Project description:To identify conserved TNFα-induced changes in chromatin-accessibility in mammals, we performed ATAC-seq in primary vascular endothelial cells (ECs) isolated from the aortas of human (HAEC), mouse (MAEC) and cow (BAEC), before and after TNFα. We overlay our data with multi-species NF-κB binding data and identify multiple modes of NF-κB-chromatin interactions that are conserved during mammalian TNFα response. Our cross-species approach identifies conserved changes in chromatin-accessibility at NF-κB binding sites that are disease-relevant and essential during mammalian acute inflammation.
Project description:Regulatory B cells (Breg) play a critical role in the control of autoimmunity and inflammation. Although IL-10 is considered the hallmark for the identification of Bregs, the molecular programme that controls IL-10 production in Bregs is yet to be defined. Here, we demonstrate that aryl hydrocarbon receptor (AhR) controls the differentiation and function of IL-10-producing Bregs. Deficiency of AhR-expressing B cells drastically reduces IL-10 production by B cells. This leads to the unrestrained differentiation of T helper (Th)17 cells and a significant reduction in the percentage of regulatory T cells (Treg), which increases the severity of experimental arthritis when compared to control animals with AhR-sufficient B cells. A combination of chromatin-landscape profiling by ATAC-seq and transcriptome analyses by RNA-seq demonstrated that a loss of AhR expression in B cells not only reduces IL-10 expression by Bregs, defined as CD21hiCD24hi B cells, but also promotes a pro-inflammatory programme in CD21hiCD24hi B cells, even under Breg inducing conditions. Thus, AhR acts as a master transcriptional regulator of Breg differentiation by implementing a molecular programme that controls IL-10 production and represses pro-inflammatory cytokine production.
Project description:Cardiac fibroblasts convert to myofibroblasts with injury to mediate healing after acute myocardial infarction and to mediate long-standing fibrosis with chronic disease. Myofibroblasts remain a poorly defined cell-type in terms of their origins and functional effects in vivo. Methods: Here we generate Postn (periostin) gene-targeted mice containing a tamoxifen inducible Cre for cellular lineage tracing analysis. This Postn allele identifies essentially all myofibroblasts within the heart and multiple other tissues. Results: Lineage tracing with 4 additional Cre-expressing mouse lines shows that periostin-expressing myofibroblasts in the heart derive from tissue-resident fibroblasts of the Tcf21 lineage, but not endothelial, immune/myeloid or smooth muscle cells. Deletion of periostin+ myofibroblasts reduces collagen production and scar formation after myocardial infarction. Periostin-traced myofibroblasts also revert back to a less activated state upon injury resolution. Conclusions: Our results define the myofibroblast as a periostin-expressing cell-type necessary for adaptive healing and fibrosis in the heart, which arises from Tcf21+ tissue-resident fibroblasts. Fluidigm C1 whole genome transcriptome analysis of lineage mapped cardiac myofibroblasts
Project description:A subset of adipocytes residing within the inguinal white adipose tissue (ingWAT) of mice exhibit thermogenic activity in response to various external stimuli, including cold exposure. The inducible nature of this thermogenic response, coupled with its robust energy-depleting capacity have prompted investigation into the adipose precursor cells (APCs) from which thermogenic adipocytes derive. To this end, we performed single-cell transcriptomics on cells derived from ingWAT, interscapular brown adipose tissue (iBAT) and epididymal WAT. A subset of single cells collected from ingWAT and epiWAT of mice were chronically (4 days) treated with CL-316,243 (dose at 1 mg kg -1). Tissues of n=28, 10 week-old mice were digested and stromal cells were subsequently purified via differential centrifugation. Single-cell RNA extraction and mRNA amplification were performed on the C1™ Single-Cell Auto Prep Integrated Fluidic Circuit (IFC) following the protocol (PN 100-7168, http://www.fluidigm.com/). Following centrifugation and removal of the medium, cells were resuspended at a concentration of 150–500 cells/μL. This cell suspension was mixed with C1 Cell Suspension Reagent (Fluidigm, Cat # 634833) at the recommended ratio of 3:2 immediately before loading 5 μL of this final mix on the C1 IFC. We obtained, on average, 2.5 million mapped reads per one single cell and successfully reconstructed single-cell expression of ~9,000 genes. Our results revealed a unique cluster of cells that exhibited enriched expression of canonical thermogenic and adipogenic gene markers. Notably, we identified tetraspanin CD81 as a discretely expressed membrane-bound protein conserved specifically within this population. All experiments were performed at our facility at the University of California, San Francisco.
Project description:Mutations in isocitrate dehydrogenase 2 (IDH2) occur in many cancers including Acute Myeloid Leukemia (AML). Recently, we showed that single agent Enasidenib, a first-in-class, selective mutant IDH2 inhibitor, produces a 40% response in relapsed/refractory AML patients by promoting differentiation of leukaemic cells. In this current study, we describe two patients who responded to Enasidenib treatment but subsequently relapsed with an IDH2-mutant subclone which had acquired mutations in DHX15 and DDX1 genes. These genes have putative functions in regulating splicing. We have studied the alternative splicing events using RNASeq in the sample pre-relapse (before acquisition of DHX15 and DDX1 mutations) and at relapse (after acquisition of DHX15 and DDX1 mutations).
Project description:The assay for transposase-accessible chromatin using sequencing (ATAC-seq) is widely used to identify regulatory regions throughout the genome. However, only a few studies have been done at the single cell level (scATAC-seq) due to technical difficulties. Here we developed a simple and robust plate-based scATAC-seq method, combining upfront bulk tagmentation with single-nuclei sorting, to investigate open chromatin regions. We applied this method on mouse splenocytes and unbiasedly revealed key regulatory regions and transcription factors that define each cell (sub)type.