Person: Beliveau, Brian
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Beliveau
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Brian
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Beliveau, Brian
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Publication Germline Progenitors Escape the Widespread Phenomenon of Homolog Pairing during Drosophila Development(Public Library of Science, 2013) Joyce, Eric F.; Apostolopoulos, N; Beliveau, Brian; Wu, C. -tingHomolog pairing, which plays a critical role in meiosis, poses a potential risk if it occurs in inappropriate tissues or between nonallelic sites, as it can lead to changes in gene expression, chromosome entanglements, and loss-of-heterozygosity due to mitotic recombination. This is particularly true in Drosophila, which supports organismal-wide pairing throughout development. Discovered over a century ago, such extensive pairing has led to the perception that germline pairing in the adult gonad is an extension of the pairing established during embryogenesis and, therefore, differs from the mechanism utilized in most species to initiate pairing specifically in the germline. Here, we show that, contrary to long-standing assumptions, Drosophila meiotic pairing in the gonad is not an extension of pairing established during embryogenesis. Instead, we find that homologous chromosomes are unpaired in primordial germ cells from the moment the germline can be distinguished from the soma in the embryo and remain unpaired even in the germline stem cells of the adult gonad. We further establish that pairing originates immediately after the stem cell stage. This pairing occurs well before the initiation of meiosis and, strikingly, continues through the several mitotic divisions preceding meiosis. These discoveries indicate that the spatial organization of the Drosophila genome differs between the germline and the soma from the earliest moments of development and thus argue that homolog pairing in the germline is an active process as versus a passive continuation of pairing established during embryogenesis.Publication Scalable amplification of strand subsets from chip-synthesized oligonucleotide libraries(Nature Pub. Group, 2015) Schmidt, Thorsten L.; Beliveau, Brian; Uca, Yavuz O.; Theilmann, Mark; Da Cruz, Felipe; Wu, Chao-ting; Shih, WilliamSynthetic oligonucleotides are the main cost factor for studies in DNA nanotechnology, genetics and synthetic biology, which all require thousands of these at high quality. Inexpensive chip-synthesized oligonucleotide libraries can contain hundreds of thousands of distinct sequences, however only at sub-femtomole quantities per strand. Here we present a selective oligonucleotide amplification method, based on three rounds of rolling-circle amplification, that produces nanomole amounts of single-stranded oligonucleotides per millilitre reaction. In a multistep one-pot procedure, subsets of hundreds or thousands of single-stranded DNAs with different lengths can selectively be amplified and purified together. These oligonucleotides are used to fold several DNA nanostructures and as primary fluorescence in situ hybridization probes. The amplification cost is lower than other reported methods (typically around US$ 20 per nanomole total oligonucleotides produced) and is dominated by the use of commercial enzymes.Publication Super-resolution imaging reveals distinct chromatin folding for different epigenetic states(2015) Boettiger, Alistair; Bintu, Bogdan; Moffitt, Jeffrey; Wang, Siyuan; Beliveau, Brian; Fudenberg, Geoffrey; Imakaev, Maxim; Mirny, Leonid A.; Wu, Chao-ting; Zhuang, XiaoweiMetazoan genomes are spatially organized at multiple scales, from packaging of DNA around individual nucleosomes to segregation of whole chromosomes into distinct territories1–5. At the intermediate scale of kilobases to megabases, which encompasses the sizes of genes, gene clusters and regulatory domains, the three-dimensional (3D) organization of DNA is implicated in multiple gene regulatory mechanisms2–4,6–8, but understanding this organization remains a challenge. At this scale, the genome is partitioned into domains of different epigenetic states that are essential for regulating gene expression9–11. Here, we investigate the 3D organization of chromatin in different epigenetic states using super-resolution imaging. We classified genomic domains in Drosophila cells into transcriptionally active, inactive, or Polycomb-repressed states and observed distinct chromatin organizations for each state. Remarkably, all three types of chromatin domains exhibit power-law scaling between their physical sizes in 3D and their domain lengths, but each type has a distinct scaling exponent. Polycomb-repressed chromatin shows the densest packing and most intriguing folding behaviour in which packing density increases with domain length. Distinct from the self-similar organization displayed by transcriptionally active and inactive chromatin, the Polycomb-repressed domains are characterized by a high degree of chromatin intermixing within the domain. Moreover, compared to inactive domains, Polycomb-repressed domains spatially exclude neighbouring active chromatin to a much stronger degree. Computational modelling and knockdown experiments suggest that reversible chromatin interactions mediated by Polycomb-group proteins plays an important role in these unique packaging properties of the repressed chromatin. Taken together, our super-resolution images reveal distinct chromatin packaging for different epigenetic states at the kilobase-to-megabase scale, a length scale that is directly relevant to genome regulation.Publication Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes(Nature Pub. Group, 2015) Beliveau, Brian; Boettiger, Alistair; Avendaño, Maier S.; Jungmann, Ralf; McCole, Ruth; Joyce, Eric F.; Kim-Kiselak, Caroline; Bantignies, Frédéric; Fonseka, Chamith; Erceg, Jelena; Hannan, Mohammed; Hoang, Hien G.; Colognori, David; Lee, Jeannie; Shih, William; Yin, Peng; Zhuang, Xiaowei; Wu, Chao-tingFluorescence in situ hybridization (FISH) is a powerful single-cell technique for studying nuclear structure and organization. Here we report two advances in FISH-based imaging. We first describe the in situ visualization of single-copy regions of the genome using two single-molecule super-resolution methodologies. We then introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems. Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals. These advances should substantially expand the capability to query parent-of-origin-specific chromosome positioning and gene expression on a cell-by-cell basis.Publication Oligopaints: Highly Programmable Oligonucleotide Probes for Visualizing Genomes in Situ(2015-01-29) Beliveau, Brian; Gaudet, Suzanne; Seidman, Jonathan; Bosco, GiovanniFluorescence in situ hybridization (FISH) is a powerful assay that can visualize the position of DNA and RNA molecules in individual cells. Here, I describe the development of a method that utilizes complex oligonucleotide (oligo) libraries as a renewable source of FISH probes, which we term ‘Oligopaints’. Our novel FISH platform includes a reliable and robust protocol for the bulk production of fluorescently labeled, strand-specific, single-stranded DNA (ssDNA) probe sets and a bioinformatic pipeline able to identify optimal target sequences for in situ hybridization on a genome-wide scale. A key advantage of Oligopaints is that it permits the researcher to precisely define the genomic sequence contained within each probe molecule, specify the placement of fluorophores, and engineer ssDNA overhangs to which activities can be targeted. We harness this control to make two significant technological advances in FISH- based imaging. In one, Oligopaint probes are programmed to carry 5’ ssDNA overhangs that enable stochastic super-resolution microscopy via two methodologies, STORM and DNA- PAINT. We have used these probes to produce <25 nm resolution images of developmentally regulated chromatin in Drosophila and mouse, which are to our knowledge the first images at this resolution of single-copy chromosomal regions produced by FISH. In the second, we utilize single nucleotide polymorphism (SNP) data to generate FISH probes that can for the first time visually distinguish single-copy regions of the maternal and paternal homologous chromosomes, thus allowing the examination of parent-of-origin dependent effects on chromosome positioning and gene expression in individual cells.Publication Oligopaints: highly efficient, bioinformatically designed probes for fluorescence in situ hybridization(BioMed Central, 2013) Beliveau, Brian; Joyce, Eric F.; Apostolopoulosa, Nicholas; Yilmaza, Feyza; Fonseka, Chamith Y; McCole, Ruth; Chang, Yiming; Li, Jin Billy; Senaratne, Niroshi Niroshini; Williams, Benjamin R; Rouillard, Jean-Marie; Wu, Chao-tingPublication Multiplexed 3D super-resolution imaging of whole cells using spinning disk confocal microscopy and DNA-PAINT(Nature Publishing Group UK, 2017) Schueder, Florian; Lara-Gutiérrez, Juanita; Beliveau, Brian; Saka, Sinem K.; Sasaki, Hiroshi; Woehrstein, Johannes B.; Strauss, Maximilian T.; Grabmayr, Heinrich; Yin, Peng; Jungmann, RalfSingle-molecule localization microscopy (SMLM) can visualize biological targets on the nanoscale, but complex hardware is required to perform SMLM in thick samples. Here, we combine 3D DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) with spinning disk confocal (SDC) hardware to overcome this limitation. We assay our achievable resolution with two- and three-dimensional DNA origami structures and demonstrate the general applicability by imaging a large variety of cellular targets including proteins, DNA and RNA deep in cells. We achieve multiplexed 3D super-resolution imaging at sample depths up to ~10 µm with up to 20 nm planar and 80 nm axial resolution, now enabling DNA-based super-resolution microscopy in whole cells using standard instrumentation.Publication OligoMiner provides a rapid, flexible environment for the design of genome-scale oligonucleotide in situ hybridization probes(National Academy of Sciences, 2018) Beliveau, Brian; Kishi, Jocelyn; Nir, Guy; Sasaki, Hiroshi; Saka, Sinem K.; Nguyen, Son C.; Wu, Chao-ting; Yin, PengOligonucleotide (oligo)-based FISH has emerged as an important tool for the study of chromosome organization and gene expression and has been empowered by the commercial availability of highly complex pools of oligos. However, a dedicated bioinformatic design utility has yet to be created specifically for the purpose of identifying optimal oligo FISH probe sequences on the genome-wide scale. Here, we introduce OligoMiner, a rapid and robust computational pipeline for the genome-scale design of oligo FISH probes that affords the scientist exact control over the parameters of each probe. Our streamlined method uses standard bioinformatic file formats, allowing users to seamlessly integrate new and existing utilities into the pipeline as desired, and introduces a method for evaluating the specificity of each probe molecule that connects simulated hybridization energetics to rapidly generated sequence alignments using supervised machine learning. We demonstrate the scalability of our approach by performing genome-scale probe discovery in numerous model organism genomes and showcase the performance of the resulting probes with diffraction-limited and single-molecule superresolution imaging of chromosomal and RNA targets. We anticipate that this pipeline will make the FISH probe design process much more accessible and will more broadly facilitate the design of pools of hybridization probes for a variety of applications.