Project description:Immunoproteasomes are specialized multiprotein proteases, that degrade intracellular proteins thereby generating antigenic peptides for HLA class I antigen presentation. Mutations in immunoproteasomal genes are associated with systemic autoinflammatory diseases. We identified a newborn with severe T lymphopenia and found a de novo heterozygous PSMB10 p.Gly209Arg variant. Molecular dynamics analysis predicted and biochemical studies confirmed the variant to impact assembly and function of the immunoproteasome resulting in impaired cytokine responses to interferons. Skin fibroblasts obtained from the patient (PSMB10G209R) and a healthy individual of comparative age (PSMB10WT) were treated with or without 75U/ml recombinant Human IFN-γ. 5 different cell passages for independent treatments to assess significance of alterations. RNA was isolated from cells to investigate the alteration in IFN response between PSMB10WT and PSMB10G209R. Skin fibroblasts obtained from the patient (PSMB10G209R) and a healthy individual of comparative age (PSMB10WT) were treated with or without 75U/ml recombinant Human IFN-γ. 5 different cell passages for independent treatments to assess significance of alterations. RNA was isolated from cells to investigate the difference of IFN response between PSMB10WT and PSMB10G209R.
Project description:We performed RNA sequencing to assess changes in gene expression in lung cancer cell lines. Total RNA was extracted from the cells, before and after stimulation with IFNγ, using standard approaches. RNA-sequencing data performed in this work was performed and analyzed at the CNAG-CRG.
Project description:PRC2-isogenic human malignant peripheral nerve sheath tumor (MPNST) M3 cells were generated through CRISPR/Cas9-mediated knockout of the PRC2 core component, SUZ12. PRC2 loss led to not only significant increase, but also significant decrease of chromatin accessibility at 15,346 (16% of all ATAC peaks) and 20,099 genomic loci (21% of all ATAC peaks), respectively. PRC2 loss decreased the chromatin accessibility for IFNγ-responsive loci in M3 cells, resulting in dampened response to IFNγ stimulation.
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:chromatin accessibility (ATAC-seq) experiment. HeLa cells were primed with IFNγ for 24 hours, followed by IFNγ washout. After 48h, naïve and primed cells were induced by IFNγ for 1h and 3h. Cells were harvested at indicated time points and processed for ATAC-seq.
Project description:Random monoallelic expression is defined by the allele-specific expression of genes, and by the fact that for an individual cell this monoallelic expression is neither obligate nor necessarily coordinated with the allelic expression in other cells. In order to find novel examples of random monoallelic expression in mouse, we did a transcriptome-wide survey of allele-specific gene expression in two different immortalized cell types. Lymphoblast cell lines and fibroblast cell lines were established (both clonal and nonclonal) and were used as a source of both nuclear RNA and genomic DNA. These samples were assessed for allele-specific gene expression using a custom-designed Mouse SNP Chip. A large number of genes (over 10% of those that were assessed in lymphoblast clones) displayed random monoallelic expression. For each cell line, two replicate samples of ds-cDNA were assessed for monoallelic expression, while genomic DNA was assessed as a control for possible LOH events. Nonclonal samples were used as controls for cis-acting allelic bias.
Project description:STAT1 and IRF1 transcription factor enrichment by CUT&RUN. HeLa cells were primed with IFNγ for 24 hours, followed with IFNγ washout. After 48h, naïve and primed cells were induced by IFNγ for 1h and 3h. Cells were harvested at indicated time points and processed for CUT&RUN