 02-04-2011
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Evc is essential for Indian Hedgehog (Hh) signalling in the cartilage growth plate. The gene encoding Evc2 is in close proximity in divergent orientation to Evc and mutations in both human genes lead to the chondrodysplasia Ellis-van Creveld syndrome.
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What are the genomic drivers of the rapid evolution of PRDM9?
Trends Genet.
PUBLISHED: 01-31-2011
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Mammalian Prdm9 has been proposed to be a key determinant of the positioning of chromosome double-strand breaks during meiosis, a contributor to speciation processes, and the most rapidly evolving gene in human, and other animal, genomes. Prdm9 genes often exhibit substantial variation in their numbers of encoded zinc fingers (ZFs), not only between closely related species but also among individuals of a species. The near-identity of these ZF sequences appears to render them very unstable in copy number. The rare sequence differences, however, cluster within ZF sites that determine the DNA-binding specificity of PRDM9, and these substitutions are frequently positively selected. Here, possible drivers of the rapid evolution of Prdm9 are discussed, including selection for efficient pairing of homologous chromosomes or for recombination of deleterious linked alleles, and selection against depletion of recombination hotspots or against disease-associated genome rearrangement.
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Comparative and demographic analysis of orang-utan genomes.
Devin P Locke, LaDeana W Hillier, Wesley C Warren, Kim C Worley, Lynne V Nazareth, Donna M Muzny, Shiaw-Pyng Yang, Zhengyuan Wang, Asif T Chinwalla, Pat Minx, Makedonka Mitreva, Lisa Cook, Kim D Delehaunty, Catrina Fronick, Heather Schmidt, Lucinda A Fulton, Robert S Fulton, Joanne O Nelson, Vincent Magrini, Craig Pohl, Tina A Graves, Chris Markovic, Andy Cree, Huyen H Dinh, Jennifer Hume, Christie L Kovar, Gerald R Fowler, Gerton Lunter, Stephen Meader, Andreas Heger, Chris P Ponting, Tomas Marques-Bonet, Can Alkan, Lin Chen, Ze Cheng, Jeffrey M Kidd, Evan E Eichler, Simon White, Stephen Searle, Albert J Vilella, Yuan Chen, Paul Flicek, Jian Ma, Brian Raney, Bernard Suh, Richard Burhans, Javier Herrero, David Haussler, Rui Faria, Olga Fernando, Fleur Darré, Domènec Farré, Elodie Gazave, Meritxell Oliva, Arcadi Navarro, Roberta Roberto, Oronzo Capozzi, Nicoletta Archidiacono, Giuliano Della Valle, Stefania Purgato, Mariano Rocchi, Miriam K Konkel, Jerilyn A Walker, Brygg Ullmer, Mark A Batzer, Arian F A Smit, Robert Hubley, Claudio Casola, Daniel R Schrider, Matthew W Hahn, Víctor Quesada, Xose S Puente, Gonzalo R Ordoñez, Carlos Lopez-Otin, Tomás Vinar, Brona Brejova, Aakrosh Ratan, Robert S Harris, Webb Miller, Carolin Kosiol, Heather A Lawson, Vikas Taliwal, André L Martins, Adam Siepel, Arindam RoyChoudhury, Xin Ma, Jeremiah Degenhardt, Carlos D Bustamante, Ryan N Gutenkunst, Thomas Mailund, Julien Y Dutheil, Asger Hobolth, Mikkel H Schierup, Oliver A Ryder, Yuko Yoshinaga, Pieter J de Jong, George M Weinstock, Jeffrey Rogers, Elaine R Mardis, Richard A Gibbs, Richard K Wilson.
