Project description:The durability of interferon signaling in human aortic endothelial activation was tested. Pro-adhesive and costimulatory gene expression, phenotype, secretome and JAK/STAT phosphorylation in human primary endothelial cells were measured under chronic and transient IFNγ stimulation, with various JAK inhibitors.
Project description:While it is clear that T cell derived IFNγ has to act on tumor stroma cells for rejection of solid tumors, it is not clear which tumor stroma cells are targets. We studied how IFNγ affects gene expression in tumor blood vessels in vivo. To study the effect on endothelial cells, we either used a model of ectopic IFNγ (MCA313 tumors) or IFNγ-GFP fusion protein (J558L tumors) expression in tumors, or we used T cell derived IFNγ in large vascularized 16.113 tumours. Tumors were grown in mice that were expressing the IFNγ receptor ubiquitously (J558L tumors + IFNγ-GFP treatment and 16.113 tumors + T cell treatment) or in some experiments the IFNγ-receptor was expressed exclusively in endothelial cells (MCA313 tumor + IFNγ treatment). We compared RNA expression profiles from mouse endothelial cells exposed to IFNγ (IFNγ-GFP) or not exposed to IFNγ (IFNγ-GFP). Endothlial cells were drived from tumors (or in one experimantal situation -J558L- as a control also from kidney)
Project description:To confirm the effect of Bcl6 on the liver sex difference, we performed the gene expression analysis in male and female wild type mice and male and female liver specific Bcl-6 deficient mice.
Project description:To confirm the effect of Bcl6 on the liver sex differrence, we performed the gene expression analysis in male and female wile type mice and male and female liver specific Bcl-6 deficient mice.
Project description:This experiment tested whether pretreatment of human microvascular endothelial cells (HMECs) with interferon-gamma (IFNγ) for 2h affected the transcriptional response to 1h exposure to interleukin-6 (IL-6).
Project description:Cytokine activation of cells induces gene networks involved in inflammation and immunity. Transient gene activation can have a lasting impact even in the absence of ongoing transcription, known as long-term transcriptional memory. Here we explore the nature of the establishment and maintenance of IFNγ-induced priming of human cells. We find that while both ongoing transcription and local chromatin signatures are short-lived, the IFNγ-primed state stably propagates through at least 14 cell division cycles. Single cell analysis reveals that memory is manifested by an increased probability of primed cells to engage in target gene expression, correlating with the strength of initial gene activation. Further, we find that strongly memorized genes tend to reside in genomic clusters and that long-term memory of these genes is locally restricted by cohesin. We define the duration, stochastic nature and molecular mechanisms of IFNγ-induced transcriptional memory, relevant to understanding of enhanced innate immune signaling. This SuperSeries is composed of the SubSeries listed below.
Project description:The cytokine IFNγ differentially impacts on tumors upon immune checkpoint blockade (ICB). Despite our understanding of downstream signaling events, less is known about 36 regulation of its receptor (IFNγ-R1). With an unbiased genome-wide CRISPR/Cas9 screen for critical regulators of IFNγ-R1 cell surface abundance, we identified STUB1 as an E3 ubiquitin ligase for IFNγ-R1 in complex with its signal-relaying kinase JAK1. STUB1 mediates ubiquitination-dependent proteasomal degradation of IFNγ-R1/JAK1 complex through IFNγ-R1K285 and JAK1K249. Conversely, STUB1 inactivation amplifies IFNγ signaling, sensitizing tumor cells to cytotoxic T cells in vitro. This was corroborated by an anticorrelation between STUB1 expression and IFNγ response in ICB-treated patients. Consistent with the context-dependent effects of IFNγ in vivo, anti-PD-1 response was increased in heterogenous tumors comprising both wildtype and STUB1-deficient cells but not full STUB1 knockout tumors. These results uncover STUB1 as a critical regulator of IFNγ-R1, and highlight the context-dependency of STUB1-regulated IFNγ signaling for ICB outcome.
Project description:Most small-molecule protein degraders act as interface stabilizers ‘molecular glues’ between E3 ubiquitin ligases and target proteins to induce ternary complex formation and ubiquitin-dependent target protein degradation. Here we report polymerization as a novel mechanism for small molecule-induced degradation. Using functional screens in combination with molecular and biochemical assays, we found that BI-3802, which binds to the BTB domain of the oncogenic transcription factor BCL6, induces polymerization of BCL6 into regular helical structures in vitro and foci in vivo. Polymerization precedes degradation by the SIAH1 E3 ubiquitin ligase. Hereby, a VxP amino acid motif on BCL6, distal from the drug-binding BTB domain, is required for SIAH1 binding, ubiquitination and BI-3802-induced degradation. Our findings propose that small molecule-induced polymerization is not only a new modality for targeted protein degradation, but also provides synthetic biology with a tool for tunable protein polymerization and opens new avenues for future drug design.