ATAC-seq of wildtype or Dppa2 knockout ESC upon release of the epigenetic editing trigger
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ABSTRACT: Chromatin accessibility was assayed in wildtype or Dppa2 knockout ESC after 26 days of release of the trigger imposed by epigenetic editing. Samples were collected in two clonal knockout and wildtype lines after sorting at FACS of cells which maintained a repressive Esg1-tdTomato (TOMneg) reporter expression after 26 days of DOX washout (release of the trigger).
Project description:Chromatin accessibility was assayed in ESC after epigenetic editing with dCas9::KRAB (or dCas9::GFP as a control) and subsequently in ESC and Endoderm differentiated cells upon release of the trigger. Samples were collected in two biological replicates after 7 days of DOX induction in ESC (epigenetic editing) and after 7 days of DOX washout (release of the trigger) in ESC and Endoderm cells.
Project description:A genome-wide CRISPR screen was combined with a tdTomato reporter-based epigenetic memory assay to identify factors that erase epigenetic memory in ESC. After introducing genome wide perturbation and dCas9::KRAB-mediated epigenetic editing of the Esg1-tdTomato reporter, the trigger was released and cells that maintained the silencing sorted at FACS. Samples were collected out of sorted tdTomato negative (TOMminus) and positive (TOMplus) cells after 6 days of DOX treatment (epigenetic editing) and 3 or 7 days of DOX washout (release of the trigger), using a gating strategy to separate the bottom 2.5% negative cells (2.5%gate) and cells ranging from mildly to fully repressed (widegate).
Project description:Enrichment of histone marks was assessed by CUT&RUN-sequencing after epigenetic editing with dCas9::KRAB (or dCas9::GFP as a control) and subsequently upon release of the trigger. Samples were collected after 7 days of DOX induction (epigenetic editing) and after 4 and 7 days of DOX washout (release of the trigger).
Project description:Recent reports have proposed a new paradigm for obtaining mature somatic cell types from fibroblasts without going through a pluripotent state, by briefly expressing canonical iPSC reprogramming factors Oct4, Sox2, Klf4 and c-Myc (abbreviated as OSKM), in cells expanded in lineage differentiation promoting conditions. Here we apply genetic lineage tracing for endogenous Nanog, Oct4 and X chromosome reactivation during OSKM induced trans-differentiation, as these molecular events mark final stages for acquisition of induced pluripotency. Remarkably, the vast majority of reprogrammed cardiomyocytes or neural stem cells derived from mouse fibroblasts via OSKM mediated trans-differentiation were attained after transient acquisition of pluripotency, and followed by rapid differentiation. Our findings underscore a molecular and functional coupling between inducing pluripotency and obtaining “trans-differentiated” somatic cells via OSKM induction, and have implications on defining molecular trajectories assumed during different cell reprogramming methods. WGBS (Whole-Genome-Bisulfite-sequencing) were measured during conversion of mouse embryonic fibroblasts to neural stem cells using OSKM trans-differentiation method, as well as in mouse emrbyonic fibroblasts, and mouse ESCs.
Project description:Recent reports have proposed a new paradigm for obtaining mature somatic cell types from fibroblasts without going through a pluripotent state, by briefly expressing canonical iPSC reprogramming factors Oct4, Sox2, Klf4 and c-Myc (abbreviated as OSKM), in cells expanded in lineage differentiation promoting conditions. Here we apply genetic lineage tracing for endogenous Nanog, Oct4 and X chromosome reactivation during OSKM induced trans-differentiation, as these molecular events mark final stages for acquisition of induced pluripotency. Remarkably, the vast majority of reprogrammed cardiomyocytes or neural stem cells derived from mouse fibroblasts via OSKM mediated trans-differentiation were attained after transient acquisition of pluripotency, and followed by rapid differentiation. Our findings underscore a molecular and functional coupling between inducing pluripotency and obtaining “trans-differentiated” somatic cells via OSKM induction, and have implications on defining molecular trajectories assumed during different cell reprogramming methods. poly RNA-Seq was measured before, during and after conversion of mouse embryonic fibroblasts to neural stem cells using OSKM trans-differentiation method.
Project description:Recent reports have proposed a new paradigm for obtaining mature somatic cell types from fibroblasts without going through a pluripotent state, by briefly expressing canonical iPSC reprogramming factors Oct4, Sox2, Klf4 and c-Myc (abbreviated as OSKM), in cells expanded in lineage differentiation promoting conditions. Here we apply genetic lineage tracing for endogenous Nanog, Oct4 and X chromosome reactivation during OSKM induced trans-differentiation, as these molecular events mark final stages for acquisition of induced pluripotency. Remarkably, the vast majority of reprogrammed cardiomyocytes or neural stem cells derived from mouse fibroblasts via OSKM mediated trans-differentiation were attained after transient acquisition of pluripotency, and followed by rapid differentiation. Our findings underscore a molecular and functional coupling between inducing pluripotency and obtaining “trans-differentiated” somatic cells via OSKM induction, and have implications on defining molecular trajectories assumed during different cell reprogramming methods. poly RNA-Seq and Chromatin accesibility (ATAC-seq) were measured during conversion of mouse embryonic fibroblasts to neural stem cells using OSKM trans-differentiation method, as well as in mouse emrbyonic fibroblasts, iPSCs and mouse ESCs.
Project description:Functional studies using genetically modified mice with COLVI-specific STAT3 loss- or gain-of function demonstrated a critical role of STAT3 activation through IL-6 and IL-11 in fibroblasts during colorectal tumorigenesis in vivo. To reveal molecular mechanism specifically involved the STAT3 driven colorectal cancer development, we performed a comparative gene expression profiling by whole genome RNA-sequencing of fibroblasts subpopulations (COLVI+ vs. COLVI-) upon STAT3 activation under different conditions (IL-6 vs. IL-11) uncovering the regulation of transcriptional patterns associated with fibroblast activation, cytokine signaling and angiogenesis.