Nature
PUBLISHED: 01-29-2011
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Orang-utan is derived from a Malay term meaning man of the forest and aptly describes the southeast Asian great apes native to Sumatra and Borneo. The orang-utan species, Pongo abelii (Sumatran) and Pongo pygmaeus (Bornean), are the most phylogenetically distant great apes from humans, thereby providing an informative perspective on hominid evolution. Here we present a Sumatran orang-utan draft genome assembly and short read sequence data from five Sumatran and five Bornean orang-utan genomes. Our analyses reveal that, compared to other primates, the orang-utan genome has many unique features. Structural evolution of the orang-utan genome has proceeded much more slowly than other great apes, evidenced by fewer rearrangements, less segmental duplication, a lower rate of gene family turnover and surprisingly quiescent Alu repeats, which have played a major role in restructuring other primate genomes. We also describe a primate polymorphic neocentromere, found in both Pongo species, emphasizing the gradual evolution of orang-utan genome structure. Orang-utans have extremely low energy usage for a eutherian mammal, far lower than their hominid relatives. Adding their genome to the repertoire of sequenced primates illuminates new signals of positive selection in several pathways including glycolipid metabolism. From the population perspective, both Pongo species are deeply diverse; however, Sumatran individuals possess greater diversity than their Bornean counterparts, and more species-specific variation. Our estimate of Bornean/Sumatran speciation time, 400,000?years ago, is more recent than most previous studies and underscores the complexity of the orang-utan speciation process. Despite a smaller modern census population size, the Sumatran effective population size (N(e)) expanded exponentially relative to the ancestral N(e) after the split, while Bornean N(e) declined over the same period. Overall, the resources and analyses presented here offer new opportunities in evolutionary genomics, insights into hominid biology, and an extensive database of variation for conservation efforts.
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Identification and functional analysis of an ovarian form of the egg activation factor phospholipase C zeta (PLC?) in pufferfish.
Mol. Reprod. Dev.
PUBLISHED: 01-14-2011
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Recent studies suggest that egg activation in mammals is triggered by a sperm-specific phospholipase C, PLCzeta. In other vertebrate species such as medaka fish, chickens, and quail, PLCzeta is also expressed as a testis-specific mRNA. Functional studies suggest that PLCzeta plays a similar role as a trigger of egg activation in these species. Here, we report the identification of PLCzeta orthologues in pufferfish species Takifugu rubripes (Fugu) and Tetraodon nigroviridis (Tetraodon). Unexpectedly in these species PLCzeta is expressed not in the testis, but in ovary and brain. Injection of pufferfish PLCzeta copy ribonucleic acid (cRNA) into mouse eggs failed to trigger calcium oscillations, unlike medaka PLCzeta cRNA. Our findings provide the first evidence that PLCzeta may be expressed in the egg, rather than the sperm, in some vertebrate species, and that its mechanism of action and physiologic role at fertilization may differ in different vertebrate species.
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Genes and biological processes commonly disrupted in rare and heterogeneous developmental delay syndromes.
Hum. Mol. Genet.
PUBLISHED: 12-08-2010
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Rare copy number variations (CNVs) are a recognized cause of common human disease. Predicting the genetic element(s) within a small CNV whose copy number loss or gain underlies a specific phenotype might be achieved reasonably rapidly for single patients. Identifying the biological processes that are commonly disrupted within a large patient cohort which possess larger CNVs, however, requires a more objective approach that exploits genomic resources. In this study, we first identified 98 large, rare CNVs within patients exhibiting multiple congenital anomalies. All patients presented with global developmental delay (DD), while other secondary symptoms such as cardiac defects, craniofacial features and seizures were varyingly presented. By applying a robust statistical procedure that matches patients clinical phenotypes to laboratory mouse gene knockouts, we were able to strongly implicate anomalies in brain morphology and, separately, in long-term potentiation as manifestations of these DD patients disorders. These and other significantly enriched model phenotypes provide insights into the pathoetiology of human DD and behavioral and anatomical secondary symptoms that are specific to DD patients. These enrichments set apart 103 genes, from among thousands overlapped by these CNVs, as strong candidates whose copy number change causally underlies approximately 46% of the cohorts DD syndromes and between 59 and 80% of the cohorts secondary symptoms. We also identified significantly enriched model phenotypes among genes overlapped by CNVs in both DD and learning disability cohorts, indicating a congruent etiology. These results demonstrate the high predictive potential of model organism phenotypes when implicating candidate genes for rare genomic disorders.
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Transcribed dark matter: meaning or myth?
Hum. Mol. Genet.
