Ulf Nehrbass

Frequent ectopic recombination of virulence factor genes in telomeric chromosome clusters of P. falciparum.

Persistent and recurrent infections by Plasmodium falciparum malaria parasites result from the ability of the parasite to undergo antigenic variation and evade host immune attack. P. falciparum parasites generate high levels of variability in gene families that comprise virulence determinants of cytoadherence and antigenic variation, such as the var genes. These genes encode the major variable parasite protein (PfEMP-1), and are expressed in a mutually exclusive manner at the surface of the erythrocyte infected by P. falciparum. Here we identify a mechanism by which var gene sequences undergo recombination at frequencies much higher than those expected from homologous crossover events alone. These recombination events occur between subtelomeric regions of heterologous chromosomes, which associate in clusters near the nuclear periphery in asexual blood-stage parasites or in bouquet-like configurations near one pole of the elongated nuclei in sexual parasite forms. We propose that the alignment of var genes in heterologous chromosomes facilitates gene conversion and promotes the diversity of antigenic and adhesive phenotypes. The association of virulence factors with a specific nuclear subcompartment may also have implications for variation during mitotic recombination in asexual blood stages.

SAGA interacting factors confine sub-diffusion of transcribed genes to the nuclear envelope.

Changes in the transcriptional state of genes have been correlated with their repositioning within the nuclear space. Tethering reporter genes to the nuclear envelope alone can impose repression and recent reports have shown that, after activation, certain genes can also be found closer to the nuclear periphery. The molecular mechanisms underlying these phenomena have remained elusive. Here, with the use of dynamic three-dimensional tracking of a single locus in live yeast (Saccharomyces cerevisiae) cells, we show that the activation of GAL genes (GAL7, GAL10 and GAL1) leads to a confinement in dynamic motility. We demonstrate that the GAL locus is subject to sub-diffusive movement, which after activation can become constrained to a two-dimensional sliding motion along the nuclear envelope. RNA-fluorescence in situ hybridization analysis after activation reveals a higher transcriptional activity for the peripherally constrained GAL genes than for loci remaining intranuclear. This confinement was mediated by Sus1 and Ada2, members of the SAGA histone acetyltransferase complex, and Sac3, a messenger RNA export factor, physically linking the activated GAL genes to the nuclear-pore-complex component Nup1. Deleting ADA2 or NUP1 abrogates perinuclear GAL confinement without affecting GAL1 transcription. Accordingly, transcriptional activation is necessary but not sufficient for the confinement of GAL genes at the nuclear periphery. The observed real-time dynamic mooring of active GAL genes to the inner side of the nuclear pore complex is in accordance with the ‘gene gating’ hypothesis.

Nuclear pore complexes in the organization of silent telomeric chromatin.

The functional regulation of chromatin is closely related to its spatial organization within the nucleus. In yeast, perinuclear chromatin domains constitute areas of transcriptional repression. These ‘silent’ domains are defined by the presence of perinuclear telomere clusters. The only protein found to be involved in the peripheral localization of telomeres is Yku70/Yku80 (ref. 5). This conserved heterodimer can bind telomeres and functions in both repair of DNA double-strand breaks and telomere maintenance. These findings, however, do not address the underlying structural basis of perinuclear silent domains. Here we show that nuclear-pore-complex extensions formed by the conserved TPR homologues Mlp1 and Mlp2 are responsible for the structural and functional organization of perinuclear chromatin. Loss of MLP2 results in a severe deficiency in the repair of double-strand breaks. Furthermore, double deletion of MLP1 and MLP2 disrupts the clustering of perinuclear telomeres and releases telomeric gene repression. These effects are probably mediated through the interaction with Yku70. Mlp2 physically tethers Yku70 to the nuclear periphery, thus forming a link between chromatin and the nuclear envelope. We show, moreover, that this structural link is docked to nuclear-pore complexes through a cleavable nucleoporin, Nup145. We propose that, through these interactions, nuclear-pore complexes organize a nuclear subdomain that is intimately involved in the regulation of chromatin metabolism.

High content screening identifies decaprenyl-phosphoribose 2' epimerase as a target for intracellular antimycobacterial inhibitors.

A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2′ epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials.

Nuclear architecture and spatial positioning help establish transcriptional states of telomeres in yeast.

Recent experiments have shown that gene repression can be correlated with relocation of genes to heterochromatin-rich silent domains. Here, we investigate whether nuclear architecture and spatial positioning can contribute directly to the transcriptional activity of a genetic locus in Saccharomyces cerevisiae. By disassembling telomeric silent domains without altering the chromatin-mediated silencing machinery, we show that the transcriptional activity of silencer–reporter constructs depends on intranuclear position. This demonstrates that telomeric silent domains are actively involved in transcriptional silencing. Employing fluorescent in situ hybridization (FISH) in combination with genetic assays, we demonstrate that telomeres control the establishment of transcriptional states by reversible partitioning with the perinuclear silencing domains. Anchoring telomeres interferes with their ability to assume an active state, whereas disassembly of silencing domains prevents telomeres from assuming a repressed state. Our data support a model in which domains of enriched transcriptional regulators allow genes to determine transcriptional states by spatial positioning.

