Lana Schaffer

Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes

Vaccinia virus is the prototypic orthopoxvirus and was the vaccine used to eradicate smallpox, yet the expression profiles of many of its genes remain unknown. Using a genome tiling array approach, we simultaneously measured the expression levels of all 223 annotated vaccinia virus genes during infection and determined their kinetics. For 95% of these genes, significant transcript levels were detected. Most remarkably, classification of the genes by their expression profiles revealed 35 genes exhibiting immediate-early expression. Although a similar kinetic class has been described for other virus families, to our knowledge, this is the first demonstration of its existence in orthopoxviruses. Despite expression levels higher than for genes in the other three kinetic classes, the functions of more than half of these remain unknown. Additionally, genes within each kinetic class were spatially grouped together in the genome. This genome-wide picture of transcription alters our understanding of how orthopoxviruses regulate gene expression.

Selectin ligand sialyl-Lewis x antigen drives metastasis of hormone-dependent breast cancers

The glycome acts as an essential interface between cells and the surrounding microenvironment. However, changes in glycosylation occur in nearly all breast cancers, which can alter this interaction. Here, we report that profiles of glycosylation vary between ER-positive and ER-negative breast cancers. We found that genes involved in the synthesis of sialyl-Lewis x (sLe(x); FUT3, FUT4, and ST3GAL6) are significantly increased in estrogen receptor alpha-negative (ER-negative) tumors compared with ER-positive ones. SLe(x) expression had no influence on the survival of patients whether they had ER-negative or ER-positive tumors. However, high expression of sLe(x) in ER-positive tumors was correlated with metastasis to the bone where sLe(x) receptor E-selectin is constitutively expressed. The ER-positive ZR-75-1 and the ER-negative BT20 cell lines both express sLe(x) but only ZR-75-1 cells could adhere to activated endothelial cells under dynamic flow conditions in a sLe(x) and E-selectin-dependent manner. Moreover, L/P-selectins bound strongly to ER-negative MDA-MB-231 and BT-20 cell lines in a heparan sulfate (HS)-dependent manner that was independent of sLe(x) expression. Expression of glycosylation genes involved in heparan biosynthesis (EXT1 and HS3ST1) was increased in ER-negative tumors. Taken together, our results suggest that the context of sLe(x) expression is important in determining its functional significance and that selectins may promote metastasis in breast cancer through protein-associated sLe(x) and HS glycosaminoglycans.

Regulated and aberrant glycosylation modulate cardiac electrical signaling

Millions afflicted with Chagas disease and other disorders of aberrant glycosylation suffer symptoms consistent with altered electrical signaling such as arrhythmias, decreased neuronal conduction velocity, and hyporeflexia. Cardiac, neuronal, and muscle electrical signaling is controlled and modulated by changes in voltage-gated ion channel activity that occur through physiological and pathological processes such as development, epilepsy, and cardiomyopathy. Glycans attached to ion channels alter channel activity through isoform-specific mechanisms. Here we show that regulated and aberrant glycosylation modulate cardiac ion channel activity and electrical signaling through a cell-specific mechanism. Data show that nearly half of 239 glycosylation-associated genes (glycogenes) were significantly differentially expressed among neonatal and adult atrial and ventricular myocytes. The N-glycan structures produced among cardiomyocyte types were markedly variable. Thus, the cardiac glycome, defined as the complete set of glycan structures produced in the heart, is remodeled. One glycogene, ST8sia2, a polysialyltransferase, is expressed only in the neonatal atrium. Cardiomyocyte electrical signaling was compared in control and ST8sia2(−/−) neonatal atrial and ventricular myocytes. Action potential waveforms and gating of less sialylated voltage-gated Na+ channels were altered consistently in ST8sia2(−/−) atrial myocytes. ST8sia2 expression had no effect on ventricular myocyte excitability. Thus, the regulated (between atrium and ventricle) and aberrant (knockout in the neonatal atrium) expression of a single glycogene was sufficient to modulate cardiomyocyte excitability. A mechanism is described by which cardiac function is controlled and modulated through physiological and pathological processes that involve regulated and aberrant glycosylation.

Noncoding transcription within the Igh distal V(H) region at PAIR elements affects the 3D structure of the Igh locus in pro-B cells.

