Methylation profiling by array of mouse wild type and C9a knockout embyronic stem cells uisng antibodies against 5mC
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Jose de las Heras
PROVIDER: E-MEXP-2872 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Chromatin profiling of nuclei isolated from genetically defined neuronal subpopulations of the adult Drosophila brain. Cell type-specific histone modification maps were generated from nuclei isolated from all neurons (R57C10-GAL4), Kenyon cells (OK107-GAL4), and octopaminergic (Tdc2-GAL4) neurons using a method similar to INTACT (Deal and Henikoff, 2010; Steinner et al., 2012). Three histone modifications were profiled: H3K4me3, H3K27ac, and H3K27me3. Sequencing was performed with an Illumina HiSeq 2000.
2012-08-02 | E-GEOD-37032 | biostudies-arrayexpress
Project description:The epigenetic determinants driving the rapid responses of memory CD4 T cells to antigen are currently an area of active research. While much has been done to characterize various Th subsets and their associated genome-wide epigenetic patterns, the dynamics of histone modifications during CD4 T cell activation and the differential kinetics of these epigenetic marks between naïve and memory T cells have not been evaluated. In this study we have detailed the dynamics of genome-wide promoter H3K4me2 and H3K4me3 over a time course during activation of human naïve and memory CD4 T cells. Our results demonstrate that changes to H3K4 methylation predominantly occur relatively late after activation (120 hours) and reinforce activation-induced upregulation of gene expression affecting multiple pathways important to T cell activation, differentiation, and function. The dynamics and mapped pathways of H3K4 methylation are distinctly different in memory cells. Memory CD4 have substantially more promoters marked by H3K4me3 alone, and that is influenced by promoter CpG content, reinforcing their more differentiated state. Our study provides the first data examining genome-wide histone modification dynamics during T cell activation, providing insight into the cross-talk between H3K4 methylation and gene expression, and underscoring the impact of these marks upon key pathways integral to CD4 T cell activation and function. ChIP-Seq for H3K4me2, H3K4me3, and H3K27me3 in naïve and memory CD4 T cells at rest and at 3 time points after activation with anti-CD3/CD28.
2016-06-01 | E-GEOD-73212 | biostudies-arrayexpress
Project description:The patterning of Drosophila retina occurs both very fast and with high precision. This process is driven by the dynamic changes in signalling activity of the conserved Hedgehog (Hh) pathway, which coordinates cell fate determination, cell cycle and tissue morphogenesis. Here we show that during Drosophila retinogenesis, the retinal determination gene dachshund (dac) is not only a target of the Hh signaling pathway, but is also a modulator of its activity. Using developmental genetics techniques, we demonstrate that dac enhances Hh signaling by promoting the accumulation of the Gli transcription factor Cubitus interruptus (Ci) parallel to or downstream of fused. In the absence of dac, all Hh-mediated events associated to the morphogenetic furrow are delayed. One of the consequences is that, posterior to the furrow, dac- cells cannot activate a Roadkill-Cullin3 negative feedback loop that attenuates Hh signaling and which is necessary for retinal cells to continue normal differentiation. Therefore, dac is part of an essential positive feedback loop in the Hh pathway, guaranteeing the speed and the accuracy of Drosophila retinogenesis. ChIP-seq against Dachshund vs input ChIP-seq. Eye-antennal imaginal discs are dissected from Grh-GFP (Bloomington stock 42269) 3rd instar larvae and fixed with formaldehyde. Chromatin is prepared and sonicated until fragments reach an average size of 500 bp. Chromatin is immunoprecipitated with an anti-GFP Ab (ab290, Abcam) and the immunocomplexes are recovered with protein A/G magnetic beads (Millipore).