NSP1: A yeast nuclear envelope protein localized at the nuclear pores exerts its essential function by its carboxy-terminal domain

NSP1 is located at the nuclear periphery in yeast and is essential for cell growth. Employing immunoelectron microscopy on yeast cells, we show that NSP1 is located at the nuclear pores. The molecular analysis of the NSP1 protein points to a two domain model: a nonessential domain (the first 603 amino acids) composed of repetitive sequences common to other nuclear proteins and an essential, carboxy-terminal domain (residues 604-823) mediating the vital function of NSP1. The NSP1 carboxy-terminal domain, which shows a heptad repeat organization, affected the correct location of two nuclear proteins: site-specific amino acid substitutions within a predicted alpha-helical structure of this domain caused a temperature-sensitive growth arrest at 37 degrees C and the appearance of NSP1 and NOP1, a nucleolar protein, in the cytosol.

Yeast Ataxin-7 links histone deubiquitination with gene gating and mRNA export.

Targeting of a gene to the nuclear pore complexes (NPCs), known as gene gating, can affect its transcriptional state. However, the mechanism underlying gene gating is poorly understood. Here, we have identified SAGA-associated Sgf73 (ref. 10), the yeast orthologue of human Ataxin-7 (ref. 11), as a regulator of histone H2B ubiquitin levels, a modification linked to both transcription initiation and elongation. Sgf73 is a key component of a minimal histone-deubiquitinating complex. Activation of the H2B deubiquitinating protease, Ubp8, is cooperative and requires complex formation with the amino-terminal zinc-finger-containing domain of Sgf73 and Sgf11–Sus1. Through a separate domain, Sgf73 mediates recruitment of the TREX-2 mRNA export factors Sac3 and Thp1 to SAGA and their stable interaction with Sus1–Cdc31. This latter step is crucial to target TREX-2 to the NPC. Loss of Sgf73 from SAGA abrogates gene gating of GAL1 and causes a GAL1 mRNA export defect. Thus, Sgf73 provides a molecular scaffold to integrate the regulation of H2B ubiquitin levels, tethering of a gene to the NPC and export of mRNA.

Hmo1 Is Required for TOR-Dependent Regulation of Ribosomal Protein Gene Transcription

ABSTRACT Ribosome biogenesis requires equimolar amounts of four rRNAs and all 79 ribosomal proteins (RP). Coordinated regulation of rRNA and RP synthesis by eukaryotic RNA polymerases (Pol) I, III, and II is a key requirement for growth control. Using a novel global genetic approach, we showed that the absence of Hmo1 becomes lethal when combined with mutations of components of either the RNA Pol II or Pol I transcription machineries, of specific RP, or of the TOR pathway. Hmo1 directly interacts with both the region transcribed by Pol I and a subset of RP gene promoters. Down-regulation of Hmo1 expression affects RP gene expression. Upon TORC1 inhibition, Hmo1 dissociates from ribosomal DNA (rDNA) and some RP gene promoters simultaneously. Finally, in the absence of Hmo1, TOR-dependent repression of RP genes is alleviated. Therefore, we show here that Saccharomyces cerevisiae Hmo1 is directly involved in coordinating rDNA transcription by Pol I and RP gene expression by Pol II under the control of the TOR pathway.

Automated Genome-Wide Visual Profiling of Cellular Proteins Involved in HIV Infection

Recent genome-wide RNAi screens have identified >842 human genes that affect the human immunodeficiency virus (HIV) cycle. The list of genes implicated in infection differs between screens, and there is minimal overlap. A reason for this variance is the interdependence of HIV infection and host cell function, producing a multitude of indirect or pleiotropic cellular effects affecting the viral infection during RNAi screening. To overcome this, the authors devised a 15-dimensional phenotypic profile to define the viral infection block induced by CD4 silencing in HeLa cells. They demonstrate that this phenotypic profile excludes nonspecific, RNAi-based side effects and viral replication defects mediated by silencing of housekeeping genes. To achieve statistical robustness, the authors used automatically annotated RNAi arrays for seven independent genome-wide RNAi screens. This identified 56 host genes, which reliably reproduced CD4-like phenotypes upon HIV infection. The factors include 11 known HIV interactors and 45 factors previously not associated with HIV infection. As proof of concept, the authors confirmed that silencing of PAK1, Ku70, and RNAseH2A impaired HIV replication in Jurkat cells. In summary, multidimensional, visual profiling can identify genes required for HIV infection.

Borna Disease Virus Glycoprotein Is Required for Viral Dissemination in Neurons

ABSTRACT Borna disease virus (BDV) is a nonsegmented negative-strand RNA virus with a tropism for neurons. Infection with BDV causes neurological diseases in a wide variety of animal species. Although it is known that the virus spreads from neuron to neuron, assembled viral particles have never been visualized in the brains of infected animals. This has led to the hypothesis that BDV spreads as nonenveloped ribonucleoproteins (RNP) rather than as enveloped viral particles. We assessed whether the viral envelope glycoprotein (GP) is required for neuronal dissemination of BDV by using primary cultures of rat hippocampal neurons. We show that upon in vitro infection, BDV replicated and spread efficiently in this system. Despite rapid virus dissemination, very few infectious viral particles were detectable in the culture. However, neutralizing antibodies directed against BDV-GP inhibited BDV spread. In addition, interference with BDV-GP processing by inhibiting furin-mediated cleavage of the glycoprotein blocked virus spread. Finally, antisense treatment with peptide nucleic acids directed against BDV-GP mRNA inhibited BDV dissemination, marking BDV-GP as an attractive target for antiviral therapy against BDV. Together, our results demonstrate that the expression and correct processing of BDV-GP are necessary for BDV dissemination in primary cultures of rat hippocampal neurons, arguing against the hypothesis that the virus spreads from neuron to neuron in the form of nonenveloped RNP.