Gilgi Friedlander

Macrophage-Restricted Interleukin-10 Receptor Deficiency, but Not IL-10 Deficiency, Causes Severe Spontaneous Colitis.

Interleukin-10 (IL-10) is a pleiotropic anti-inflammatory cytokine produced and sensed by most hematopoietic cells. Genome-wide association studies and experimental animal models point at a central role of the IL-10 axis in inflammatory bowel diseases. Here we investigated the importance of intestinal macrophage production of IL-10 and their IL-10 exposure, as well as the existence of an IL-10-based autocrine regulatory loop in the gut. Specifically, we generated mice harboring IL-10 or IL-10 receptor (IL-10Rα) mutations in intestinal lamina propria-resident chemokine receptor CX3CR1-expressing macrophages. We found macrophage-derived IL-10 dispensable for gut homeostasis and maintenance of colonic T regulatory cells. In contrast, loss of IL-10 receptor expression impaired the critical conditioning of these monocyte-derived macrophages and resulted in spontaneous development of severe colitis. Collectively, our results highlight IL-10 as a critical homeostatic macrophage-conditioning agent in the colon and define intestinal CX3CR1(hi) macrophages as a decisive factor that determines gut health or inflammation.

Sphingolipid Depletion Increases Formation of the Scrapie Prion Protein in Neuroblastoma Cells Infected with Prions

Sphingolipid-rich rafts play an essential role in the posttranslational (Borchelt, D. R., Scott, M., Taraboulos, A., Stahl, N., and Prusiner, S. B. (1990) J. Cell Biol. 110, 743–752)) formation of the scrapie prion protein PrPSc from its normal conformer PrPC(Taraboulos, A., Scott, M., Semenov, A., Avrahami, D., Laszlo, L., Prusiner, S. B., and Avraham, D. (1995) J. Cell Biol. 129, 121–132). We investigated the importance of sphingolipids in the metabolism of the PrP isoforms in scrapie-infected ScN2a cells. The ceramide synthase inhibitor fumonisin B1(FB1) reduced both sphingomyelin (SM) and ganglioside GM1 in cells by up to 50%, whereas PrPSc increased by 3–4-fold. Whereas FB1 profoundly altered the cell lipid composition, the raft residents PrPC, PrPSc, caveolin 1, and GM1 remained insoluble in Triton X-100. Metabolic radiolabeling demonstrated that PrPC production was either unchanged or slightly reduced in FB1-treated cells, whereas PrPSc formation was augmented by 3–4-fold. To identify the sphingolipid species the decrease of which correlates with increased PrPSc, we used two other reagents. When cells were incubated with sphingomyelinase for 3 days, SM levels decreased, GM1 was unaltered, and PrPSc increased by 3–4-fold. In contrast, the glycosphingolipid inhibitor PDMP reduced PrPSc while increasing SM. Thus, PrPSc seems to correlate inversely with SM levels. The effects of SM depletion contrasted with those previously obtained with the cholesterol inhibitor lovastatin, which reduced PrPSc and removed it from detergent-insoluble complexes.

Failure of the Tomato Trans-Acting Short Interfering RNA Program to Regulate AUXIN RESPONSE FACTOR3 and ARF4 Underlies the Wiry Leaf Syndrome

Tomato mutants defective in ta-siRNA production have narrow shoestring leaves reminiscent of virus-infected plants. The tomato leaf phenotype is due to deregulated ARF gene expression, but ectopic expression of the same ARF genes in related species fails to recapitulate these developmental defects, analogous to species-specific viral infection symptoms. Interfering with small RNA production is a common strategy of plant viruses. A unique class of small RNAs that require microRNA and short interfering (siRNA) biogenesis for their production is termed trans-acting short interfering RNAs (ta-siRNAs). Tomato (Solanum lycopersicum) wiry mutants represent a class of phenotype that mimics viral infection symptoms, including shoestring leaves that lack leaf blade expansion. Here, we show that four WIRY genes are involved in siRNA biogenesis, and in their corresponding mutants, levels of ta-siRNAs that regulate AUXIN RESPONSE FACTOR3 (ARF3) and ARF4 are reduced, while levels of their target ARFs are elevated. Reducing activity of both ARF3 and ARF4 can rescue the wiry leaf lamina, and increased activity of either can phenocopy wiry leaves. Thus, a failure to negatively regulate these ARFs underlies tomato shoestring leaves. Overexpression of these ARFs in Arabidopsis thaliana, tobacco (Nicotiana tabacum), and potato (Solanum tuberosum) failed to produce wiry leaves, suggesting that the dramatic response in tomato is exceptional. As negative regulation of orthologs of these ARFs by ta-siRNA is common to land plants, we propose that ta-siRNA levels serve as universal sensors for interference with small RNA biogenesis, and changes in their levels direct species-specific responses.

