ChIP-seq of H3K4me3 and H3K27ac of dermal fibroblasts with and without dsRNA (poly I:C) stimulation
ABSTRACT: Dermal fibroblasts from human, rhesus macaque, mouse and rat with and without dsRNA (poly I:C) stimulation (1ug/mL for 4 hours). 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 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: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:HNF1A and UTX are putative tumor suppressors in pancreatic cancer. In this study, we have combined mouse genetics, transcriptomics and genome binding studies to link HNF1A and UTX in a molecular mechanism that suppresses pancreatic cancer. In this session, we have profiled UTX, HNF1A, H3K27me3 and H3K27ac in normal and UTX- or HNF1A-deficient mouse pancreas by ChIP-seq experiments. We show that HNF1A recruits UTX to its genomic targets in pancreatic acinar cells, which results in remodeling of the chromatin landscape and activation of a broad transcriptional program of differentiated acinar cells, which in turn indirectly suppresses tumor suppressor pathways.
Project description:In neuroblastoma, amplification of the oncogenic basic helix-loop-helix (bHLH) transcription factor (TF) MYCN is the defining prognosticator of high-risk disease, occurs in one-third of neuroblastoma, and drastically reduces overall survival rates1,2. As a proto-oncogene, targeted MYCN overexpression in peripheral neural crest is sufficient to initiate disease in mouse models3. In MYCN amplified neuroblastoma, elevated expression of the factor is crucial to maintain tumor stemness4,5 and is associated with increased proliferation and aberrant cell cycle progression, as these tumors lack the ability to arrest in G1 in response to irradiation6-9. MYCN down-regulation broadly reverses these oncogenic phenotypes in a variety of neuroblastoma models10-12 and recent thereapeutic strategies to indirectly target MYCN production or protein stability have reduced tumor growth in vivo13-15. These observations motivate an investigation of MYCN binding in MYCN amplified tumors as it remains fundamentally unclear how elevated levels of the factor occupy the genome and alter transcriptional programs in neuroblastoma. Here we present the first dynamic chromatin and transcriptional landscape of direct MYCN perturbation in neuroblastoma. We find that at oncogenic levels, MYCN associates with E-box (CANNTG) binding motifs in an affinity dependent manner across most active cis-regulatory promoters and enhancers. MYCN shutdown globally reduces histone acetylation and transcription, consistent with prior descriptions of MYC proteins as non-linear amplifiers of gene expression. We establish that MYCN load at the promoter and proximal enhancers predicts transcriptional responsiveness to MYCN shutdown and that MYCN enhancer binding occurs prominently at the most strongly occupied and down-regulated genes, suggesting a role for these tissue specific elements in predicating MYCN responsive “target” genes. At these invaded enhancers, we identify the lineage specific bHLH TWIST1 as a key collaborator and dependency of oncogenic MYCN. These data suggest that MYCN enhancer invasion helps shape transcriptional amplification of the neuroblastoma gene expression program to promote tumorigenesis. ATAC-Seq in SHEP21, BE2C, KELLY, NGP, and MM1S cell lines
Project description:The experiment was designed to look into chromatin accessibility changes during cell cycle progression in human embryonic stem cells (hESCs) during definitive endoderm differentiation. For this, FUCCI hESCs were sorted in Early G1 (EG1), and differentiation into endoderm was performed for up to 72 hours with a combination of cytokines as described in Pauklin and Vallier (2013) and Pauklin et al. (2016). Samples at 0, 12, 24, 36, 48 and 72 hours were generated from two independent experiments, with 100,000 cells per sample, as previously described in Kumasaka et al. (2016). Library preparation and sequencing were performed at the Wellcome Sanger Institute next-generation sequencing facility. ATAC-seq libraries were prepared with one of i5 and i7 Nextera tags combination (see protocol details), and pooled equimolarly. Sequencing was performed on Illumina HiSeq 2000, 2 x 75bp paired-end reads.
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.