PUBLISHED: 08-25-2010
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Genomic tiling arrays, cDNA sequencing and, more recently, RNA-Seq have provided initial insights into the extent and depth of transcribed sequence across human and other genomes. These methods have led to greatly improved annotations of protein-coding genes, but have also identified transcription outside of annotated exons. One resultant issue that has aroused dispute is the balance of transcription of known exons against transcription outside of known exons. While non-genic dark matter transcription was found by tiling arrays to be pervasive, it was seen to contribute only a small percentage of the polyadenylated transcriptome in some RNA-Seq experiments. This apparent contradiction has been compounded by a lack of clarity about what exactly constitutes a protein-coding gene. It remains unclear, for example, whether or not all transcripts that overlap on either strand within a genomic locus should be assigned to a single gene locus, including those that fail to share promoters, exons and splice junctions. The inability of tiling arrays and RNA-Seq to count transcripts, rather than exons or exon pairs, adds to these difficulties. While there is agreement that thousands of apparently non-coding loci are present outside of protein-coding genes in the human genome, there is vigorous debate of what constitutes evidence for their functionality. These issues will only be resolved upon the demonstration, or otherwise, that organismal or cellular phenotypes frequently result when non-coding RNA loci are disrupted.
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A ChIP-seq defined genome-wide map of vitamin D receptor binding: associations with disease and evolution.
Genome Res.
PUBLISHED: 08-24-2010
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Initially thought to play a restricted role in calcium homeostasis, the pleiotropic actions of vitamin D in biology and their clinical significance are only now becoming apparent. However, the mode of action of vitamin D, through its cognate nuclear vitamin D receptor (VDR), and its contribution to diverse disorders, remain poorly understood. We determined VDR binding throughout the human genome using chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq). After calcitriol stimulation, we identified 2776 genomic positions occupied by the VDR and 229 genes with significant changes in expression in response to vitamin D. VDR binding sites were significantly enriched near autoimmune and cancer associated genes identified from genome-wide association (GWA) studies. Notable genes with VDR binding included IRF8, associated with MS, and PTPN2 associated with Crohns disease and T1D. Furthermore, a number of single nucleotide polymorphism associations from GWA were located directly within VDR binding intervals, for example, rs13385731 associated with SLE and rs947474 associated with T1D. We also observed significant enrichment of VDR intervals within regions of positive selection among individuals of Asian and European descent. ChIP-seq determination of transcription factor binding, in combination with GWA data, provides a powerful approach to further understanding the molecular bases of complex diseases.
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Massive turnover of functional sequence in human and other mammalian genomes.
Genome Res.
PUBLISHED: 08-06-2010
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Despite the availability of dozens of animal genome sequences, two key questions remain unanswered: First, what fraction of any species genome confers biological function, and second, are apparent differences in organismal complexity reflected in an objective measure of genomic complexity? Here, we address both questions by applying, across the mammalian phylogeny, an evolutionary model that estimates the amount of functional DNA that is shared between two species genomes. Our main findings are, first, that as the divergence between mammalian species increases, the predicted amount of pairwise shared functional sequence drops off dramatically. We show by simulations that this is not an artifact of the method, but rather indicates that functional (and mostly noncoding) sequence is turning over at a very high rate. We estimate that between 200 and 300 Mb (?6.5%-10%) of the human genome is under functional constraint, which includes five to eight times as many constrained noncoding bases than bases that code for protein. In contrast, in D. melanogaster we estimate only 56-66 Mb to be constrained, implying a ratio of noncoding to coding constrained bases of about 2. This suggests that, rather than genome size or protein-coding gene complement, it is the number of functional bases that might best mirror our naïve preconceptions of organismal complexity.
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Identification of the MMS22L-TONSL complex that promotes homologous recombination.
Mol. Cell
PUBLISHED: 06-18-2010
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Budding yeast Mms22 is required for homologous recombination (HR)-mediated repair of stalled or broken DNA replication forks. Here we identify a human Mms22-like protein (MMS22L) and an MMS22L-interacting protein, NF?BIL2/TONSL. Depletion of MMS22L or TONSL from human cells causes a high level of double-strand breaks (DSBs) during DNA replication. Both proteins accumulate at stressed replication forks, and depletion of MMS22L or TONSL from cells causes hypersensitivity to agents that cause S phase-associated DSBs, such as topoisomerase (TOP) inhibitors. In this light, MMS22L and TONSL are required for the HR-mediated repair of replication fork-associated DSBs. In cells depleted of either protein, DSBs induced by the TOP1 inhibitor camptothecin are resected normally, but the loading of the RAD51 recombinase is defective. Therefore, MMS22L and TONSL are required for the maintenance of genome stability when unscheduled DSBs occur in the vicinity of DNA replication forks.