Global Aspects of pacC Regulation of Pathogenicity Genes in Colletotrichum gloeosporioides as Revealed by Transcriptome Analysis

Colletotrichum gloeosporioides alkalinizes its surroundings during colonization of host tissue. The transcription factor pacC is a regulator of pH-controlled genes and is essential for successful colonization. We present here the sequence assembly of the Colletotrichum fruit pathogen and use it to explore the global regulation of pathogenicity by ambient pH. The assembled genome size was 54 Mb, encoding 18,456 genes. Transcriptomes of the wild type and ΔpacC mutant were established by RNA-seq and explored for their global pH-dependent gene regulation. The analysis showed that pacC upregulates 478 genes and downregulates 483 genes, comprising 5% of the fungal genome, including transporters, antioxidants, and cell-wall-degrading enzymes. Interestingly, gene families with similar functionality are both up- and downregulated by pacC. Global analysis of secreted genes showed significant pacC activation of degradative enzymes at alkaline pH and during fruit infection. Select genes from alkalizing-type pathogen C. gloeosporioides and from acidifying-type pathogen Sclerotinia sclerotiorum were verified by quantitative reverse-transcription polymerase chain reaction analysis at different pH values. Knock out of several pacC-activated genes confirmed their involvement in pathogenic colonization of alkalinized surroundings. The results suggest a global regulation by pacC of key pathogenicity genes during pH change in alkalinizing and acidifying pathogens.

Modulation of the transcription regulatory program in yeast cells committed to sporulation.

BackgroundMeiosis in budding yeast is coupled to the process of sporulation, where the four haploid nuclei are packaged into a gamete. This differentiation process is characterized by a point of transition, termed commitment, when it becomes independent of the environment. Not much is known about the mechanisms underlying commitment, but it is often assumed that positive feedback loops stabilize the underlying gene-expression cascade.ResultsWe describe the gene-expression program of committed cells. Sporulating cells were transferred back to growth medium at different stages of the process, and their transcription response was characterized. Most sporulation-induced genes were immediately downregulated upon transfer, even in committed cells that continued to sporulate. Focusing on the metabolic-related transcription response, we observed that pre-committed cells, as well as mature spores, responded to the transfer to growth medium in essentially the same way that vegetative cells responded to glucose. In contrast, committed cells elicited a dramatically different response.ConclusionOur results suggest that cells ensure commitment to sporulation not by stabilizing the process, but by modulating their gene-expression program in an active manner. This unique transcriptional program may optimize sporulation in an environment-specific manner.

The Transcription and Translation Landscapes during Human Cytomegalovirus Infection Reveal Novel Host-Pathogen Interactions.

Viruses are by definition fully dependent on the cellular translation machinery, and develop diverse mechanisms to co-opt this machinery for their own benefit. Unlike many viruses, human cytomegalovirus (HCMV) does suppress the host translation machinery, and the extent to which translation machinery contributes to the overall pattern of viral replication and pathogenesis remains elusive. Here, we combine RNA sequencing and ribosomal profiling analyses to systematically address this question. By simultaneously examining the changes in transcription and translation along HCMV infection, we uncover extensive transcriptional control that dominates the response to infection, but also diverse and dynamic translational regulation for subsets of host genes. We were also able to show that, at late time points in infection, translation of viral mRNAs is higher than that of cellular mRNAs. Lastly, integration of our translation measurements with recent measurements of protein abundance enabled comprehensive identification of dozens of host proteins that are targeted for degradation during HCMV infection. Since targeted degradation indicates a strong biological importance, this approach should be applicable for discovering central host functions during viral infection. Our work provides a framework for studying the contribution of transcription, translation and degradation during infection with any virus.

