Structural chromosome rearrangements may result in the exchange of coding or regulatory DNA sequences between genes. Many such gene fusions are strong driver mutations in neoplasia and have provided ...fundamental insights into the disease mechanisms that are involved in tumorigenesis. The close association between the type of gene fusion and the tumour phenotype makes gene fusions ideal for diagnostic purposes, enabling the subclassification of otherwise seemingly identical disease entities. In addition, many gene fusions add important information for risk stratification, and increasing numbers of chimeric proteins encoded by the gene fusions serve as specific targets for treatment, resulting in dramatically improved patient outcomes. In this Timeline article, we describe the spectrum of gene fusions in cancer and how the methods to identify them have evolved, and also discuss conceptual implications of current, sequencing-based approaches for detection.
Chromatin immunoprecipitation (ChIP-chip and ChIP-seq) assays identify where proteins bind throughout a genome. However, DNA contamination and DNA fragmentation heterogeneity produce false positives ...(erroneous calls) and imprecision in mapping. Consequently, stringent data filtering produces false negatives (missed calls). Here we describe ChIP-exo, where an exonuclease trims ChIP DNA to a precise distance from the crosslinking site. Bound locations are detectable as peak pairs by deep sequencing. Contaminating DNA is degraded or fails to form complementary peak pairs. With the single bp accuracy provided by ChIP-exo, we show an unprecedented view into genome-wide binding of the yeast transcription factors Reb1, Gal4, Phd1, Rap1, and human CTCF. Each of these factors was chosen to address potential limitations of ChIP-exo. We found that binding sites become unambiguous and reveal diverse tendencies governing in vivo DNA-binding specificity that include sequence variants, functionally distinct motifs, motif clustering, secondary interactions, and combinatorial modules within a compound motif.
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► ChIP-exo defines genomic binding locations with few false positives or negatives ► Binding-site complexity is revealed at single-nucleotide resolution ► Low-occupancy sites are reproducible and likely to be functional
ChIP-exo identifies DNA-binding sites across the whole genome at single base-pair resolution, revealing the diverse ways in which transcription factors achieve specificity.
About the Authors: Didier Y. R. Stainier * E-mail: didier.stainier@mpi-bn.mpg.de (DYRS); cmoens@fredhutch.org (CBM) Affiliation: Department of Developmental Genetics, Max Planck Institute for Heart ...and Lung Research, Bad Nauheim, Germany ORCID http://orcid.org/0000-0002-0382-0026 Erez Raz Affiliation: Institute of Cell Biology, ZBME, University of Münster, Münster, Germany ORCID http://orcid.org/0000-0002-6347-3302 Nathan D. Lawson Affiliation: Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America Stephen C. Ekker Affiliation: Mayo Clinic, Rochester, Minnesota, United States of America Rebecca D. Burdine Affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America Judith S. Eisen Affiliation: Institute of Neuroscience, University of Oregon, Eugene, Oregon, United States of America ORCID http://orcid.org/0000-0003-1229-1696 Philip W. Ingham Affiliations Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, The Living Systems Institute, University of Exeter, Exeter, United Kingdom Stefan Schulte-Merker Affiliation: Institute of Cardiovascular Organogenesis and Regeneration, WWU Münster, Faculty of Medicine, Münster, Germany Deborah Yelon Affiliation: Division of Biological Sciences, University of California, San Diego, La Jolla, California, United States of America Brant M. Weinstein Affiliation: Division of Developmental Biology, NICHD, NIH, Bethesda, Maryland, United States of America Mary C. Mullins Affiliation: Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States of America ORCID http://orcid.org/0000-0002-9979-1564 Stephen W. Wilson Affiliation: Department of Cell and Developmental Biology, University College London, London, United Kingdom ORCID http://orcid.org/0000-0002-8557-5940 Lalita Ramakrishnan Affiliation: Molecular Immunity Unit, Department of Medicine, University of Cambridge, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom Sharon L. Amacher Affiliation: Departments of Molecular Genetics and Biological Chemistry and Pharmacology, Ohio State University, Columbus, Ohio, United States of America Stephan C. F. Neuhauss Affiliation: Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland ORCID http://orcid.org/0000-0002-9615-480X Anming Meng Affiliation: School of Life Sciences, Tsinghua University, Beijing, China Naoki Mochizuki Affiliation: National Cerebral and Cardiovascular Center Research Institute, Osaka, Japan ORCID http://orcid.org/0000-0002-3938-9602 Pertti Panula Affiliation: Department of Anatomy and Neuroscience Center, University of Helsinki, Helsinki, Finland Cecilia B. Moens * E-mail: didier.stainier@mpi-bn.mpg.de (DYRS); cmoens@fredhutch.org (CBM) Affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of AmericaCitation: Stainier DYR, Raz E, Lawson ND, Ekker SC, Burdine RD, Eisen JS, et al. Additionally, mutant alleles for many genes are now readily available through zebrafish community resource centers. ...MOs should be used alongside mutant(s) for the corresponding gene. ...a word of caution that previous publication of MOs is not a guarantee of their fidelity, particularly if a new phenotype is being described. ...we hope that these brief and mostly conceptual guidelines will assist scientists working with zebrafish as well as those assessing manuscripts and grant proposals based on experiments using zebrafish.
