ABSTRACT: CD4+ T cells were extracted from mouse and human. They were activated in vitro with CD3/28 and cultured with Il4. ChIPmentation was then performed
Project description:The IRE1a-XBP1 pathway, a conserved adaptive response to the unfolded protein response, is indispensable for development of the secretory cells. It maintains endoplasmic reticulum homeostasis by enhancing protein folding and the secretory capacity of the cells. Here, we used a modified ChIP-seq protocol (ChIPmentation) to investigate the genome-wide binding events of the transcription factor XBP1 in differentiated mouse Th2 cells.
Project description:Dermal fibroblasts from human, rhesus macaque, mouse and rat with and without dsRNA (poly I:C) stimulation (1ug/mL for 4 hours).<br>The innate immune response - the expression programme that is initiated once a pathogen is sensed - is known to be variable among responding cells, as well as to rapidly evolve in the course of mammal evolution. To study the transcriptional divergence and cell-to-cell variability of this response, we stimulated dermal fibroblast cells from two primates (human and macaque) and two rodents (mouse and rat) with dsRNA - a mimic of viral RNA that elicits a rapid innate immune response. Subsequently, we profiled the response using bulk RNA-seq, scRNA-seq and ChIP-seq across the four species and across different time points.
Project description:CD4+ T cells were extracted from mouse and human. They were activated in vitro with CD3/28 and cultured with Il4. ATAC-seq was then performed at different time points
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:Objective: Although glucagon-secreting α-cells and insulin-secreting β-cells have opposing functions in regulating plasma glucose levels, the two cell types share a common developmental origin and have overlaps in their transcriptome and epigenome profiles. Notably, destruction of one of these cell populations can stimulate repopulation via transdifferentiation of the other cell type, at least in mice, suggesting plasticity between these cell fates. Furthermore, dysfunction of both α- and β-cells contributes to the pathophysiology of type 1 and type 2 diabetes, and β-cell de-differentiation has been proposed to contribute to type 2 diabetes. Our objective was to delineate the molecular properties that maintain islet cell type specification yet allow for cellular plasticity. We hypothesized that correlating cell type-specific transcriptomes with an atlas of open chromatin will identify novel genes and transcriptional regulatory elements such as enhancers involved in α- and β-cell specification and plasticity. Methods: We sorted human a- and b-cells and performed the â??Assay for Transposase-Accessible Chromatin with high throughput sequencingâ?? (ATAC-seq) and mRNA-seq, followed by integrative analysis to identify cell type-selective gene regulatory regions. Results: We identified numerous transcripts with either α-cell- or β-cell-selective expression and discovered the cell type-selective open chromatin regions that correlate with these gene activation patterns. We confirmed cell type-selective expression on the protein level for two of the top hits from our screen. The â??group specific proteinâ?? (GC; or vitamin D binding protein) was restricted to a-cells, while CHODL (chondrolectin) immunoreactivity was only present in b-cells. Furthermore, α-cell- and β-cell-selective ATAC-seq peaks were identified to overlap with known binding sites for islet transcription factors, as well as with common single nucleotide polymorphisms (SNPs) previously identified as risk loci for type 2 diabetes. Conclusions: We have determined the genetic landscape of human α- and β-cells based on chromatin accessibility and transcript levels, which allowed for detection of novel α- and β-cell signature genes not previously known to be expressed in islets. Using fine-mapping of open chromatin, we have identified thousands of potential cis-regulatory elements that operate in an endocrine cell type-specific fashion. ATAC-seq on 3 human alpha cell samples, 3 human beta cell samples, and 2 human acinar cell samples. RNA-seq on 7 human alpha cell samples and 8 human beta cell samples.
Project description:Investigations of 5-hydroxymethylcytosine (5hmC) in biologically and clinically samples and models with low cell numbers have been hampered by the low sensitivity and reproducibility using current 5hmC mapping approaches. Here, we develop a selective 5hmC chemical labeling approach using tagmentation-based library preparation in order to profile nanogram levels of 5hmC isolated from ~1,000 cells (nano-hmC-Seal). Using this technology, we profiled the dynamics of 5hmC across different stages of mouse hematopoietic differentiation. Additionally, applying nano-hmC-Seal to the hematopoietic multipotent progenitor cells in an acute myeloid leukemia (AML) mouse model, we identified leukemia-specific, differentially hydroxymethylated regions that harbor previously reported and as-yet-unidentified functionally relevant factors. The change of 5hmC patterns in AML strongly correlates with the altered gene expression on a global scale. Together, our new approach offers a highly sensitive and robust method to study and detect DNA methylation dynamics from in vivo model and clinical samples. Selective 5hmC chemical labeling approach using tagmentation-based library preparation in order to profile nanogram levels of 5hmC isolated from ~1,000 cells
Project description:Cohesin complex members have recently been identified as putative tumor suppressors in hematologic and epithelial malignancies. The cohesin complex guides chromosome segregation, however cohesin-mutant leukemias do not show genomic instability. We hypothesized reduced cohesin function alters chromatin structure and disrupts cis-regulatory architecture of hematopoietic progenitors. We investigated the consequences of Smc3 deletion in normal and malignant hematopoiesis. Bi-allelic Smc3 loss induced bone marrow aplasia with premature sister chromatid separation, and revealed an absolute requirement for cohesin in hematopoietic stem cell function. In contrast, Smc3 haploinsufficiency increased self-renewal in vitro and in vivo including competitive transplantation. Smc3 haploinsufficiency reduced coordinated transcriptional output, including reduced expression of transcription factors and other genes associated with lineage commitment. Smc3 haploinsufficiency cooperated with Flt3-ITD to induce acute leukemia in vivo, with potentiated Stat5 signaling and altered nucleolar topology. These data establish a dose-dependency for cohesin in regulating chromatin structure and hematopoietic stem cell function. ATAC-seq in murine c-kit+ cells for the following genotypes: Smc3 fl/+, Smc3 del/+, Flt3-ITD, Smc3 fl/del Flt3-ITD
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.
Project description:Plasmodium-specific CD4+ T cells from mice infected with Plasmodium chabaudi chabaudi AS parasites were recovered at Days 0, 4, 7, and 32 to undergo processing and to generate scATAC-seq dataset. At Day 7, CXCR5+ and CXCR6+ cells were recovered separately. At Day 32, mice were administered with either saline or artesunate (intermittent artesunate therapy - IAT). scATAC-seq dataset was analysed to investigate epigenomic landscapes of CD4+ T cells from effector to memory states.