Resolving the 3D landscape of transcription-linked mammalian chromatin folding, bioRxiv, 2019-05-17
ABSTRACTChromatin folding below the scale of topologically associating domains (TADs) remains largely unexplored in mammals. Here, we used a high-resolution 3C-based method, Micro-C, to probe links between 3D-genome organization and transcriptional regulation in mouse stem cells. Combinatorial binding of transcription factors, cofactors, and chromatin modifiers spatially segregate TAD regions into “microTADs” with distinct regulatory features. Enhancer-promoter and promoter-promoter interactions extending from the edge of these domains predominantly link co-regulated loci, often independently of CTCFCohesin. Acute inhibition of transcription disrupts the gene-related folding features without altering higher-order chromatin structures. Intriguingly, we detect “two-start” zig-zag 30-nanometer chromatin fibers. Our work uncovers the finer-scale genome organization that establishes novel functional links between chromatin folding and gene regulation.ONE SENTENCE SUMMARYTranscriptional regulatory elements shape 3D genome architecture of microTADs.
biorxiv genomics 100-200-users 2019Tracking of antibiotic resistance transfer and rapid plasmid evolution in a hospital setting by Nanopore sequencing, bioRxiv, 2019-05-17
AbstractBackgroundInfection of patients with multidrug-resistant (MDR) bacteria often leave very limited or no treatment options. The transfer of antimicrobial resistance genes (ARG) carrying plasmids between bacterial species by horizontal gene transfer represents an important mode of expansion of ARGs. Here, we evaluated the application of Nanopore sequencing technology in a hospital setting for monitoring the transfer and rapid evolution of antibiotic resistance plasmids within and across multiple species.ResultsIn 2009 we experienced an outbreak with an extensively multidrug resistant P. aeruginosa harboring the carbapenemase enzyme blaIMP-8, and in 2012 the first Citrobacter freundii and Citrobacter werkmanii harboring the same enzyme were detected. Using Nanopore and Illumina sequencing we conducted a comparative analysis of all blaIMP-8 bacteria isolated in our hospital over a 6-year period (n = 54). We developed the computational platforms pathoLogic and plasmIDent for Nanopore-based characterization of clinical isolates and monitoring of ARG transfer, comprising de-novo assembly of genomes and plasmids, polishing, QC, plasmid circularization, ARG annotation, comparative genome analysis of multiple isolates and visualization of results. Using plasmIDent we identified a 40 kb plasmid carrying blaIMP-8 in P. aeruginosa and C. freundii, verifying that plasmid transfer had occurred. Within C. freundii the plasmid underwent further evolution and plasmid fusion, resulting in a 164 kb mega-plasmid, which was transferred to C. werkmanii. Moreover, multiple rearrangements of the multidrug resistance gene cassette were detected in P. aeruginosa, including deletions and translocations of complete ARGs.ConclusionPlasmid transfer, plasmid fusion and rearrangement of the multidrug resistance gene cassette mediated the rapid evolution of opportunistic pathogens in our hospital. We demonstrated the feasibility of tracking plasmid evolution dynamics and ARG transfer in clinical settings in a timely manner. The approach will allow for successful countermeasures to contain not only clonal, but also plasmid mediated outbreaks.
biorxiv genomics 100-200-users 2019Ultrastructural details of mammalian chromosome architecture, bioRxiv, 2019-05-17
ABSTRACTOver the past decade, 3C-related methods, complemented by increasingly detailed microscopic views of the nucleus, have provided unprecedented insights into chromosome folding in vivo. Here, to overcome the resolution limits inherent to the majority of genome-wide chromosome architecture mapping studies, we extend a recently-developed Hi-C variant, Micro-C, to map chromosome architecture at nucleosome resolution in human embryonic stem cells and fibroblasts. Micro-C maps robustly capture well-described features of mammalian chromosome folding including AB compartment organization, topologically associating domains (TADs), and cis interaction peaks anchored at CTCF binding sites, while also providing a detailed 1-dimensional map of nucleosome positioning and phasing genome-wide. Compared to high-resolution in situ Hi-C, Micro-C exhibits substantially improved signal-to-noise with an order of magnitude greater dynamic range, enabling not only localization of domain boundaries with single-nucleosome accuracy, but also resolving more than 20,000 additional looping interaction peaks in each cell type. Intriguingly, many of these newly-identified peaks are localized along stripe patterns and form transitive grids, consistent with their anchors being pause sites impeding the process of cohesin-dependent loop extrusion. Together, our analyses provide the highest resolution maps of chromosome folding in human cells to date, and provide a valuable resource for studies of chromosome folding mechanisms.