A Metabolic Gene Cluster in the Wheat W1 and the Barley Cer-cqu Loci Determines β-Diketone Biosynthesis and Glaucousness

A metabolic gene cluster underlying the known glaucousness loci W1 in wheat and Cer-cqu in barley establishes a novel molecular pathway for β-diketone wax biosynthesis The glaucous appearance of wheat (Triticum aestivum) and barley (Hordeum vulgare) plants, that is the light bluish-gray look of flag leaf, stem, and spike surfaces, results from deposition of cuticular β-diketone wax on their surfaces; this phenotype is associated with high yield, especially under drought conditions. Despite extensive genetic and biochemical characterization, the molecular genetic basis underlying the biosynthesis of β-diketones remains unclear. Here, we discovered that the wheat W1 locus contains a metabolic gene cluster mediating β-diketone biosynthesis. The cluster comprises genes encoding proteins of several families including type-III polyketide synthases, hydrolases, and cytochrome P450s related to known fatty acid hydroxylases. The cluster region was identified in both genetic and physical maps of glaucous and glossy tetraploid wheat, demonstrating entirely different haplotypes in these accessions. Complementary evidence obtained through gene silencing in planta and heterologous expression in bacteria supports a model for a β-diketone biosynthesis pathway involving members of these three protein families. Mutations in homologous genes were identified in the barley eceriferum mutants defective in β-diketone biosynthesis, demonstrating a gene cluster also in the β-diketone biosynthesis Cer-cqu locus in barley. Hence, our findings open new opportunities to breed major cereal crops for surface features that impact yield and stress response.

Runx3-mediated transcriptional program in cytotoxic lymphocytes.

The transcription factor Runx3 is highly expressed in CD8+ T and NK cytotoxic lymphocytes and is required for their effective activation and proliferation but molecular insights into the transcription program regulated by Runx3 in these cells are still missing. Using Runx3-ChIP-seq and transcriptome analysis of wild type vs. Runx3-/- primary cells we have now identified Runx3-regulated genes in the two cell types at both resting and IL-2-activated states. Runx3-bound genomic regions in both cell types were distantly located relative to gene transcription start sites and were enriched for RUNX and ETS motifs. Bound genomic regions significantly overlapped T-bet and p300-bound enhancer regions in Runx3-expressing Th1 helper cells. Compared to resting cells, IL-2-activated CD8+ T and NK cells contain three times more Runx3-regulated genes that are common to both cell types. Functional annotation of shared CD8+ T and NK Runx3-regulated genes revealed enrichment for immune-associated terms including lymphocyte activation, proliferation, cytotoxicity, migration and cytokine production, highlighting the role of Runx3 in CD8+ T and NK activated cells.

Mutant p53 Attenuates the Anti-Tumorigenic Activity of Fibroblasts-Secreted Interferon Beta

Mutations in the p53 tumor suppressor protein are highly frequent in tumors and often endow cells with tumorigenic capacities. We sought to examine a possible role for mutant p53 in the cross-talk between cancer cells and their surrounding stroma, which is a crucial factor affecting tumor outcome. Here we present a novel model which enables individual monitoring of the response of cancer cells and stromal cells (fibroblasts) to co-culturing. We found that fibroblasts elicit the interferon beta (IFNβ) pathway when in contact with cancer cells, thereby inhibiting their migration. Mutant p53 in the tumor was able to alleviate this response via SOCS1 mediated inhibition of STAT1 phosphorylation. IFNβ on the other hand, reduced mutant p53 RNA levels by restricting its RNA stabilizer, WIG1. These data underscore mutant p53 oncogenic properties in the context of the tumor microenvironment and suggest that mutant p53 positive cancer patients might benefit from IFNβ treatment.

Divergent Levels of LBP and TGFβ1 in Murine MSCs Lead to Heterogenic Response to TLR and Proinflammatory Cytokine Activation

The outstanding heterogeneity of stem cell populations is a major obstacle on the way to their clinical application. It is therefore paramount to identify the molecular mechanisms that underlay this heterogeneity. Individually derived bone marrow mesenchymal stromal cells (MSCs) preparations, studied here, diverged markedly in various properties, despite of being all tripotent in their differentiation potential. Microarray analysis showed that MSC diversity is evident also in highly variable gene expression patterns. Differentially expressed genes were significantly enriched in toll-like receptors (TLRs) and differentiation pathways. Marked differences were observed in LPS binding protein (LBP) and transforming growth factor (TGF)β1 expression. These differences correlated with MSC functionality. Therefore, the possible contribution of these molecules to MSC diversity was examined. In the TLR signaling pathway, LBP levels predicted the ability of specific MSCs to secrete interleukin (IL)-6 in response to LPS. A relatively higher expression of TGFβ1 endowed MSCs with a capacity to respond to IL-1β by reduced osteogenic differentiation. This study thus demonstrates major diversity within MSC isolates, which appears early on following derivation and persists following long–term culture. MSC heterogeneity results from highly variable transcriptome. Differential expression of LBP and TGFβ1, along with other genes, in different MSC preparations, produces the variable responses to external stimuli.