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: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: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: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:Understanding the regulatory genome remains a significant challenge. Annotation of regulatory elements and identification of the transcription factors (TFs) targeting these elements are key steps in understanding how a given cell interprets its genetic blueprint. One goal of the modENCODE (model organism Encyclopedia of DNA Elements) project is to survey a diverse sampling of TFs, both DNA-binding and non-DNA binding factors, to provide a framework for the subsequent study of the mechanisms by which transcriptional regulators target the genome. Here we provide an updated map of the Drosophila melanogaster regulatory genome based on the location of 84 TFs at various stages of development. This regulatory map reveals a variety of genomic targeting patterns, including factors with strong preferences toward proximal promoter binding, factors that target intergenic and intronic DNA, and factors with distinct chromatin state preferences. The data also suggest the existence of a partially self-contained Polycomb regulatory network, and highlight the importance of Trithorax-like (Trl) in maintaining hotspots of DNA binding throughout development. Furthermore, the data identify over 5,800 instances in which TFs target DNA regions with demonstrated enhancer activity. Regions of high TF co-occupancy are more likely to be associated with open enhancers used across cell types, while lower TF occupancy regions are associated with complex enhancers that are also regulated at the epigenetic level. A putative regulatory network generated based on these 84 regulators reveals hundreds of co-binding events, thousands of potential regulatory interactions, and distinct regulatory strategies at developmental and housekeeping genes. These data serve as a resource for the research community in the continued effort to dissect transcriptional regulatory mechanisms directing Drosophila development. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf This is a dataset generated by the Drosophila Regulatory Elements modENCODE Project led by Kevin P. White at the University of Chicago.
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:We over-expressed an epigenetic regulator in a glioblastoma (GBM) primary culture from an adult patient. These GBM cells have cancer stem cell phenotypes, as they have self-renewal properties and tumor initiation potential when transplanted in immunocompromised mice. ATAC-seq was performed on cells over-expressing the epigenetic regulator and control cells expressing EGFP. ATAC-Seq on glioblastoma cells that over-express EGFP or an epigenetic regulator.