Imaging Poliovirus Entry in Live Cells

Viruses initiate infection by transferring their genetic material across a cellular membrane and into the appropriate compartment of the cell. The mechanisms by which animal viruses, especially nonenveloped viruses, deliver their genomes are only poorly understood. This is due in part to technical difficulties involved in direct visualization of viral gene delivery and to uncertainties in distinguishing productive and nonproductive pathways caused by the high particle-to–plaque forming unit ratio of most animal viruses. Here, we combine an imaging assay that simultaneously tracks the viral capsid and genome in live cells with an infectivity-based assay for RNA release to characterize the early events in the poliovirus (PV) infection. Effects on RNA genome delivery from inhibitors of cell trafficking pathways were probed systematically by both methods. Surprisingly, we observe that genome release by PV is highly efficient and rapid, and thus does not limit the overall infectivity or the infection rate. The results define a pathway in which PV binds to receptors on the cell surface and enters the cell by a clathrin-, caveolin-, flotillin-, and microtubule-independent, but tyrosine kinase- and actin-dependent, endocytic mechanism. Immediately after the internalization of the virus particle, genome release takes place from vesicles or tightly sealed membrane invaginations located within 100–200 nm of the plasma membrane. These results settle a long-lasting debate of whether PV directly breaks the plasma membrane barrier or relies on endocytosis to deliver its genome into the cell. We expect this imaging assay to be broadly applicable to the investigation of entry mechanisms for nonenveloped viruses

Drosophila Carrying Pex3 or Pex16 Mutations Are Models of Zellweger Syndrome That Reflect Its Symptoms Associated with the Absence of Peroxisomes

The peroxisome biogenesis disorders (PBDs) are currently difficult-to-treat multiple-organ dysfunction disorders that result from the defective biogenesis of peroxisomes. Genes encoding Peroxins, which are required for peroxisome biogenesis or functions, are known causative genes of PBDs. The human peroxin genes PEX3 or PEX16 are required for peroxisomal membrane protein targeting, and their mutations cause Zellweger syndrome, a class of PBDs. Lack of understanding about the pathogenesis of Zellweger syndrome has hindered the development of effective treatments. Here, we developed potential Drosophila models for Zellweger syndrome, in which the Drosophila pex3 or pex16 gene was disrupted. As found in Zellweger syndrome patients, peroxisomes were not observed in the homozygous Drosophila pex3 mutant, which was larval lethal. However, the pex16 homozygote lacking its maternal contribution was viable and still maintained a small number of peroxisome-like granules, even though PEX16 is essential for the biosynthesis of peroxisomes in humans. These results suggest that the requirements for pex3 and pex16 in peroxisome biosynthesis in Drosophila are different, and the role of PEX16 orthologs may have diverged between mammals and Drosophila. The phenotypes of our Zellweger syndrome model flies, such as larval lethality in pex3, and reduced size, shortened longevity, locomotion defects, and abnormal lipid metabolisms in pex16, were reminiscent of symptoms of this disorder, although the Drosophila pex16 mutant does not recapitulate the infant death of Zellweger syndrome. Furthermore, pex16 mutants showed male-specific sterility that resulted from the arrest of spermatocyte maturation. pex16 expressed in somatic cyst cells but not germline cells had an essential role in the maturation of male germline cells, suggesting that peroxisome-dependent signals in somatic cyst cells could contribute to the progression of male germ-cell maturation. These potential Drosophila models for Zellweger syndrome should contribute to our understanding of its pathology

The peroxisome: still a mysterious organelle

More than half a century of research on peroxisomes has revealed unique features of this ubiquitous subcellular organelle, which have often been in disagreement with existing dogmas in cell biology. About 50 peroxisomal enzymes have so far been identified, which contribute to several crucial metabolic processes such as β-oxidation of fatty acids, biosynthesis of ether phospholipids and metabolism of reactive oxygen species, and render peroxisomes indispensable for human health and development. It became obvious that peroxisomes are highly dynamic organelles that rapidly assemble, multiply and degrade in response to metabolic needs. However, many aspects of peroxisome biology are still mysterious. This review addresses recent exciting discoveries on the biogenesis, formation and degradation of peroxisomes, on peroxisomal dynamics and division, as well as on the interaction and cross talk of peroxisomes with other subcellular compartments. Furthermore, recent advances on the role of peroxisomes in medicine and in the identification of novel peroxisomal proteins are discussed