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The protein composition of mitotic chromosomes determined using multiclassifier combinatorial proteomics.
Cell
PUBLISHED: 05-20-2010
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Despite many decades of study, mitotic chromosome structure and composition remain poorly characterized. Here, we have integrated quantitative proteomics with bioinformatic analysis to generate a series of independent classifiers that describe the approximately 4,000 proteins identified in isolated mitotic chromosomes. Integrating these classifiers by machine learning uncovers functional relationships between protein complexes in the context of intact chromosomes and reveals which of the approximately 560 uncharacterized proteins identified here merits further study. Indeed, of 34 GFP-tagged predicted chromosomal proteins, 30 were chromosomal, including 13 with centromere-association. Of 16 GFP-tagged predicted nonchromosomal proteins, 14 were confirmed to be nonchromosomal. An unbiased analysis of the whole chromosome proteome from genetic knockouts of kinetochore protein Ska3/Rama1 revealed that the APC/C and RanBP2/RanGAP1 complexes depend on the Ska complex for stable association with chromosomes. Our integrated analysis predicts that up to 97 new centromere-associated proteins remain to be discovered in our data set.
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Functional impact of global rare copy number variation in autism spectrum disorders.
Dalila Pinto, Alistair T Pagnamenta, Lambertus Klei, Richard Anney, Daniele Merico, Regina Regan, Judith Conroy, Tiago R Magalhães, Catarina Correia, Brett S Abrahams, Joana Almeida, Elena Bacchelli, Gary D Bader, Anthony J Bailey, Gillian Baird, Agatino Battaglia, Tom Berney, Nadia Bolshakova, Sven Bölte, Patrick F Bolton, Thomas Bourgeron, Sean Brennan, Jessica Brian, Susan E Bryson, Andrew R Carson, Guillermo Casallo, Jillian Casey, Brian H Y Chung, Lynne Cochrane, Christina Corsello, Emily L Crawford, Andrew Crossett, Cheryl Cytrynbaum, Geraldine Dawson, Maretha de Jonge, Richard Delorme, Irene Drmic, Eftichia Duketis, Frederico Duque, Annette Estes, Penny Farrar, Bridget A Fernandez, Susan E Folstein, Eric Fombonne, Christine M Freitag, John Gilbert, Christopher Gillberg, Joseph T Glessner, Jeremy Goldberg, Andrew Green, Jonathan Green, Stephen J Guter, Hakon Hakonarson, Elizabeth A Heron, Matthew Hill, Richard Holt, Jennifer L Howe, Gillian Hughes, Vanessa Hus, Roberta Igliozzi, Cecilia Kim, Sabine M Klauck, Alexander Kolevzon, Olena Korvatska, Vlad Kustanovich, Clara M Lajonchere, Janine A Lamb, Magdalena Laskawiec, Marion Leboyer, Ann Le Couteur, Bennett L Leventhal, Anath C Lionel, Xiao-Qing Liu, Catherine Lord, Linda Lotspeich, Sabata C Lund, Elena Maestrini, William Mahoney, Carine Mantoulan, Christian R Marshall, Helen McConachie, Christopher J McDougle, Jane McGrath, William M McMahon, Alison Merikangas, Ohsuke Migita, Nancy J Minshew, Ghazala K Mirza, Jeff Munson, Stanley F Nelson, Carolyn Noakes, Abdul Noor, Gudrun Nygren, Guiomar Oliveira, Katerina Papanikolaou, Jeremy R Parr, Barbara Parrini, Tara Paton, Andrew Pickles, Marion Pilorge, Joseph Piven, Chris P Ponting, David J Posey, Annemarie Poustka, Fritz Poustka, Aparna Prasad, Jiannis Ragoussis, Katy Renshaw, Jessica Rickaby, Wendy Roberts, Kathryn Roeder, Bernadette Rogé, Michael L Rutter, Laura J Bierut, John P Rice, Jeff Salt, Katherine Sansom, Daisuke Sato, Ricardo Segurado, Ana F Sequeira, Lili Senman, Naisha Shah, Val C Sheffield, Latha Soorya, Inês Sousa, Olaf Stein, Nuala Sykes, Vera Stoppioni, Christina Strawbridge, Raffaella Tancredi, Katherine Tansey, Bhooma Thiruvahindrapduram, Ann P Thompson, Susanne Thomson, Ana Tryfon, John Tsiantis, Herman van Engeland, John B Vincent, Fred Volkmar, Simon Wallace, Kai Wang, Zhouzhi Wang, Thomas H Wassink, Caleb Webber, Rosanna Weksberg, Kirsty Wing, Kerstin Wittemeyer, Shawn Wood, Jing Wu, Brian L Yaspan, Danielle Zurawiecki, Lonnie Zwaigenbaum, Joseph D Buxbaum, Rita M Cantor, Edwin H Cook, Hilary Coon, Michael L Cuccaro, Bernie Devlin, Sean Ennis, Louise Gallagher, Daniel H Geschwind, Michael Gill, Jonathan L Haines, Joachim Hallmayer, Judith Miller, Anthony P Monaco, John I Nurnberger, Andrew D Paterson, Margaret A Pericak-Vance, Gerard D Schellenberg, Peter Szatmari, Astrid M Vicente, Veronica J Vieland, Ellen M Wijsman, Stephen W Scherer, James S Sutcliffe, Catalina Betancur.
