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  • Computational Methods  (4)
  • Protein-nucleic acid interaction, Chromatin and Epigenetics  (4)
  • Oxford University Press  (8)
  • Hindawi
  • International Union of Crystallography (IUC)
  • 2010-2014  (8)
  • 2013  (8)
  • 1
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    TrAp: a tree approach for fingerprinting subclonal tumor composition (2013)
    Oxford University Press
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    Publication Date: 2013-09-26
    Description: Revealing the clonal composition of a single tumor is essential for identifying cell subpopulations with metastatic potential in primary tumors or with resistance to therapies in metastatic tumors. Sequencing technologies provide only an overview of the aggregate of numerous cells. Computational approaches to de-mix a collective signal composed of the aberrations of a mixed cell population of a tumor sample into its individual components are not available. We propose an evolutionary framework for deconvolving data from a single genome-wide experiment to infer the composition, abundance and evolutionary paths of the underlying cell subpopulations of a tumor. We have developed an algorithm (TrAp) for solving this mixture problem. In silico analyses show that TrAp correctly deconvolves mixed subpopulations when the number of subpopulations and the measurement errors are moderate. We demonstrate the applicability of the method using tumor karyotypes and somatic hypermutation data sets. We applied TrAp to Exome-Seq experiment of a renal cell carcinoma tumor sample and compared the mutational profile of the inferred subpopulations to the mutational profiles of single cells of the same tumor. Finally, we deconvolve sequencing data from eight acute myeloid leukemia patients and three distinct metastases of one melanoma patient to exhibit the evolutionary relationships of their subpopulations.
    Keywords: Computational Methods
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 2
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    Reconstitution of hemisomes on budding yeast centromeric DNA (2013)
    Oxford University Press
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    Publication Date: 2013-06-08
    Description: The structure of nucleosomes that contain the cenH3 histone variant has been controversial. In budding yeast, a single right-handed cenH3/H4/H2A/H2B tetramer wraps the ~80-bp Centromere DNA Element II (CDE II) sequence of each centromere into a ‘hemisome’. However, attempts to reconstitute cenH3 particles in vitro have yielded exclusively ‘octasomes’, which are observed in vivo on chromosome arms only when Cse4 (yeast cenH3) is overproduced. Here, we show that Cse4 octamers remain intact under conditions of low salt and urea that dissociate H3 octamers. However, particles consisting of two DNA duplexes wrapped around a Cse4 octamer and separated by a gap efficiently split into hemisomes. Hemisome dimensions were confirmed using a calibrated gel-shift assay and atomic force microscopy, and their identity as tightly wrapped particles was demonstrated by gelFRET. Surprisingly, Cse4 hemisomes were stable in 4 M urea. Stable Cse4 hemisomes could be reconstituted using either full-length or tailless histones and with a 78-bp CDEII segment, which is predicted to be exceptionally stiff. We propose that CDEII DNA stiffness evolved to favor Cse4 hemisome over octasome formation. The precise correspondence between Cse4 hemisomes resident on CDEII in vivo and reconstituted on CDEII in vitro without any other factors implies that CDEII is sufficient for hemisome assembly.
    Keywords: Protein-nucleic acid interaction, Chromatin and Epigenetics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 3
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    Global profiling of miRNAs and the hairpin precursors: insights into miRNA processing and novel miRNA discovery (2013)
    Oxford University Press
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    Publication Date: 2013-04-02
    Description: MicroRNAs (miRNAs) constitute an important class of small regulatory RNAs that are derived from distinct hairpin precursors (pre-miRNAs). In contrast to mature miRNAs, which have been characterized in numerous genome-wide studies of different organisms, research on global profiling of pre-miRNAs is limited. Here, using massive parallel sequencing, we have performed global characterization of both mouse mature and precursor miRNAs. In total, 87 369 704 and 252 003 sequencing reads derived from 887 mature and 281 precursor miRNAs were obtained, respectively. Our analysis revealed new aspects of miRNA/pre-miRNA processing and modification, including eight Ago2-cleaved pre-miRNAs, eight new instances of miRNA editing and exclusively 5' tailed mirtrons. Furthermore, based on the sequences of both mature and precursor miRNAs, we developed a miRNA discovery pipeline, miRGrep, which does not rely on the availability of genome reference sequences. In addition to 239 known mouse pre-miRNAs, miRGrep predicted 41 novel ones with high confidence. Similar as known ones, the mature miRNAs derived from most of these novel loci showed both reduced abundance following Dicer knockdown and the binding with Argonaute2. Evaluation on data sets obtained from Caenorhabditis elegans and Caenorhabditis sp.11 demonstrated that miRGrep could be widely used for miRNA discovery in metazoans, especially in those without genome reference sequences.
    Keywords: Computational Methods
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 4
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    Utilizing sequence intrinsic composition to classify protein-coding and long non-coding transcripts (2013)
    Oxford University Press
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    Publication Date: 2013-09-26
    Description: It is a challenge to classify protein-coding or non-coding transcripts, especially those re-constructed from high-throughput sequencing data of poorly annotated species. This study developed and evaluated a powerful signature tool, Coding-Non-Coding Index (CNCI), by profiling adjoining nucleotide triplets to effectively distinguish protein-coding and non-coding sequences independent of known annotations. CNCI is effective for classifying incomplete transcripts and sense–antisense pairs. The implementation of CNCI offered highly accurate classification of transcripts assembled from whole-transcriptome sequencing data in a cross-species manner, that demonstrated gene evolutionary divergence between vertebrates, and invertebrates, or between plants, and provided a long non-coding RNA catalog of orangutan. CNCI software is available at http://www.bioinfo.org/software/cnci .