Coordinate Regulation of DNA Methylation and H3K27me3 in Mouse Embryonic Stem Cells

<div><p>Chromatin is separated into functional domains distinguished by combinatorial patterns of post-translational histone modifications and DNA methylation. Recent studies examining multiple histone modifications have found numerous chromatin states with distinct profiles of chromatin marks and functional enrichments. There are data showing coordinate regulation between DNAme and H3K27me3, which are both involved in the establishment and maintenance of epigenetic gene silencing, but the data are conflicting. Multiple studies have presented evidence to support the theory that PRC2 and DNAme cooperate to achieve silencing, or alternatively that H3K27me3 and DNAme act antagonistically. Here we examine the effect loss of either PRC2 or DNA methyltransferase activity has on the placement of the reciprocal mark in mouse ES cells. We find that DNAme is acting globally to antagonize the placement of H3K27me3, in accordance with recently published results. At least 471,011 domains in the mouse genome acquire H3K27me3 when DNAme is diminished. Of these 466,563 have been shown to be fully methylated in wildtype ES cells, indicating the effects of DNAme on H3K27me3 are direct. In a reciprocal experiment, we examine the effect loss of PRC2 has on the placement of DNAme. In contrast to the global antagonism DNAme has on the placement of H3K27me3, loss of H3K27me3 has a modest effect on DNAme, with only 4% of genes undergoing changes in DNAme, including 861 showing increases and 552 showing losses of overall DNAme. We anticipate that integrating genomic datasets where the effect of loss of a particular epigenetic mark has on the placement of other marks will help elucidate the rules governing epigenetic regulation and what role coordinate regulation of epigenetic marks plays in development and disease.</p> </div

Loss of PRC2 activity leads to changes in DNA methylation. a,

<p>Relative fluorescence ratios for each probe from three independent MeDIP-chip experiments across the Nkx2-1 promoter. The peak of increased DNA methylation is indicated under the probes (grey bar) and the first 1 kb of the gene is indicated on the bottom. <b>b,</b> Validation of the peak of increased DNA methylation by bisulfite PCR. Each line represents an individual clone. Methylated CpGs are indicated by filled-in circles. <b>c,</b> A Fisher’s exact test was conducted for each CpG in (b) (** p<.01, *** p<.001). <b>d,</b> Profile of average DNA methylation relative to TSS calculated in 100 bp bins. <b>e,</b> Hierarchical clustering was performed on MeDIP-chip enrichment profiles to identify genes with similar profiles. 1,282 genes that passed the filtering step of the clustering software are on the y-axis. The x-axis is based on average fluorescent ratios in 100 bp bins from −3 kb (left) to +1 kb (right). Red indicates increased DNA methylation and green indicates decreased DNA methylation while black indicates unchanged DNA methylation. <b>f,</b> Gene ontology classifications for genes with increased (red) or decreased (green) DNAme. <b>g,</b> Gene ontology classifications for genes with increased DNAme upstream of the TSS (cluster 1, white) or across the entire promoter (cluster 2, grey). <b>h,</b> Classification of promoters based on CpG content. HCP, ICP & LCP, High-, Intermediate- & low CpG content promoter. <b>i,</b> Classification of promoters based on presence of H3K27me3 & H3K4me3. CpG and bivalent data used in (h) and (i) from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053880#pone.0053880-Mikkelsen1" target="_blank">[5] </a><b>j,</b> Boxplot of expression level change for genes enriched or depleted for DNAme in <i>Eed^−/−</i> cells as well as for each of the two clusters described in (e).</p

Global antagonism to H3K27me3 in <i>Dnmt^TKO</i> cells. a,

<p>Classification of promoters identified in ChIP-seq experiment based on presence of H3K27me3 and H3K4me3 in wildtype cells. H3K4 and H3K27 methylation data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053880#pone.0053880-Mikkelsen1" target="_blank">[5]</a>. <b>b,</b> Profile of enrichment of ChIP-seq tags in 100 bp bins across the promoter for all genes with or without peaks of increased H3K27me3 in <i>Dnmt^TKO</i> cells. <b>c,</b> Distribution of ChIP-seq reads according to genomic features. <b>d,</b> Number of ChIP-seq peaks intersecting with either fully-, low- or unmethylated regions according to data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053880#pone.0053880-Stadler1" target="_blank">[26]</a>. <b>e,</b> Expression level of <i>Eed</i> in v6.5 and <i>Dnmt^TKO</i> cells by qRT-PCR. <b>f,</b> Western blot analysis of EZH2 in v6.5 and <i>Dnmt^TKO</i> cells. Relative intensity of EZH2 band from calculated using ImageJ is shown on the bottom. Intensity levels of EZH2 are normalized to Tubulin. <b>h,</b> Boxplot of expression level change for genes enriched in H3K27me3 in <i>Dnmt^TKO</i> cells.</p

<i>Eed^−/−</i> and <i>Dnmt^TKO</i> cells have similar gene expression changes relative to wildtype cells by RNA-seq.

