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Boettiger, Alistair

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Boettiger

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Alistair

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Boettiger, Alistair
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    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, Xiaowei
    Metazoan 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.
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    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-ting
    Fluorescence 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.
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    Chromatin topology is coupled to Polycomb group protein subnuclear organization
    (Nature Publishing Group, 2016) Wani, Ajazul H.; Boettiger, Alistair; Schorderet, Patrick; Ergun, Ayla; Münger, Christine; Sadreyev, Ruslan; Zhuang, Xiaowei; Kingston, Robert; Francis, Nicole J.
    The genomes of metazoa are organized at multiple scales. Many proteins that regulate genome architecture, including Polycomb group (PcG) proteins, form subnuclear structures. Deciphering mechanistic links between protein organization and chromatin architecture requires precise description and mechanistic perturbations of both. Using super-resolution microscopy, here we show that PcG proteins are organized into hundreds of nanoscale protein clusters. We manipulated PcG clusters by disrupting the polymerization activity of the sterile alpha motif (SAM) of the PcG protein Polyhomeotic (Ph) or by increasing Ph levels. Ph with mutant SAM disrupts clustering of endogenous PcG complexes and chromatin interactions while elevating Ph level increases cluster number and chromatin interactions. These effects can be captured by molecular simulations based on a previously described chromatin polymer model. Both perturbations also alter gene expression. Organization of PcG proteins into small, abundant clusters on chromatin through Ph SAM polymerization activity may shape genome architecture through chromatin interactions.
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    Spatial organization shapes the turnover of a bacterial transcriptome
    (eLife Sciences Publications, Ltd, 2016) Moffitt, Jeffrey; Pandey, Shristi; Boettiger, Alistair; Wang, Siyuan; Zhuang, Xiaowei
    Spatial organization of the transcriptome has emerged as a powerful means for regulating the post-transcriptional fate of RNA in eukaryotes; however, whether prokaryotes use RNA spatial organization as a mechanism for post-transcriptional regulation remains unclear. Here we used super-resolution microscopy to image the E. coli transcriptome and observed a genome-wide spatial organization of RNA: mRNAs encoding inner-membrane proteins are enriched at the membrane, whereas mRNAs encoding outer-membrane, cytoplasmic and periplasmic proteins are distributed throughout the cytoplasm. Membrane enrichment is caused by co-translational insertion of signal peptides recognized by the signal-recognition particle. Time-resolved RNA-sequencing revealed that degradation rates of inner-membrane-protein mRNAs are on average greater that those of the other mRNAs and that this selective destabilization of inner-membrane-protein mRNAs is abolished by dissociating the RNA degradosome from the membrane. Together, these results demonstrate that the bacterial transcriptome is spatially organized and suggest that this organization shapes the post-transcriptional dynamics of mRNAs. DOI: http://dx.doi.org/10.7554/eLife.13065.001