    Keywords: Computational Methods
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 5
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    r3Cseq: an R/Bioconductor package for the discovery of long-range genomic interactions from chromosome conformation capture and next-generation sequencing data (2013)
    Oxford University Press
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    Publication Date: 2013-07-16
    Description: The coupling of chromosome conformation capture (3C) with next-generation sequencing technologies enables the high-throughput detection of long-range genomic interactions, via the generation of ligation products between DNA sequences, which are closely juxtaposed in vivo . These interactions involve promoter regions, enhancers and other regulatory and structural elements of chromosomes and can reveal key details of the regulation of gene expression. 3C-seq is a variant of the method for the detection of interactions between one chosen genomic element (viewpoint) and the rest of the genome. We present r3Cseq , an R/Bioconductor package designed to perform 3C-seq data analysis in a number of different experimental designs. The package reads a common aligned read input format, provides data normalization, allows the visualization of candidate interaction regions and detects statistically significant chromatin interactions, thus greatly facilitating hypothesis generation and the interpretation of experimental results. We further demonstrate its use on a series of real-world applications.
    Keywords: Computational Methods
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 6
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    Vika/vox, a novel efficient and specific Cre/loxP-like site-specific recombination system (2013)
    Oxford University Press
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    Publication Date: 2013-01-20
    Description: Targeted genome engineering has become an important research area for diverse disciplines, with site-specific recombinases (SSRs) being among the most popular genome engineering tools. Their ability to trigger excision, integration, inversion and translocation has made SSRs an invaluable tool to manipulate DNA in vitro and in vivo . However, sophisticated strategies that combine different SSR systems are ever increasing. Hence, the demand for additional precise and efficient recombinases is dictated by the increasing complexity of the genetic studies. Here, we describe a novel site-specific recombination system designated Vika/ vox . Vika originates from a degenerate bacteriophage of Vibrio coralliilyticus and shares low sequence similarity to other tyrosine recombinases, but functionally carries out a similar type of reaction. We demonstrate that Vika is highly specific in catalyzing vox recombination without recombining target sites from other SSR systems. We also compare the recombination activity of Vika/ vox with other SSR systems, providing a guideline for deciding on the most suitable enzyme for a particular application and demonstrate that Vika expression does not cause cytotoxicity in mammalian cells. Our results show that Vika/ vox is a novel powerful and safe instrument in the ‘genetic toolbox’ that can be used alone or in combination with other SSRs in heterologous hosts.
    Keywords: Protein-nucleic acid interaction, Chromatin and Epigenetics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 7
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    A multi-parametric flow cytometric assay to analyze DNA-protein interactions (2013)
    Oxford University Press
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    Publication Date: 2013-01-20
    Description: Interactions between DNA and transcription factors (TFs) guide cellular function and development, yet the complexities of gene regulation are still far from being understood. Such understanding is limited by a paucity of techniques with which to probe DNA–protein interactions. We have devised magnetic protein immobilization on enhancer DNA (MagPIE), a simple, rapid, multi-parametric assay using flow cytometric immunofluorescence to reveal interactions among TFs, chromatin structure and DNA. In MagPIE, synthesized DNA is bound to magnetic beads, which are then incubated with nuclear lysate, permitting sequence-specific binding by TFs, histones and methylation by native lysate factors that can be optionally inhibited with small molecules. Lysate protein–DNA binding is monitored by flow cytometric immunofluorescence, which allows for accurate comparative measurement of TF-DNA affinity. Combinatorial fluorescent staining allows simultaneous analysis of sequence-specific TF-DNA interaction and chromatin modification. MagPIE provides a simple and robust method to analyze complex epigenetic interactions in vitro .
    Keywords: Protein-nucleic acid interaction, Chromatin and Epigenetics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 8
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    Elucidating combinatorial histone modifications and crosstalks by coupling histone-modifying enzyme with biotin ligase activity (2013)
    Oxford University Press
    Details
    Publication Date: 2013-02-02
    Description: Histone post-translational modifications (PTMs) often form complex patterns of combinations and cooperate to specify downstream biological processes. In order to systemically analyse combinatorial PTMs and crosstalks among histone PTMs, we have developed a novel nucleosome purification method called B iotinylation-assisted I solation of CO -modified N ucleosomes (BICON). This technique is based on physical coupling of the enzymatic activity of a histone-modifying enzyme with in vivo biotinylation by the biotin ligase BirA, and using streptavidin to purify the co-modified nucleosomes. Analysing the nucleosomes isolated by BICON allows the identification of PTM combinations that are enriched on the modified nucleosomes and function together within the nucleosome context. We used this new approach to study MSK1-mediated H3 phosphorylation and found that MSK1 not only directly phosphorylated H3, but also induced hyperacetylation of both histone H3 and H4 within the nucleosome. Moreover, we identified a novel crosstalk pathway between H3 phosphorylation and H4 acetylation on K12. Involvement of these acetyl marks in MSK1-mediated transcription was further confirmed by chromatin immunoprecipitation assays, thus validating the biological relevance of the BICON results. These studies serve as proof-of-principle for this new technical approach, and demonstrate that BICON can be further adapted to study PTMs and crosstalks associated with other histone-modifying enzymes.
    Keywords: Protein-nucleic acid interaction, Chromatin and Epigenetics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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