Noncoding sense and antisense germ-line transcription within the Ig heavy chain locus precedes V(D)J recombination and has been proposed to be associated with Igh locus accessibility, although its precise role remains elusive. However, no global analysis of germ-line transcription throughout the Igh locus has been done. Therefore, we performed directional RNA-seq, demonstrating the locations and extent of both sense and antisense transcription throughout the Igh locus. Surprisingly, the majority of antisense transcripts are localized around two Pax5-activated intergenic repeat (PAIR) elements in the distal IghV region. Importantly, long-distance loops measured by chromosome conformation capture (3C) are observed between these two active PAIR promoters and Eμ, the start site of Iμ germ-line transcription, in a lineage- and stage-specific manner, even though this antisense transcription is Eμ-independent. YY1−/− pro-B cells are greatly impaired in distal VH gene rearrangement and Igh locus compaction, and we demonstrate that YY1 deficiency greatly reduces antisense transcription and PAIR-Eμ interactions. ChIP-seq shows high level YY1 binding only at Eμ, but low levels near some antisense promoters. PAIR–Eμ interactions are not disrupted by DRB, which blocks transcription elongation without disrupting transcription factories once they are established, but the looping is reduced after heat-shock treatment, which disrupts transcription factories. We propose that transcription-mediated interactions, most likely at transcription factories, initially compact the Igh locus, bringing distal VH genes close to the DJH rearrangement which is adjacent to Eμ. Therefore, we hypothesize that one key role of noncoding germ-line transcription is to facilitate locus compaction, allowing distal VH genes to undergo efficient rearrangement.

Rad3ATR Decorates Critical Chromosomal Domains with γH2A to Protect Genome Integrity during S-Phase in Fission Yeast

Schizosaccharomyces pombe Rad3 checkpoint kinase and its human ortholog ATR are essential for maintaining genome integrity in cells treated with genotoxins that damage DNA or arrest replication forks. Rad3 and ATR also function during unperturbed growth, although the events triggering their activation and their critical functions are largely unknown. Here, we use ChIP-on-chip analysis to map genomic loci decorated by phosphorylated histone H2A (γH2A), a Rad3 substrate that establishes a chromatin-based recruitment platform for Crb2 and Brc1 DNA repair/checkpoint proteins. Unexpectedly, γH2A marks a diverse array of genomic features during S-phase, including natural replication fork barriers and a fork breakage site, retrotransposons, heterochromatin in the centromeres and telomeres, and ribosomal RNA (rDNA) repeats. γH2A formation at the centromeres and telomeres is associated with heterochromatin establishment by Clr4 histone methyltransferase. We show that γH2A domains recruit Brc1, a factor involved in repair of damaged replication forks. Brc1 C-terminal BRCT domain binding to γH2A is crucial in the absence of Rqh1Sgs1, a RecQ DNA helicase required for rDNA maintenance whose human homologs are mutated in patients with Werner, Bloom, and Rothmund–Thomson syndromes that are characterized by cancer-predisposition or accelerated aging. We conclude that Rad3 phosphorylates histone H2A to mobilize Brc1 to critical genomic domains during S-phase, and this pathway functions in parallel with Rqh1 DNA helicase in maintaining genome integrity.

Localization of Smc5/6 to centromeres and telomeres requires heterochromatin and SUMO, respectively.

The Smc5/6 holocomplex executes key functions in genome maintenance that include ensuring the faithful segregation of chromosomes at mitosis and facilitating critical DNA repair pathways. Smc5/6 is essential for viability and therefore, dissecting its chromosome segregation and DNA repair roles has been challenging. We have identified distinct epigenetic and post‐translational modifications that delineate roles for fission yeast Smc5/6 in centromere function, versus replication fork‐associated DNA repair. We monitored Smc5/6 subnuclear and genomic localization in response to different replicative stresses, using fluorescence microscopy and chromatin immunoprecipitation (ChIP)‐on‐chip methods. Following hydroxyurea treatment, and during an unperturbed S phase, Smc5/6 is transiently enriched at the heterochromatic outer repeats of centromeres in an H3‐K9 methylation‐dependent manner. In contrast, methyl methanesulphonate treatment induces the accumulation of Smc5/6 at subtelomeres, in an Nse2 SUMO ligase‐dependent, but H3‐K9 methylation‐independent manner. Finally, we determine that Smc5/6 loads at all genomic tDNAs, a phenomenon that requires intact consensus TFIIIC‐binding sites in the tDNAs.

Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins

BackgroundLipopolysaccharide (LPS), also referred to as endotoxin, is the major constituent of the outer leaflet of the outer membrane of virtually all Gram-negative bacteria. The lipid A moiety, which anchors the LPS molecule to the outer membrane, acts as a potent agonist for Toll-like receptor 4/myeloid differentiation factor 2-mediated pro-inflammatory activity in mammals and, thus, represents the endotoxic principle of LPS. Recombinant proteins, commonly manufactured in Escherichia coli, are generally contaminated with endotoxin. Removal of bacterial endotoxin from recombinant therapeutic proteins is a challenging and expensive process that has been necessary to ensure the safety of the final product.ResultsAs an alternative strategy for common endotoxin removal methods, we have developed a series of E. coli strains that are able to grow and express recombinant proteins with the endotoxin precursor lipid IVA as the only LPS-related molecule in their outer membranes. Lipid IVA does not trigger an endotoxic response in humans typical of bacterial LPS chemotypes. Hence the engineered cells themselves, and the purified proteins expressed within these cells display extremely low endotoxin levels.ConclusionsThis paper describes the preparation and characterization of endotoxin-free E. coli strains, and demonstrates the direct production of recombinant proteins with negligible endotoxin contamination.

