ATAC-seq in the differentiation system ESC to NPC upon O-GlcNAc removal
Ontology highlight
ABSTRACT: This experiment uses a transgenic cell line expressing bacterial OGA BtGH84 fused to a localization peptide (NLS) and regulated by Tet-On system. OGA is a glycosidase that removes O-GlcNAc modifications. We evaluated the changes in chromatin openness before and after O-GlcNac removal by OGA.
Project description:This experiment aimed at investigating how O-GlcNac occupancy sites are impacted by RNA Polymerase II removal upon doxycycline stimulation. These human colon adenocarcinoma DLD-1 cells express OsTIR and a cassette encoding mini-AID (mAID) and fluorescent protein mClover (mAID+mClover) at the initiation site of the endogenous Rpb1 gene locus (POLR2A) (Nagashima 2019).
Project description:Beneficial effects of SIRT1 on healthspan are likely to be pleiotropic and may include effects on DNA methylation. We demonstrated recently that manipulating SIRT1 in human cells affected DNA methylation of a panel of test genes, and that genes with expression modified by dietary restriction corresponded with genes that underwent changes in DNA methylation during ageing. Here we tested the hypothesis that genes particularly susceptible to SIRT1-induced effects on DNA methylation across the genome map to genes for which DNA methylation changes during ageing. We increased or reduced SIRT1 expression in human intestinal (Caco-2) and vascular endothelial (HuVEC) cells by transient transfection with an expression construct or with siRNA respectively. Effects on DNA methylation were measured by enriching for the methylated faction then either sequencing (HuVEC) or hybridising to a human promoter microarray (Caco-2). Effects using these two different cell lines and techniques for analysis were remarkably consistent. Genes with a DNA methylation status affected by SIRT1 manipulation were enriched for those that undergo age-dependent changes in DNA methylation, thus supporting our hypothesis. Polycomb group protein target genes (PCGTs), which are suppressed by epigenetic mechanisms in stem cells and have been shown previously to correspond with loci particularly susceptible to age-related changes in DNA methylation, were over-represented within the set of genes showing altered DNA methylation in response to SIRT1 manipulation in both cell lines. We thus propose that effects of SIRT1 to extend healthspan include influences on the DNA methylation status of genes affected during ageing, in particular PCGTs. MBD-Sequencing to ascertain effects of SIRT1 over & under expression on methylation, in presence and absence of TNF-alpha. One sample per condition.
Project description:Chromatin transactions are typically studied in vivo, or in vitro using artificial chromatin lacking the epigenetic complexity of the natural material. Attempting to bridge the gap between these approaches, we established a system for isolating the yeast genome as a library of mono-nucleosomes harbouring the natural epigenetic signature, suitable for biochemical manipulation. Combined with deep sequencing, this library was used to investigate the stability of individual nucleosomes, and – as proof of principle - the nucleosome preference of the chromatin remodeling complex, RSC. In order to generate a library of native yeast nucleosomes, we developed a three-step purification protocol: first, purified yeast nuclei were incubated with micrococcal nuclease (MNase), which preferentially digests naked DNA to generate short chromatin fragments. The resulting fragments were extracted from the nuclei, then bound to and eluted from DEAE sepharose. This was followed by ultracentrifugation through a sucrose gradient to separate the fragments by length to further remove contaminating proteins and free DNA. We chose a simple disassembly assay, which involves incubating the nucleosome library with ATP and the histone chaperone Nap1, with or without RSC. In this assay, RSC binds to nucleosomes and transfers the histones to Nap1, thereby releasing ‘naked’ DNA. Under certain conditions, reaction intermediates can be observed (tetramers or hexasomes), but for simplicity we chose to compare the input nucleosomes with the final naked DNA product. To separate the RSC-dependent released DNA from the non-remodeled nucleosomes, the reactions were subjected to native agarose gel electrophoresis, and DNA of the four bands isolated by gel-extraction. The upper bands, harboring nucleosomes, were named NUC (no RSC) and NUCR (with RSC), whereas the lower, ‘naked’ DNA bands were named DNA (no RSC) and DNAR (with RSC).
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:Analysis of the RNA-seq data performed in IR vs NIR hematopoietic stem cells show the loss of the TNF_via_NFKB signature. We showed that the loss of this signature could be associated with H3K9me3 loss at specific retrotransposable elements . To validate this association, we tested if TNFa treatment before irradiation was able to prevent IR-effect on H3K9me3 loss at retrotransposable elements. For this purpose, we treated mice with TNFa 1h before irradiation (IR_TNF) and performed H3K9me3 cut&tag experiments on hematopoietic stem cells 1 month after irradiation and compared them to hematopoietic stem cells sorted from non irradiated mice (NIR) and from non-treated irradiated mice (IR).
Project description:Normal thymic T cell development is enabled by a stromal microenvironment most importantly composed of distinct epithelial cell populations in cortex and medulla. Their differentiation, growth and function require the expression of the transcription factor Foxn1. Direct targets of Foxn1 have, however, remained largely undefined. Utilizing newly created static and inducible genetic model systems, we now provide a genome wide map of Foxn1 target genes and the sequences bound by this master regulator. Foxn1 controls not only essential steps early in intrathymic lymphoid development including T cell lineage commitment but is also indispensable for later stages in T cell maturation such as the selection of CD4 and CD8 T cells. Thus, Foxn1 function critically choreographs both early and late events in thymic lympho-stromal cross-talk. Foxn1 ChIP-seq and RNA-seq in mouse models of hypofunctional or conditional knock-out of Foxn1 Brief sample descriptions are shown below: Foxn1 ChIP-seq (GSM1945905) - chromatin immunoprecipitated using an antibody against FOXN1-FLAG (wt*); 2 samples Input ChIP-seq (GSM1945906) - input chromatin; 2 samples 42cT (GSM1945907) - RNA-seq on wt*/-::nu/nu cTEC; 3 samples 42mT (GSM1945908) - RNA-seq on wt*/-::nu/nu mTEC; 2 samples 96cT (GSM1945909) - RNA-seq on wt*/wt*::nu/nu cTEC; 3 samples 96mT (GSM1945910) - RNA-seq on wt*/wt*::nu/nu mTEC; 2 samples NcT (GSM1945911) - RNA-seq on Dox-treated TetO- iFoxn1(del7,8) cTEC; 5 samples PcT (GSM1945912) - RNA-seq on Dox-treated TetO+ iFoxn1(del7,8) cTEC; 5 samples C57BL/6 mice (GSM1945913) - ATAC-seq on wild-type cTEC; 1 sample Please see each sample for more detailed information.
Project description:The HASTER promoter region is a cis-regulatory element that stabilizes the transcription of HNF1A, preventing silencing or overexpression. We have generated a mouse model where the promoter of Haster has been specifically deleted in liver (Haster loxP/loxP; AlbCre). In liver the prevailing consequence is upregulation of HNF1A. We performed HNF1A, H3K4me3 and H3K27ac ChIP-seq to assess the impact of HNF1A upregulation on the chromatin landscape of Haster KO liver.