The world of molecular profiling has undergone revolutionary changes over the last few years as knowledge, technology, and even standard clinical practice have evolved. Broad molecular profiling is ...now nearly essential for all patients with metastatic solid tumors. New agents have been approved based on molecular testing instead of tumor site of origin. Molecular profiling methodologies have likewise changed such that tests that were performed on patients a few years ago are no longer complete and possibly inaccurate today. As with all rapid change, medical providers can quickly fall behind or struggle to find up‐to‐date sources to ensure he or she provides optimum care. In this review, the authors provide the current state of the art for molecular profiling/precision medicine, practice standards, and a view into the future ahead.
Adaptive evolution plays a large role in generating the phenotypic diversity observed in nature, yet current methods are impractical for characterizing the molecular basis and fitness effects of ...large numbers of individual adaptive mutations. Here, we used a DNA barcoding approach to generate the genotype-to-fitness map for adaptation-driving mutations from a Saccharomyces cerevisiae population experimentally evolved by serial transfer under limiting glucose. We isolated and measured the fitness of thousands of independent adaptive clones and sequenced the genomes of hundreds of clones. We found only two major classes of adaptive mutations: self-diploidization and mutations in the nutrient-responsive Ras/PKA and TOR/Sch9 pathways. Our large sample size and precision of measurement allowed us to determine that there are significant differences in fitness between mutations in different genes, between different paralogs, and even between different classes of mutations within the same gene.
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•High-throughput method to survey adaptive mutations in laboratory evolution•Isolation of thousands of independent single adaptive events genome-wide•Bulk fitness measurements and genotyping generates a genotype-to-fitness map•Large sample size allows sensitive detection of fitness differences even within genes
How can the entire suite of adaptive mutations that occur in an evolving population be pinpointed and each of their fitness effects measured?
CTCF and the associated cohesin complex play a central role in insulator function and higher-order chromatin organization of mammalian genomes. Recent studies identified a correlation between the ...orientation of CTCF-binding sites (CBSs) and chromatin loops. To test the functional significance of this observation, we combined CRISPR/Cas9-based genomic-DNA-fragment editing with chromosome-conformation-capture experiments to show that the location and relative orientations of CBSs determine the specificity of long-range chromatin looping in mammalian genomes, using protocadherin (Pcdh) and β-globin as model genes. Inversion of CBS elements within the Pcdh enhancer reconfigures the topology of chromatin loops between the distal enhancer and target promoters and alters gene-expression patterns. Thus, although enhancers can function in an orientation-independent manner in reporter assays, in the native chromosome context, the orientation of at least some enhancers carrying CBSs can determine both the architecture of topological chromatin domains and enhancer/promoter specificity. These findings reveal how 3D chromosome architecture can be encoded by linear genome sequences.
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•The orientation of Pcdh CBSs determines the direction of topological DNA looping•Directional CTCF binding to CBSs is crucial for loop topology and gene expression•The CTCF binding orientation functions similarly in β-globin and the whole genome•CTCF/cohesin-mediated directional DNA-looping determines chromosome architecture
The relative orientations of CTCF binding sites in enhancers and promoters determine the directionality of DNA looping and regulation of gene expression. The findings reveal how chromosome architecture is encoded by genome sequence and provoke thinking about how sequence orientation is functionally translated into a 3D genome.