2016-07-01 | E-GEOD-82151 | biostudies-arrayexpress
Project description:The plant circadian clock exerts a critical role in the regulation of multiple biological processes including responses to biotic and abiotic stresses. It is estimated that the clock regulates up to 80% of the transcriptome in Arabidopsis, thus understanding the molecular mechanisms that control this rhythmic transcriptome requires identification of the targets of each clock component. The Arabidopsis core clock is partially comprised of a transcriptional regulatory loop between the MYB domain containing transcription factors CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), and TIMING OF CAB EXPRESSION1 (TOC1). As a key component of the clock, CCA1 is able to initiate and set the phase of clock-controlled rhythms. CCA1 regulates the transcription of several genes by directly binding to the evening element (EE) motif primarily found in the promoters of evening expressed genes. Using a genome-wide approach we have identified direct targets of CCA1 in plants grown in constant (LL) and driven conditions (LD). These CCA1 targets are enriched for a myriad of biological processes and stress responses. While many of these target genes are evening phased and contain the EE in their promoter regions, a significant subset is morning phased and lack an EE. Furthermore, several CCA1 targets do not cycle in either LL or LD or both. Expression analysis in CCA1 overexpressing plants confirms CCA1 regulation of analyzed targets. Our results emphasize an expanded role for the circadian clock in regulation of key pathways in Arabidopsis, and provide a comprehensive and solid resource for future functional studies. ChIP-Seq of CCA1-GFP plants under control of the CCA1 promoter in continuous light and diel conditions
2015-07-26 | E-GEOD-70533 | biostudies-arrayexpress
Project description:Phenotypic plasticity, the ability of one genotype to express different phenotypes in response to changing environmental conditions, is one of the most common phenomena characterising the living world and is not only relevant for the ecology but also for the evolution of species. Daphnia, the waterflea, is a textbook example for predator induced phenotypic plastic defences including changes in life-history, behaviour and morphology. However, the analysis of molecular mechanisms underlying these inducible defences is still in its early stages.<br><br>We exposed Daphnia magna to chemical cues of the predator Triops cancriformis to identify key processes underlying plastic defensive trait formation. D. magna is known to develop an array of morphological changes in the presence of T. cancriformis including changes of carapace morphology and cuticle hardening. To get a more comprehensive idea of this phenomenon, we studied four different genotypes originating from habitats with different predation history, reaching from predator-free to temporary habitats containing T. cancriformis.<br><br>We analysed the morphologies as well as proteomes of predator-exposed and control animals. Three genotypes showed morphological changes when the predator was present. Using a high-throughput proteomics approach, we found 294 proteins which were significantly altered in their abundance after predator exposure in a general or genotype dependant manner. Proteins connected to genotype dependant responses were related to the cuticle, protein synthesis and calcium binding whereas the yolk protein vitellogenin increased in abundance in all genotypes, indicating their involvement in a more general response. Furthermore, genotype dependant responses at the proteome level correlated well with local adaptation to Triops predation.<br><br>Altogether, our study provides new insights concerning genotype dependant and general molecular processes involved in predator-induced phenotypic plasticity in D. magna.
2015-07-03 | PXD001787 | Pride
Project description:Purpose: The goal of this study was to compare the genome-wide promoter methylation alterations in macrophages and endothelial cells during hindlimb ischemia among normal, hyperlipidemic and type-2 diabetic mice. Methods: Unilateral hindlimb ischemia was induced by ligating femoral artery proximal to the bifurcation of superficial and deep femoral artery in mice deficient of LDL receptor and expressing only apolipoprotein B100 (LDLR-/-ApoB100/100, C57BL/6J background) (The Jackson Laboratory, Bar Harbor,USA) and mice with β-cell specific over-expression of insulin-like growth factor-2 in atherosclerotic background (IGF-II/LDLR-/-ApoB100/100, C57BL/6J background) with type 2 diabetic features on high-fat diet (TD 88173, Harlan Teklad: 42% of calories from fat and 0.15% from cholesterol, no sodium cholate) 8 weeks prior to surgery and continued throughout the study 1. C57BL/6J (WT) mice fed with regular chow-diet (R36, Lactamin) served as controls. All animals were aged between 20 to 24 weeks at the time of hindlimb operations. For sorting macrophages from ischemic muscles, ischemic gastrocnemius muscles were minced and enzymatically dissociated using a cocktail containing 450 U/mL Collagenase I, 125 U/mL Collagenase XI, 60 