Nature
PUBLISHED: 05-07-2010
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The autism spectrum disorders (ASDs) are a group of conditions characterized by impairments in reciprocal social interaction and communication, and the presence of restricted and repetitive behaviours. Individuals with an ASD vary greatly in cognitive development, which can range from above average to intellectual disability. Although ASDs are known to be highly heritable ( approximately 90%), the underlying genetic determinants are still largely unknown. Here we analysed the genome-wide characteristics of rare (<1% frequency) copy number variation in ASD using dense genotyping arrays. When comparing 996 ASD individuals of European ancestry to 1,287 matched controls, cases were found to carry a higher global burden of rare, genic copy number variants (CNVs) (1.19 fold, P = 0.012), especially so for loci previously implicated in either ASD and/or intellectual disability (1.69 fold, P = 3.4 x 10(-4)). Among the CNVs there were numerous de novo and inherited events, sometimes in combination in a given family, implicating many novel ASD genes such as SHANK2, SYNGAP1, DLGAP2 and the X-linked DDX53-PTCHD1 locus. We also discovered an enrichment of CNVs disrupting functional gene sets involved in cellular proliferation, projection and motility, and GTPase/Ras signalling. Our results reveal many new genetic and functional targets in ASD that may lead to final connected pathways.
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Molecular evolution of genes in avian genomes.
Genome Biol.
PUBLISHED: 04-30-2010
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Obtaining a draft genome sequence of the zebra finch (Taeniopygia guttata), the second bird genome to be sequenced, provides the necessary resource for whole-genome comparative analysis of gene sequence evolution in a non-mammalian vertebrate lineage. To analyze basic molecular evolutionary processes during avian evolution, and to contrast these with the situation in mammals, we aligned the protein-coding sequences of 8,384 1:1 orthologs of chicken, zebra finch, a lizard and three mammalian species.
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Accurate distinction of pathogenic from benign CNVs in mental retardation.
PLoS Comput. Biol.
PUBLISHED: 03-19-2010
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Copy number variants (CNVs) have recently been recognized as a common form of genomic variation in humans. Hundreds of CNVs can be detected in any individual genome using genomic microarrays or whole genome sequencing technology, but their phenotypic consequences are still poorly understood. Rare CNVs have been reported as a frequent cause of neurological disorders such as mental retardation (MR), schizophrenia and autism, prompting widespread implementation of CNV screening in diagnostics. In previous studies we have shown that, in contrast to benign CNVs, MR-associated CNVs are significantly enriched in genes whose mouse orthologues, when disrupted, result in a nervous system phenotype. In this study we developed and validated a novel computational method for differentiating between benign and MR-associated CNVs using structural and functional genomic features to annotate each CNV. In total 13 genomic features were included in the final version of a Naïve Bayesian Tree classifier, with LINE density and mouse knock-out phenotypes contributing most to the classifiers accuracy. After demonstrating that our method (called GECCO) perfectly classifies CNVs causing known MR-associated syndromes, we show that it achieves high accuracy (94%) and negative predictive value (99%) on a blinded test set of more than 1,200 CNVs from a large cohort of individuals with MR. These results indicate that this classification method will be of value for objectively prioritizing CNVs in clinical research and diagnostics.