biorxiv genomics 100-200-users 2019Actin chromobody imaging reveals sub-organellar actin dynamics, bioRxiv, 2019-05-16
AbstractThe actin cytoskeleton plays multiple critical roles in cells, from cell migration to organelle dynamics. The small and transient actin structures regulating organelle dynamics are difficult to detect with fluorescence microscopy. We developed an approach using fluorescent protein-tagged actin nanobodies targeted to organelle membranes to enable live cell imaging of sub-organellar actin dynamics with unprecedented spatiotemporal resolution. These probes reveal that ER-associated actin drives fission of multiple organelles including mitochondria, endosomes, lysosomes, peroxisomes, and the Golgi.<jatsfig id=ufig1 position=float fig-type=figure orientation=portrait><jatsgraphic xmlnsxlink=httpwww.w3.org1999xlink xlinkhref=639278v2_ufig1 position=float orientation=portrait >
biorxiv cell-biology 100-200-users 2019Chromatin accessibility established by Pou5f3, Sox19b and Nanog primes genes for activity during zebrafish genome activation, bioRxiv, 2019-05-16
ABSTRACTIn many organisms, early embryonic development is driven by maternally provided factors until the controlled onset of transcription during zygotic genome activation. The regulation of chromatin accessibility and its relationship to gene activity during this transition remains poorly understood. Here, we generated chromatin accessibility maps from genome activation until the onset of lineage specification. During this period, chromatin accessibility increases at regulatory elements. This increase is independent of RNA polymerase II-mediated transcription, with the exception of the hyper-transcribed miR-430 locus. Instead, accessibility often precedes the transcription of associated genes. Loss of the maternal transcription factors Pou5f3, Sox19b, and Nanog, which are known to be required for zebrafish genome activation, results in decreased accessibility at regulatory elements. Importantly, the accessibility of regulatory regions, especially when established by Pou5f3, Sox19b and Nanog, is predictive for future transcription. Our results show that the maternally provided transcription factors Pou5f3, Sox19b, and Nanog open up chromatin and prime genes for activity during zygotic genome activation in zebrafish.
biorxiv developmental-biology 100-200-users 2019Gene knock-ins in Drosophila using homology-independent insertion of universal donor plasmids, bioRxiv, 2019-05-16
AbstractSite-specific insertion of DNA into endogenous genes (knock-in) is a powerful method to study gene function. However, traditional methods for knock-in require laborious cloning of long homology arms for homology-directed repair. Here, we report a simplified method in Drosophila melanogaster to insert large DNA elements into any gene using homology-independent repair. This method, known as CRISPaint, employs CRISPR-Cas9 and non-homologous end joining (NHEJ) to linearize and insert donor plasmid DNA into a target genomic cut site. The inclusion of commonly used elements such as GFP on donor plasmids makes them universal, abolishing the need to create gene-specific homology arms and greatly reducing user workload. Using this method, we show robust gene-specific integration of donor plasmids in cultured cells and the fly germ line. Furthermore, we use this method to analyze gene function by fluorescently tagging endogenous proteins, disrupting gene function, and generating reporters of gene expression. Finally, we assemble a collection of donor plasmids for germ line knock-in that contain commonly used insert sequences. This method simplifies the generation of site-specific large DNA insertions in Drosophila cell lines and fly strains, and better enables researchers to dissect gene function in vivo.SummaryWe report a new homology-independent genomic knock-in method in Drosophila to insert large DNA elements into any target gene. Using CRISPR-Cas9 and non-homologous end joining (NHEJ), an entire donor plasmid is inserted into the genome without the need for homology arms. This approach eliminates the burden associated with designing and constructing traditional donor plasmids. We demonstrate its usefulness in cultured cells and in vivo to fluorescently tag endogenous proteins, generate reporters of gene expression, and disrupt gene function.
biorxiv genetics 0-100-users 2019