Project description:We develop a new ChIpseq method (iChIP) to profile chromatin states of low cell number samples. We use IChIP to profile the chromatin dynamics during hematopoiesis across 16 different cell types which include the principal hematopoietic progenitors 3' RNA-seq for digital gene expression quantitation across multiple cell types.
Project description:During hematopoiesis, cells originating from the same stem cell reservoir differentiate into distinct cell types. The mechanisms enabling common progenitors to differentiate into distinct cell fates are not fully understood. Here, we identify chromatin-regulating and cell-fate-determining transcription factors (TF) governing dendritic cell (DC) development by annotating the enhancer and promoter landscapes of the DC lineage. Combining these analyses with detailed over-expression, knockdown and ChIP-Seq studies, we show that Irf8 functions as a plasmacytoid DC epigenetic and fate-determining TF, regulating massive, cell-specific chromatin changes in thousands of pDC enhancers. Importantly, Irf8 forms a negative feedback loop with Cebpb, a monocyte-derived DC epigenetic fate-determining TF. We show that using this circuit logic, differential activity of TF can stably define epigenetic and transcriptional states, regardless of the microenvironment. More broadly, our study proposes a general paradigm that allows closely related cells with a similar set of signal-dependent factors to generate differential and persistent enhancer landscapes. Here analyzed 2 experiments, each one contains samples of moDC and pDC ex vivo cultured cells. The first experiment contains 32 samples of moDC and pDC following stimulation with various TLR stimulators. The second experiment contains 8 samples of moDC and pDC following perturbations; Cebpb and Irf8 knock down or over expression.
Project description:We develop a new ChIpseq method (iChIP) to profile chromatin states of low cell number samples. We use IChIP to profile the chromatin dynamics during hematopoiesis across 16 different cell types which include the principal hematopoietic progenitors Examination of 2 different histone modifications in 2 cell types.
Project description:During hematopoiesis, cells originating from the same stem cell reservoir differentiate into distinct cell types. The mechanisms enabling common progenitors to differentiate into distinct cell fates are not fully understood. Here, we identify chromatin-regulating and cell-fate-determining transcription factors (TF) governing dendritic cell (DC) development by annotating the enhancer and promoter landscapes of the DC lineage. Combining these analyses with detailed over-expression, knockdown and ChIP-Seq studies, we show that Irf8 functions as a plasmacytoid DC epigenetic and fate-determining TF, regulating massive, cell-specific chromatin changes in thousands of pDC enhancers. Importantly, Irf8 forms a negative feedback loop with Cebpb, a monocyte-derived DC epigenetic fate-determining TF. We show that using this circuit logic, differential activity of TF can stably define epigenetic and transcriptional states, regardless of the microenvironment. More broadly, our study proposes a general paradigm that allows closely related cells with a similar set of signal-dependent factors to generate differential and persistent enhancer landscapes. Here analyzed 2 experiments, each one contains samples of moDC and pDC ex vivo cultured cells. The first experiment contains 32 samples of moDC and pDC following stimulation with various TLR stimulators. The second experiment contains 8 samples of moDC and pDC following perturbations; Cebpb and Irf8 knock down or over expression.
Project description:In multi-cellular organisms, biological function emerges when cells of heterogeneous types and states are combined into complex tissues. Nevertheless unbiased dissection of tissues into coherent cell subpopulations is currently lacking. We introduce an automated, massively parallel single cell RNA sequencing method for intuitively analyzing in-vivo transcriptional states in thousands of single cells. Combined with unsupervised classification algorithms, it facilitates ab initio and marker-free characterization of classical hematopoietic cell types from splenic tissues. Importantly, modeling single cells transcriptional states in dendritic cells subpopulations, where a cell type hierarchy is difficult to define with marker-based approaches, uncovers complex combinatorial activity of multiple gene modules and capture cell-to-cell variability in steady state conditions and following pathogen activation. Massively parallel single cell RNA-seq thereby emerges as an effective tool for unbiased dissection of complex tissues. CD11c+ enriched splenocyte mRNA profiles from single cells were generated by deep sequencing of thousands of single cells, sequenced in several batches in an Illumina Hiseq 2000 The 'umitab.txt' processed data file contains the mRNA counts (post-filtering RMT counts) of a gene per each well (columns) The 'experimental_design.txt' contains a detailed information regarding each well. The 'readme0421.txt' was provided with details about each supplementary file.
Project description:To identify genome-wide, direct targets of HES1 in human pancreas progenitors, we performed Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) of endogenous HES1 in the HUES4 PDX1GFP/+ reporter cell line on Day 13 of differentiation from both unsorted bulk populations and FACS-sorted GFP+ cells.
Project description:Neutrophils rapidly respond to inflammation and infection, but to which degree their functional trajectories after mobilization from the bone marrow can be shaped within the circulation remains vague. Phenotypic changes of circulating neutrophils caused by systemic inflammation are thought to result from several factors, including a “left shift” of the neutrophil compartment towards younger bone marrow-derived subsets. However, experimental limitations have so far hampered neutrophil research in human disease. Here, using innovative fixation and single-cell-based toolsets, we profile the human and murine neutrophil transcriptome and proteome during steady state and bacterial infection. We find that peripheral priming of circulating neutrophils leads to plastic shifts dominated by conserved upregulation of antimicrobial genes across neutrophil substates, facilitating pathogen containment. We show the TLR4/NF-κB-signaling dependent upregulation of canonical neutrophil activation markers like CD177/NB-1 during acute inflammation, resulting in functional shifts in vivo. Importantly, blocking de novo RNA synthesis in circulating neutrophils abrogates these plastic shifts and prevents the adaptation of antibacterial neutrophil programs by upregulation of distinct effector molecules upon infection. In summary, these data underline transcriptional plasticity as a relevant mechanism of functional neutrophil reprogramming during acute infection to foster bacterial containment within the circulation. This adds to the understanding of competing modes of adaptation to inflammatory challenges by the neutrophil compartment.
Project description:We showed different function of monocyte derived cells in the lamina propria of the colon under steady state and inflammatory conditions. We used microarrays to detail the global programme of gene expression and identified distinct clusters of regulated genes during this process. Different subsets of mononuclear phagocytes were sorted from the colonic lamina propria as well as the spleen. Sorting was done in C57BL/6 mice in steady state and under inflammatory conditions (Dextran Sodium Sulphate induced colitis model)
Project description:HeLa cells were fracitonated into nuclear soluble, chromatin bound and cytosolic. Extracts were immunoprecipitated with a CSN3 antibody, then eluted with glycine. Proteins were digested, then enriched for phosphopeptides. Samples were analyzed on an LTQ-Orbitrap. Data was analyzed using the TRansOMics software for time alignment and feature detection. MS/MS spectra searched using Mascot.