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Genome assembly quality: assessment and improvement using the neutral indel model.
Genome Res.
PUBLISHED: 03-19-2010
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We describe a statistical and comparative-genomic approach for quantifying error rates of genome sequence assemblies. The method exploits not substitutions but the pattern of insertions and deletions (indels) in genome-scale alignments for closely related species. Using two- or three-way alignments, the approach estimates the amount of aligned sequence containing clusters of nucleotides that were wrongly inserted or deleted during sequencing or assembly. Thus, the method is well-suited to assessing fine-scale sequence quality within single assemblies, between different assemblies of a single set of reads, and between genome assemblies for different species. When applying this approach to four primate genome assemblies, we found that average gap error rates per base varied considerably, by up to sixfold. As expected, bacterial artificial chromosome (BAC) sequences contained lower, but still substantial, predicted numbers of errors, arguing for caution in regarding BACs as the epitome of genome fidelity. We then mapped short reads, at approximately 10-fold statistical coverage, from a Bornean orangutan onto the Sumatran orangutan genome assembly originally constructed from capillary reads. This resulted in a reduced gap error rate and a separation of error-prone from high-fidelity sequence. Over 5000 predicted indel errors in protein-coding sequence were corrected in a hybrid assembly. Our approach contributes a new fine-scale quality metric for assemblies that should facilitate development of improved genome sequencing and assembly strategies.
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Accelerated evolution of PAK3- and PIM1-like kinase gene families in the zebra finch, Taeniopygia guttata.
Mol. Biol. Evol.
PUBLISHED: 03-17-2010
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Genes encoding protein kinases tend to evolve slowly over evolutionary time, and only rarely do they appear as recent duplications in sequenced vertebrate genomes. Consequently, it was a surprise to find two families of kinase genes that have greatly and recently expanded in the zebra finch (Taeniopygia guttata) lineage. In contrast to other amniotic genomes (including chicken) that harbor only single copies of p21-activated serine/threonine kinase 3 (PAK3) and proviral integration site 1 (PIM1) genes, the zebra finch genome appeared at first to additionally contain 67 PAK3-like (PAK3L) and 51 PIM1-like (PIM1L) protein kinase genes. An exhaustive analysis of these gene models, however, revealed most to be incomplete, owing to the absence of terminal exons. After reprediction, 31 PAK3L genes and 10 PIM1L genes remain, and all but three are predicted, from the retention of functional sites and open reading frames, to be enzymatically active. PAK3L, but not PIM1L, gene sequences show evidence of recurrent episodes of positive selection, concentrated within structures spatially adjacent to N- and C-terminal protein regions that have been discarded from zebra finch PAK3L genes. At least seven zebra finch PAK3L genes were observed to be expressed in testis, whereas two sequences were found transcribed in the brain, one broadly including the song nuclei and the other in the ventricular zone and in cells resembling Bergmanns glia in the cerebellar Purkinje cell layer. Two PIM1L sequences were also observed to be expressed with broad distributions in the zebra finch brain, one in both the ventricular zone and the cerebellum and apparently associated with glial cells and the other showing neuronal cell expression and marked enrichment in midbrain/thalamic nuclei. These expression patterns do not correlate with zebra finch-specific features such as vocal learning. Nevertheless, our results show how ancient and conserved intracellular signaling molecules can be co-opted, following duplication, thereby resulting in lineage-specific functions, presumably affecting the zebra finch testis and brain.
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Long noncoding RNA genes: conservation of sequence and brain expression among diverse amniotes.
Genome Biol.
PUBLISHED: 03-04-2010
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Long considered to be the building block of life, it is now apparent that protein is only one of many functional products generated by the eukaryotic genome. Indeed, more of the human genome is transcribed into noncoding sequence than into protein-coding sequence. Nevertheless, whilst we have developed a deep understanding of the relationships between evolutionary constraint and function for protein-coding sequence, little is known about these relationships for non-coding transcribed sequence. This dearth of information is partially attributable to a lack of established non-protein-coding RNA (ncRNA) orthologs among birds and mammals within sequence and expression databases.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.