Abstract
Inferring changes in effective population size (Ne) in the recent past is of special interest for conservation of endangered species and for human history research. Current methods for ...estimating the very recent historical Ne are unable to detect complex demographic trajectories involving multiple episodes of bottlenecks, drops, and expansions. We develop a theoretical and computational framework to infer the demographic history of a population within the past 100 generations from the observed spectrum of linkage disequilibrium (LD) of pairs of loci over a wide range of recombination rates in a sample of contemporary individuals. The cumulative contributions of all of the previous generations to the observed LD are included in our model, and a genetic algorithm is used to search for the sequence of historical Ne values that best explains the observed LD spectrum. The method can be applied from large samples to samples of fewer than ten individuals using a variety of genotyping and DNA sequencing data: haploid, diploid with phased or unphased genotypes and pseudohaploid data from low-coverage sequencing. The method was tested by computer simulation for sensitivity to genotyping errors, temporal heterogeneity of samples, population admixture, and structural division into subpopulations, showing high tolerance to deviations from the assumptions of the model. Computer simulations also show that the proposed method outperforms other leading approaches when the inference concerns recent timeframes. Analysis of data from a variety of human and animal populations gave results in agreement with previous estimations by other methods or with records of historical events.
Sequence-specific control of gene expression on a genome-wide scale is an important approach for understanding gene functions and for engineering genetic regulatory systems. We have recently ...described an RNA-based method, CRISPR interference (CRISPRi), for targeted silencing of transcription in bacteria and human cells. The CRISPRi system is derived from the Streptococcus pyogenes CRISPR (clustered regularly interspaced palindromic repeats) pathway, requiring only the coexpression of a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA). The Cas9-sgRNA complex binds to DNA elements complementary to the sgRNA and causes a steric block that halts transcript elongation by RNA polymerase, resulting in the repression of the target gene. Here we provide a protocol for the design, construction and expression of customized sgRNAs for transcriptional repression of any gene of interest. We also provide details for testing the repression activity of CRISPRi using quantitative fluorescence assays and native elongating transcript sequencing. CRISPRi provides a simplified approach for rapid gene repression within 1-2 weeks. The method can also be adapted for high-throughput interrogation of genome-wide gene functions and genetic interactions, thus providing a complementary approach to RNA interference, which can be used in a wider variety of organisms.
High selectivity and exquisite control over the outcome of reactions entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not ...in nature’s known repertoire. In this Review, we outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progression has been recapitulated in the laboratory starting from extant enzymes. We then examine non‐native enzyme activities that have been exploited for chemical synthesis, with an emphasis on reactions that do not have natural counterparts. Non‐natural activities can be improved by directed evolution, thus mimicking the process used by nature to create new catalysts. Finally, we describe the discovery of non‐native catalytic functions that may provide future opportunities for the expansion of the enzyme universe.
Exploiting hidden talents: The engineering of enzymes to catalyze reactions not known in nature will expand the range of transformations that can be promoted by biocatalysis. This Review presents a common pathway by which new enzyme activities evolve in nature and examples of the use of a similar approach to create enzymes for non‐natural reactions (see picture).
Although studies have identified hundreds of loci associated with human traits and diseases, pinpointing causal alleles remains difficult, particularly for non-coding variants. To address this ...challenge, we adapted the massively parallel reporter assay (MPRA) to identify variants that directly modulate gene expression. We applied it to 32,373 variants from 3,642 cis-expression quantitative trait loci and control regions. Detection by MPRA was strongly correlated with measures of regulatory function. We demonstrate MPRA’s capabilities for pinpointing causal alleles, using it to identify 842 variants showing differential expression between alleles, including 53 well-annotated variants associated with diseases and traits. We investigated one in detail, a risk allele for ankylosing spondylitis, and provide direct evidence of a non-coding variant that alters expression of the prostaglandin EP4 receptor. These results create a resource of concrete leads and illustrate the promise of this approach for comprehensively interrogating how non-coding polymorphism shapes human biology.
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•A new version of MPRA with greater throughput and sensitivity•Evaluation of 32,373 variants associated with eQTLs in lymphoblastoid cell lines•842 variants showed differential gene expression between alleles•Use of CRISPR/cas9 to identify a distal eQTL causal allele for PTGER4
A massively parallel reporter assay analyzes thousands of human polymorphisms to identify alleles that impact gene expression, providing a tool with which to move from disease-associated GWAS hits to the identification of functional variants.
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