<p><b>a,</b> Number of genes in <i>Dnmt^TKO</i> and <i>Eed^−/−</i> cells with significant changes in expression relative to wildtype cells. <b>b,</b> Boxplot of mean fold change in expression level relative to wildtype. <b>c,</b> Venn diagram showing number of genes with significant expression level changes common to both <i>Eed^−/−</i> and <i>Dnmt^TKO</i> cells. Significance of common genes determined by chi-square test, df = 1 (p<.0001). <b>d,</b> Gene ontology analysis of genes commonly misregulated in both <i>Eed^−/−</i> and <i>Dnmt^TKO</i> cells. <b>e,</b> Classification of genes commonly misregulated in <i>Dnmt^TKO</i> and <i>Eed^−/−</i> cells based on promoter CpG content, or H3K4me3 and H3K27me3 marks. Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053880#pone.0053880-Mikkelsen1" target="_blank">[5]</a>.</p

High contiguity Arabidopsis thaliana genome assembly with a single nanopore flow cell

The handheld Oxford Nanopore MinION sequencer generates ultra-long reads with minimal cost and time requirements, which makes sequencing genomes at the bench feasible. Here, we sequence the gold standard Arabidopsis thaliana genome (KBS-Mac-74 accession) on the bench with the MinION sequencer, and assemble the genome using typical consumer computing hardware (4 Cores, 16 Gb RAM) into chromosome arms (62 contigs with an N50 length of 12.3 Mb). We validate the contiguity and quality of the assembly with two independent single-molecule technologies, Bionano optical genome maps and Pacific Biosciences Sequel sequencing. The new A. thaliana KBS-Mac-74 genome enables resolution of a quantitative trait locus that had previously been recalcitrant to a Sanger-based BAC sequencing approach. In summary, we demonstrate that even when the purpose is to understand complex structural variation at a single region of the genome, complete genome assembly is becoming the simplest way to achieve this goal

The complex architecture and epigenomic impact of plant T-DNA insertions.

The bacterium Agrobacterium tumefaciens has been the workhorse in plant genome engineering. Customized replacement of native tumor-inducing (Ti) plasmid elements enabled insertion of a sequence of interest called Transfer-DNA (T-DNA) into any plant genome. Although these transfer mechanisms are well understood, detailed understanding of structure and epigenomic status of insertion events was limited by current technologies. Here we applied two single-molecule technologies and analyzed Arabidopsis thaliana lines from three widely used T-DNA insertion collections (SALK, SAIL and WISC). Optical maps for four randomly selected T-DNA lines revealed between one and seven insertions/rearrangements, and the length of individual insertions from 27 to 236 kilobases. De novo nanopore sequencing-based assemblies for two segregating lines partially resolved T-DNA structures and revealed multiple translocations and exchange of chromosome arm ends. For the current TAIR10 reference genome, nanopore contigs corrected 83% of non-centromeric misassemblies. The unprecedented contiguous nucleotide-level resolution enabled an in-depth study of the epigenome at T-DNA insertion sites. SALK_059379 line T-DNA insertions were enriched for 24nt small interfering RNAs (siRNA) and dense cytosine DNA methylation, resulting in transgene silencing via the RNA-directed DNA methylation pathway. In contrast, SAIL_232 line T-DNA insertions are predominantly targeted by 21/22nt siRNAs, with DNA methylation and silencing limited to a reporter, but not the resistance gene. Additionally, we profiled the H3K4me3, H3K27me3 and H2A.Z chromatin environments around T-DNA insertions using ChIP-seq in SALK_059379, SAIL_232 and five additional T-DNA lines. We discovered various effect s ranging from complete loss of chromatin marks to the de novo incorporation of H2A.Z and trimethylation of H3K4 and H3K27 around the T-DNA integration sites. This study provides new insights into the structural impact of inserting foreign fragments into plant genomes and demonstrates the utility of state-of-the-art long-range sequencing technologies to rapidly identify unanticipated genomic changes