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

Impairment of circulating endothelial progenitors in Down syndrome

<p>Abstract</p> <p>Background</p> <p>Pathological angiogenesis represents a critical issue in the progression of many diseases. Down syndrome is postulated to be a systemic anti-angiogenesis disease model, possibly due to increased expression of anti-angiogenic regulators on chromosome 21. The aim of our study was to elucidate some features of circulating endothelial progenitor cells in the context of this syndrome.</p> <p>Methods</p> <p>Circulating endothelial progenitors of Down syndrome affected individuals were isolated, <it>in vitro </it>cultured and analyzed by confocal and transmission electron microscopy. ELISA was performed to measure SDF-1α plasma levels in Down syndrome and euploid individuals. Moreover, qRT-PCR was used to quantify expression levels of <it>CXCL12 </it>gene and of its receptor in progenitor cells. The functional impairment of Down progenitors was evaluated through their susceptibility to hydroperoxide-induced oxidative stress with BODIPY assay and the major vulnerability to the infection with human pathogens. The differential expression of crucial genes in Down progenitor cells was evaluated by microarray analysis.</p> <p>Results</p> <p>We detected a marked decrease of progenitors' number in young Down individuals compared to euploid, cell size increase and some major detrimental morphological changes. Moreover, Down syndrome patients also exhibited decreased SDF-1α plasma levels and their progenitors had a reduced expression of SDF-1α encoding gene and of its membrane receptor. We further demonstrated that their progenitor cells are more susceptible to hydroperoxide-induced oxidative stress and infection with Bartonella henselae. Further, we observed that most of the differentially expressed genes belong to angiogenesis, immune response and inflammation pathways, and that infected progenitors with trisomy 21 have a more pronounced perturbation of immune response genes than infected euploid cells.</p> <p>Conclusions</p> <p>Our data provide evidences for a reduced number and altered morphology of endothelial progenitor cells in Down syndrome, also showing the higher susceptibility to oxidative stress and to pathogen infection compared to euploid cells, thereby confirming the angiogenesis and immune response deficit observed in Down syndrome individuals.</p

Massive-Scale RNA-Seq Analysis of Non Ribosomal Transcriptome in Human Trisomy 21

Hybridization- and tag-based technologies have been successfully used in Down syndrome to identify genes involved in various aspects of the pathogenesis. However, these technologies suffer from several limits and drawbacks and, to date, information about rare, even though relevant, RNA species such as long and small non-coding RNAs, is completely missing. Indeed, none of published works has still described the whole transcriptional landscape of Down syndrome. Although the recent advances in high-throughput RNA sequencing have revealed the complexity of transcriptomes, most of them rely on polyA enrichment protocols, able to detect only a small fraction of total RNA content. On the opposite end, massive-scale RNA sequencing on rRNA-depleted samples allows the survey of the complete set of coding and non-coding RNA species, now emerging as novel contributors to pathogenic mechanisms. Hence, in this work we analysed for the first time the complete transcriptome of human trisomic endothelial progenitor cells to an unprecedented level of resolution and sensitivity by RNA-sequencing. Our analysis allowed us to detect differential expression of even low expressed genes crucial for the pathogenesis, to disclose novel regions of active transcription outside yet annotated loci, and to investigate a plethora of non-polyadenilated long as well as short non coding RNAs. Novel splice isoforms for a large subset of crucial genes, and novel extended untranslated regions for known genes—possibly novel miRNA targets or regulatory sites for gene transcription—were also identified in this study. Coupling the rRNA depletion of samples, followed by high-throughput RNA-sequencing, to the easy availability of these cells renders this approach very feasible for transcriptome studies, offering the possibility of investigating in-depth blood-related pathological features of Down syndrome, as well as other genetic disorders

Genome-Wide Analysis of Factors Affecting Transcription Elongation and DNA Repair: A New Role for PAF and Ccr4-Not in Transcription-Coupled Repair

RNA polymerases frequently deal with a number of obstacles during transcription elongation that need to be removed for transcription resumption. One important type of hindrance consists of DNA lesions, which are removed by transcription-coupled repair (TC-NER), a specific sub-pathway of nucleotide excision repair. To improve our knowledge of transcription elongation and its coupling to TC-NER, we used the yeast library of non-essential knock-out mutations to screen for genes conferring resistance to the transcription-elongation inhibitor mycophenolic acid and the DNA-damaging agent 4-nitroquinoline-N-oxide. Our data provide evidence that subunits of the SAGA and Ccr4-Not complexes, Mediator, Bre1, Bur2, and Fun12 affect transcription elongation to different extents. Given the dependency of TC-NER on RNA Polymerase II transcription and the fact that the few proteins known to be involved in TC-NER are related to transcription, we performed an in-depth TC-NER analysis of a selection of mutants. We found that mutants of the PAF and Ccr4-Not complexes are impaired in TC-NER. This study provides evidence that PAF and Ccr4-Not are required for efficient TC-NER in yeast, unraveling a novel function for these transcription complexes and opening new perspectives for the understanding of TC-NER and its functional interconnection with transcription elongation

Nutritional genomics era: opportunities toward a genome-tailored nutritional regimen

There is increasing evidence indicating that nutritional genomics represents a promise to improve public health. This goal will be reached by highlighting the mechanisms through which diet can reduce the risk of monogenic and common polygenic diseases. Indeed, nutrition is a very relevant environmental factor involved in the development and progression of metabolic disorders, as well as other kind of diseases. The revolutionary changes in the field of genomics have led to the development and implementation of new technologies and molecular tools. These technologies have a useful application in the nutritional sciences, since they allow a more precise and accurate analysis of biochemical alterations, in addition to filling fundamental gaps in the knowledge of nutrient-genome interactions in both health and disease. Overall, these advances will open undiscovered ways in genome-customized diets for disease prevention and therapy. This review summarizes the recent knowledge concerning this novel nutritional approach, paying attention to the human genome variations, such as single-nucleotide polymorphisms and copy number variations, gene expression and innovative molecular tools to reveal them. (C) 2010 Elsevier Inc. All rights reserved