U/mL DNAseI, and 60 U/mL hyaluronidase (Sigma Aldrich) for 1 h at 37°C. The cells were then counted and divided into CD31+ve and CD31-vefractions using CD31 magnetic bead enrichment (Miltenyi Biotec). For macrophage sorting CD31-ve fraction was incubated for 15 minutes with rat anti-mouse CD16/32 mAb (Fc Block, BD-pharmingen) and stained with FITC conjugated rat anti-mouse F4/80 antibody (Serotec) for 20 minutes at 4ËC. For endothelial sorting CD31+ fraction was incubated for 15 minutes with rat anti-mouse CD16/32 mAb (Fc Block, BD-pharmingen) and stained with APC conjugated rat anti-mouse CD31 antibody (BD-pharmingen) and FITC conjugated rat anti-mouse CD45 ((BD-pharmingen) for 20 minutes at 4ËC. FACS sorting was performed on FACS AriaIII (BD Biosciences). Genomic DNA was isolated from FACS sorted macrophages and endothelial cells using AllPrep DNA/RNA/Protein Mini Kit (Qiagen Finland, Helsinki, Finland) according to manufacturer's instructions. Results: The sample similarity as assessed by Pearsonâs correlation matrix and Hierarchial clustering showed high correalation among macrophages, as well as endothelial cells. There was a clear clustering of macrophages and endothelial cells as evidence by their CpG methylation clustering, furthermore macrophages from HL and T2DM mice showed clear clustering compared to control macrophages. Differential methylation analysis of RRBS methylation data from macrophages and endothelial cells was performed using Methylkit. Using a threshold of adjusted p value (Q) <0.05 and percentage methylation difference of >5%, we identified 198 and 272 genes whose promoters were hypomethylated in HL and T2DM macrophages. Similarly, there were 102 and 136 gene promoters were hypermethylated in HL and T2DM macrophages, respectively compare to control macrophages. Thus, proximal promoter methylation suggested that HL and T2DM have convergent influences on the proximal promoter methylation of numerous macrophage specific genes. In order to find out whether these genes with differential methylated promoters were differentially expressed at mRNA expression level in purified macrophages, we further compared our data with the GEO datasets as above. Of the 198 genes with promoter hypomethylation in HL macrophages 72 genes were suggested to be upregulated in M1- MÏs; whereas, of the 102 genes with promoter hypermethylation, 51 genes were suggested to be upregulated in M2- MÏs. Similarly, out of 272 genes with differentially methylated promoters in T2DM macrophages 88 genes were suggested to be upregulated in M1-MÏs; whereas, out of 136 genes with promoter hypermethylation 60 genes were suggested to be upregulated in M2- MÏs. Thus a significant promoter hypomethylation of M1-MÏ and hypermethylation of M2-MÏ genes suggested the predominance of proinflammatory M1-MÏs in ischemic muscles of HL and T2DM compared to M2-MÏs in control mice. Conclusions: We found significant promoter hypomethylation of genes typical for proinflammatory M1-MÏs and hypermethylation of anti-inflammatory, proangiogenic M2-MÏ associated genes in HL and T2DM ischemic muscles. Epigenetic alterations skewing MÏ phenotype towards proinflammatory as opposed to anti-inflammatory, proangiogenic and tissue repair phenotype may contribute to impaired adaptive vascular growth in these pathological conditions. Macrophages and endothelial whole genome DNA methylation was performed in triplicates (Each sample was pooled from 3-4 mice) by RRBS Sequencing approach using Illumina HiSeq 2500. qRTâPCR validation was performed using TaqMan assays.
2015-12-21 | E-GEOD-65759 | biostudies-arrayexpress
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2012-08-01 | E-GEOD-37033 | biostudies-arrayexpress
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2016-02-08 | PXD002425 | Pride
Project description:Epithelial cell adhesion molecule (EpCAM), a membrane protein known to modulate cell-cell adhesion, is also a signaling molecule internalized into the nucleus for transcriptional regulation. Here we demonstrate that activated EGF/EGFR is a signaling factor to drive the proteolysis of EpCAM. Cleavage of the extracellular fragment EpEX results in topographic fading of cell-surface EpCAM detected by antibody-conjugated cantilevers of atomic force microscope (AFM). As a result, internalization of the cytoplasmic domain EpICD forms a transcription factor complex with LEF1 that regulates gene transcription for enhanced cell-mobility functions. Comprehensive probing of cell surface using AFM tip (without antibody) reveals increased elasticity and non-stickiness of these cells, promoting epithelial to mesenchymal transition. While EpCAM cleavage may contribute to the loss of cell-surface adhesiveness, its internalized EpICD additionally regulates targets for promoting cell migration. Thus, this EGF/EGFR-modulated action on structural EpCAM and regulatory EpICD can enhance invasion potential of transformed cells. RL95-2 were stimulated with EGF for 0,12,24,and 48 hr.Immunoprecipitation was carried out using antibodies against EpCAM and Lef-1, sequenced by Illumina HiSeq 2000
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