Whole genome experimental maps of DNA G-quadruplexes in multiple species.

Genomic maps of DNA G-quadruplexes (G4s) can help elucidate the roles that these secondary structures play in various organisms. Herein, we employ an improved version of a G-quadruplex sequencing method (G4-seq) to generate whole genome G4 maps for 12 species that include widely studied model organisms and also pathogens of clinical relevance. We identify G4 structures that form under physiological K+ conditions and also G4s that are stabilized by the G4-targeting small molecule pyridostatin (PDS). We discuss the various structural features of the experimentally observed G-quadruplexes (OQs), highlighting differences in their prevalence and enrichment across species. Our study describes diversity in sequence composition and genomic location for the OQs in the different species and reveals that the enrichment of OQs in gene promoters is particular to mammals such as mouse and human, among the species studied. The multi-species maps have been made publicly available as a resource to the research community. The maps can serve as blueprints for biological experiments in those model organisms, where G4 structures may play a role.The S.B. research group is supported by programme grant funding from Cancer Research UK (C9681/A18618), European Research Council Advanced Grant No. 339778, a Wellcome Trust Senior Investigator Award (grant 209441/z/17/z) and by core funding from Cancer Research UK (C14303/A17197). We are grateful to the Biotechnology and Biological Sciences Research Council (BBSRC) and Illumina for the CASE studentship supporting V.S.C. (BB/I015477/1)

Learning Interpretable Rules for Multi-label Classification

Multi-label classification (MLC) is a supervised learning problem in which, contrary to standard multiclass classification, an instance can be associated with several class labels simultaneously. In this chapter, we advocate a rule-based approach to multi-label classification. Rule learning algorithms are often employed when one is not only interested in accurate predictions, but also requires an interpretable theory that can be understood, analyzed, and qualitatively evaluated by domain experts. Ideally, by revealing patterns and regularities contained in the data, a rule-based theory yields new insights in the application domain. Recently, several authors have started to investigate how rule-based models can be used for modeling multi-label data. Discussing this task in detail, we highlight some of the problems that make rule learning considerably more challenging for MLC than for conventional classification. While mainly focusing on our own previous work, we also provide a short overview of related work in this area.Comment: Preprint version. To appear in: Explainable and Interpretable Models in Computer Vision and Machine Learning. The Springer Series on Challenges in Machine Learning. Springer (2018). See http://www.ke.tu-darmstadt.de/bibtex/publications/show/3077 for further informatio

A systematic analysis of host factors reveals a Med23-interferon-λ regulatory axis against herpes simplex virus type 1 replication

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. To comprehensively investigate the complex interaction between HSV-1 and its host we combined two genome-scale screens for host factors (HFs) involved in virus replication. A yeast two-hybrid screen for protein interactions and a RNA interference (RNAi) screen with a druggable genome small interfering RNA (siRNA) library confirmed existing and identified novel HFs which functionally influence HSV-1 infection. Bioinformatic analyses found the 358 HFs were enriched for several pathways and multi-protein complexes. Of particular interest was the identification of Med23 as a strongly anti-viral component of the largely pro-viral Mediator complex, which links specific transcription factors to RNA polymerase II. The anti-viral effect of Med23 on HSV-1 replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to HSV-1, as a range of other viruses including Vaccinia virus and Semliki Forest virus were unaffected by Med23 depletion. We found Med23 significantly upregulated expression of the type III interferon family (IFN-λ) at the mRNA and protein level by directly interacting with the transcription factor IRF7. The synergistic effect of Med23 and IRF7 on IFN-λ induction suggests this is the major transcription factor for IFN-λ expression. Genotypic analysis of patients suffering recurrent orofacial HSV-1 outbreaks, previously shown to be deficient in IFN-λ secretion, found a significant correlation with a single nucleotide polymorphism in the IFN-λ3 (IL28b) promoter strongly linked to Hepatitis C disease and treatment outcome. This paper describes a link between Med23 and IFN-λ, provides evidence for the crucial role of IFN-λ in HSV-1 immune control, and highlights the power of integrative genome-scale approaches to identify HFs critical for disease progression and outcome

Mutation analysis of CBP and PCAF reveals rare inactivating mutations in cancer cell lines but not in primary tumours

In this study we screened the histone acetyltransferases CBP and PCAF for mutations in human epithelial cancer cell lines and primary tumours. We identified two CBP truncations (both in cell lines), seven PCAF missense variants and four CBP intronic microdeletions. These data suggest that neither gene is commonly inactivated in human epithelial cancers

Single-genome analysis reveals heterogeneous association of the herpes simplex virus genome with H3K27me2 and the reader PHF20L1 following infection of human fibroblasts

The fate of herpesvirus genomes following entry into different cell types is thought to regulate the outcome of infection. For the Herpes simplex virus 1 (HSV-1), latent infection of neurons is characterized by association with repressive heterochromatin marked with Polycomb silencing-associated lysine 27 methylation on histone H3 (H3K27me). However, whether H3K27 methylation plays a role in repressing lytic gene expression in non-neuronal cells is unclear. To address this gap in knowledge, and with consideration that the fate of the viral genome and outcome of HSV-1 infection could be heterogeneous, we developed an assay to quantify the abundance of histone modifications within single viral genome foci of infected fibroblasts. Using this approach, combined with bulk epigenetic techniques, we were unable to detect any role for H3K27me3 during HSV-1 lytic infection of fibroblasts. By contrast, we could detect the lesser studied H3K27me2 on a subpopulation of viral genomes, which was consistent with a role for H3K27 demethylases in promoting lytic gene expression. In addition, viral genomes co-localized with the H3K27me2 reader protein PHF20L1, and this association was enhanced by inhibition of the H3K27 demethylases UTX and JMJD3. Notably, targeting of H3K27me2 to viral genomes was enhanced following infection with a transcriptionally defective virus in the absence of Promyelocytic leukemia nuclear bodies. Collectively, these studies implicate a role for H3K27me2 in fibroblast-associated HSV genome silencing in a manner dependent on genome sub-nuclear localization and transcriptional activity