ATP-sensitive potassium channels mediate survival during infection in mammals and insects

Specific homeostatic mechanisms confer stability in innate immune responses, preventing injury or death from infection. Here we identify, from a screen of N-ethyl-N-nitrosourea–mutagenized mice, a mutation causing both profound susceptibility to infection by mouse cytomegalovirus and ∼20,000-fold sensitization to lipopolysaccharide (LPS), poly(I·C) and immunostimulatory (CpG) DNA. The LPS hypersensitivity phenotype is not suppressed by mutations in Myd88, Trif, Tnf, Tnfrsf1a, Ifnb, Ifng or Stat1, genes contributing to LPS responses, and results from an abnormality extrinsic to hematopoietic cells. The phenotype is due to a null allele of Kcnj8, encoding Kir6.1, a protein that combines with SUR2 to form an ATP-sensitive potassium channel (KATP) expressed in coronary artery smooth muscle and endothelial cells. In Drosophila melanogaster, suppression of dSUR by RNA interference similarly causes hypersensitivity to infection by flock house virus. Thus, KATP evolved to serve a homeostatic function during infection, and in mammals it prevents coronary artery vasoconstriction induced by cytokines dependent on TLR and/or MDA5 immunoreceptors.

Peracetylated 4-fluoro-glucosamine reduces the content and repertoire of N- and O-glycans without direct incorporation.

Prior studies have shown that treatment with the peracetylated 4-fluorinated analog of glucosamine (4-F-GlcNAc) elicits anti-skin inflammatory activity by ablating N-acetyllactosamine (LacNAc), sialyl Lewis X (sLeX), and related lectin ligands on effector leukocytes. Based on anti-sLeX antibody and lectin probing experiments on 4-F-GlcNAc-treated leukocytes, it was hypothesized that 4-F-GlcNAc inhibited sLeX formation by incorporating into LacNAc and blocking the addition of galactose or fucose at the carbon 4-position of 4-F-GlcNAc. To test this hypothesis, we determined whether 4-F-GlcNAc is directly incorporated into N- and O-glycans released from 4-F-GlcNAc-treated human sLeX (+) T cells and leukemic KG1a cells. At concentrations that abrogated galectin-1 (Gal-1) ligand and E-selectin ligand expression and related LacNAc and sLeX structures, MALDI-TOF and MALDI-TOF/TOF mass spectrometry analyses showed that 4-F-GlcNAc 1) reduced content and structural diversity of tri- and tetra-antennary N-glycans and of O-glycans, 2) increased biantennary N-glycans, and 3) reduced LacNAc and sLeX on N-glycans and on core 2 O-glycans. Moreover, MALDI-TOF MS did not reveal any m/z ratios relating to the presence of fluorine atoms, indicating that 4-F-GlcNAc did not incorporate into glycans. Further analysis showed that 4-F-GlcNAc treatment had minimal effect on expression of 1200 glycome-related genes and did not alter the activity of LacNAc-synthesizing enzymes. However, 4-F-GlcNAc dramatically reduced intracellular levels of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), a key precursor of LacNAc synthesis. These data show that Gal-1 and E-selectin ligand reduction by 4-F-GlcNAc is not caused by direct 4-F-GlcNAc glycan incorporation and consequent chain termination but rather by interference with UDP-GlcNAc synthesis.

Reciprocal patterns of methylation of H3K36 and H3K27 on proximal vs. distal IgVH genes are modulated by IL-7 and Pax5

The usage of >100 functional murine Ig heavy chain VH genes, when rearranged to DHJH genes, generates a diverse antibody repertoire. The VH locus encompasses 2.5 Mb, and rearrangement of VH genes in the DH-distal half of the locus are controlled very differently from the VH genes in the proximal end of the locus. The rearrangement of distal but not proximal VH genes is impaired in mice deficient in the cytokine IL-7 or its receptor, in the transcription factor Pax5, or in Ezh2, a histone methyltransferase for Lys-27 of histone H3 (H3K27). The relative role of IL-7, Pax5, and Ezh2 in regulating distal vs. proximal VH rearrangement is not clear. Here, we show by ChIP and ChIP-on-chip that the active histone modification H3K36me2 is most highly associated with distal VH segments and the repressive histone modification H3K27me3 is exclusively present on proximal VH segments. We observed an absence of H3K27me3 in fetal pro-B cells, which predominantly rearrange proximal VH genes. Absence of IL-7 signaling reduces H3K36me2, and overexpression of IL-7 increases H3K36me2. In contrast, the major effect of the absence of Pax5 is the reduction in H3K27me3. Our data indicate that the cytokine IL-7 and the transcription factor Pax5 influence the rearrangement of the two regions of the VH locus by differentially modulating two reciprocal histone modifications during B lymphocyte development.