Disruption of PML Nuclear Bodies Is Mediated by ORF61 SUMO-Interacting Motifs and Required for Varicella-Zoster Virus Pathogenesis in Skin

Promyelocytic leukemia protein (PML) has antiviral functions and many viruses encode gene products that disrupt PML nuclear bodies (PML NBs). However, evidence of the relevance of PML NB modification for viral pathogenesis is limited and little is known about viral gene functions required for PML NB disruption in infected cells in vivo. Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes cutaneous lesions during primary and recurrent infection. Here we show that VZV disrupts PML NBs in infected cells in human skin xenografts in SCID mice and that the disruption is achieved by open reading frame 61 (ORF61) protein via its SUMO-interacting motifs (SIMs). Three conserved SIMs mediated ORF61 binding to SUMO1 and were required for ORF61 association with and disruption of PML NBs. Mutation of the ORF61 SIMs in the VZV genome showed that these motifs were necessary for PML NB dispersal in VZV-infected cells in vitro. In vivo, PML NBs were highly abundant, especially in basal layer cells of uninfected skin, whereas their frequency was significantly decreased in VZV-infected cells. In contrast, mutation of the ORF61 SIMs reduced ORF61 association with PML NBs, most PML NBs remained intact and importantly, viral replication in skin was severely impaired. The ORF61 SIM mutant virus failed to cause the typical VZV lesions that penetrate across the basement membrane into the dermis and viral spread in the epidermis was limited. These experiments indicate that VZV pathogenesis in skin depends upon the ORF61-mediated disruption of PML NBs and that the ORF61 SUMO-binding function is necessary for this effect. More broadly, our study elucidates the importance of PML NBs for the innate control of a viral pathogen during infection of differentiated cells within their tissue microenvironment in vivo and the requirement for a viral protein with SUMO-binding capacity to counteract this intrinsic barrier

3D Reconstruction of VZV Infected Cell Nuclei and PML Nuclear Cages by Serial Section Array Scanning Electron Microscopy and Electron Tomography

Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level

Ebola Zaire Virus Blocks Type I Interferon Production by Exploiting the Host SUMO Modification Machinery

Ebola Zaire virus is highly pathogenic for humans, with case fatality rates approaching 90% in large outbreaks in Africa. The virus replicates in macrophages and dendritic cells (DCs), suppressing production of type I interferons (IFNs) while inducing the release of large quantities of proinflammatory cytokines. Although the viral VP35 protein has been shown to inhibit IFN responses, the mechanism by which it blocks IFN production has not been fully elucidated. We expressed VP35 from a mouse-adapted variant of Ebola Zaire virus in murine DCs by retroviral gene transfer, and tested for IFN transcription upon Newcastle Disease virus (NDV) infection and toll-like receptor signaling. We found that VP35 inhibited IFN transcription in DCs following these stimuli by disabling the activity of IRF7, a transcription factor required for IFN transcription. By yeast two-hybrid screens and coimmunoprecipitation assays, we found that VP35 interacted with IRF7, Ubc9 and PIAS1. The latter two are the host SUMO E2 enzyme and E3 ligase, respectively. VP35, while not itself a SUMO ligase, increased PIAS1-mediated SUMOylation of IRF7, and repressed Ifn transcription. In contrast, VP35 did not interfere with the activation of NF-κB, which is required for induction of many proinflammatory cytokines. Our findings indicate that Ebola Zaire virus exploits the cellular SUMOylation machinery for its advantage and help to explain how the virus overcomes host innate defenses, causing rapidly overwhelming infection to produce a syndrome resembling fulminant septic shock

Human BRCA1-BARD1 ubiquitin ligase activity counters chromatin barriers to DNA resection

The opposing activities of 53BP1 and BRCA1 influence pathway choice of DNA double-strand break repair. How BRCA1 counters the inhibitory effect of 53BP1 on DNA resection and homologous recombination is unknown. Here we identify the site of BRCA1-BARD1 required for priming ubiquitin transfer from E2~ubiquitin. We demonstrate that BRCA1-BARD1’s ubiquitin ligase activity is required for repositioning 53BP1 on damaged chromatin. We confirm H2A ubiquitylation by BRCA1-BARD1 and show that an H2A-ubiquitin fusion protein promotes DNA resection and repair in BARD1 deficient cells. We show BRCA1-BARD1 function in homologous recombination requires the chromatin remodeler SMARCAD1. SMARCAD1 binding to H2A-ubiquitin, optimal localization to sites of damage and activity in DNA repair requires its ubiquitin-binding CUE domains. SMARCAD1 is required for 53BP1 repositioning and the need for SMARCAD1 in Olaparib or camptothecin resistance is alleviated by 53BP1 loss. Thus BRCA1- BARD1 ligase activity and subsequent SMARCAD1-dependent chromatin remodeling are critical regulators of DNA repair