The heart is a highly specialized organ with essential function for the organism throughout life. The significance of DNA methylation in shaping the phenotype of the heart remains only partially known. Here we generate and analyse DNA methylomes from highly purified cardiomyocytes of neonatal, adult healthy and adult failing hearts. We identify large genomic regions that are differentially methylated during cardiomyocyte development and maturation. Demethylation of cardiomyocyte gene bodies correlates strongly with increased gene expression. Silencing of demethylated genes is characterized by the polycomb mark H3K27me3 or by DNA methylation. De novo methylation by DNA methyltransferases 3A/B causes repression of fetal cardiac genes, including essential components of the cardiac sarcomere. Failing cardiomyocytes partially resemble neonatal methylation patterns. This study establishes DNA methylation as a highly dynamic process during postnatal growth of cardiomyocytes and their adaptation to pathological stress in a process tightly linked to gene regulation and activity.
Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a key technique in chromatin research. Although heavily applied, existing ChIP-seq protocols are often highly fine-tuned workflows, optimized for specific experimental requirements. Especially the initial steps of ChIP-seq, particularly chromatin shearing, are deemed to be exceedingly cell-type-specific, thus impeding any protocol standardization efforts. Here we demonstrate that harmonization of ChIP-seq workflows across cell types and conditions is possible when obtaining chromatin from properly isolated nuclei. We established an ultrasound-based nuclei extraction method (NEXSON: Nuclei EXtraction by SONication) that is highly effective across various organisms, cell types and cell numbers. The described method has the potential to replace complex cell-type-specific, but largely ineffective, nuclei isolation protocols. By including NEXSON in ChIP-seq workflows, we completely eliminate the need for extensive optimization and sample-dependent adjustments. Apart from this significant simplification, our approach also provides the basis for a fully standardized ChIP-seq and yields highly reproducible transcription factor and histone modifications maps for a wide range of different cell types. Even small cell numbers (∼10 000 cells per ChIP) can be easily processed without application of modified chromatin or library preparation protocols.
Invasion, metastasis and therapy resistance are the major cause of cancer‐associated deaths, and the EMT‐inducing transcription factor ZEB1 is a crucial stimulator of these processes. While work on ZEB1 has mainly focused on its role as a transcriptional repressor, it can also act as a transcriptional activator. To further understand these two modes of action, we performed a genome‐wide ZEB1 binding study in triple‐negative breast cancer cells. We identified ZEB1 as a novel interactor of the AP‐1 factors FOSL1 and JUN and show that, together with the Hippo pathway effector YAP, they form a transactivation complex, predominantly activating tumour‐promoting genes, thereby synergising with its function as a repressor of epithelial genes. High expression of ZEB1, YAP, FOSL1 and JUN marks the aggressive claudin‐low subtype of breast cancer, indicating the translational relevance of our findings. Thus, our results link critical tumour‐promoting transcription factors: ZEB1, AP‐1 and Hippo pathway factors. Disturbing their molecular interaction may provide a promising treatment option for aggressive cancer types.
BackgroundMonocyte-to-macrophage differentiation involves major biochemical and structural changes. In order to elucidate the role of gene regulatory changes during this process, we used high-throughput sequencing to analyze the complete transcriptome and epigenome of human monocytes that were differentiated in vitro by addition of colony-stimulating factor 1 in serum-free medium.ResultsNumerous mRNAs and miRNAs were significantly up- or down-regulated. More than 100 discrete DNA regions, most often far away from transcription start sites, were rapidly demethylated by the ten eleven translocation enzymes, became nucleosome-free and gained histone marks indicative of active enhancers. These regions were unique for macrophages and associated with genes involved in the regulation of the actin cytoskeleton, phagocytosis and innate immune response.ConclusionsIn summary, we have discovered a phagocytic gene network that is repressed by DNA methylation in monocytes and rapidly de-repressed after the onset of macrophage differentiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0079-z) contains supplementary material, which is available to authorized users.
Our results demonstrate that gliotoxin and patulin increase the allergic immune response in mice by modulating the Th1/Th2 balance via direct effects on IL-12 secretion in DCs and by inducing oxidative stress.
Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is an invaluable tool for mapping chromatin-associated proteins. Current barcoding strategies aim to improve assay throughput and scalability but intense sample handling and lack of standardization over cell types, cell numbers and epitopes hinder wide-spread use in the field. Here, we present a barcoding method to enable high-throughput ChIP-seq using common molecular biology techniques. The method, called RELACS (restriction enzyme-based labeling of chromatin in situ) relies on standardized nuclei extraction from any source and employs chromatin cutting and barcoding within intact nuclei. Barcoded nuclei are pooled and processed within the same ChIP reaction, for maximal comparability and workload reduction. The innovative barcoding concept is particularly user-friendly and suitable for implementation to standardized large-scale clinical studies and scarce samples. Aiming to maximize universality and scalability, RELACS can generate ChIP-seq libraries for transcription factors and histone modifications from hundreds of samples within three days.
BackgroundEpidemiological studies suggest an association between exposure to volatile organic compounds (VOCs) and adverse allergic and respiratory symptoms. However, whether VOCs exhibit a causal role as adjuvants in asthma development remains unclear.MethodsTo investigate the effect of VOC exposure on the development of allergic airway inflammation Balb/c mice were exposed to VOCs emitted by new polyvinylchloride (PVC) flooring, sensitized with ovalbumin (OVA) and characterized in acute and chronic murine asthma models. Furthermore, prevalent evaporated VOCs were analyzed and mice were exposed to selected single VOCs.ResultsExposure of mice to PVC flooring increased eosinophilic lung inflammation and OVA-specific IgE serum levels compared to un-exposed control mice. The increased inflammation was associated with elevated levels of Th2-cytokines. Long-term exposure to PVC flooring exacerbated chronic airway inflammation. VOCs with the highest concentrations emitted by new PVC flooring were N-methyl-2-pyrrolidone (NMP) and 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (TXIB). Exposure to NMP or TXIB also increased the allergic immune response in OVA-sensitized mice. In vitro or in vivo exposure to NMP or TXIB reduced IL-12 production in maturing dendritic cells (DCs) and enhanced airway inflammation after adoptive DC transfer into Balb/c mice. At higher concentrations both VOCs induced oxidative stress demonstrated by increased isoprostane and glutathione-S-transferase-pi1 protein levels in the lung of non-sensitized mice. Treatment of PVC flooring-exposed mice with N-acetylcysteine prevented the VOC-induced increase of airway inflammation.ConclusionsOur results demonstrate that exposure to VOCs may increase the allergic immune response by interfering with DC function and by inducing oxidative stress and has therefore to be considerate as risk factor for the development of allergic diseases.
Human Thy-1 (CD90) has been shown to mediate adhesion of inflammatory cells to activated microvascular endothelial cells via interaction with Mac-1 (CD11b/CD18) in vitro. Since there are no data showing the physiological relevance of Thy-1 for the recruitment of inflammatory cells in vivo, different inflammation models were investigated in Thy-1-deficient mice and littermate controls. In thioglycollate-induced peritonitis, the number of neutrophils and monocytes was significantly diminished in Thy-1-deficient mice. During acute lung inflammation, the extravasation of eosinophils and monocytes into the lung was significantly reduced in Thy-1-deficient mice. Moreover, during chronic lung inflammation, the influx of eosinophils and monocytes was also strongly decreased. These effects were independent of Thy-1 expression on T cells, as shown by the transplantation of WT BM into the Thy-1-deficient mice. In spite of the strong Thy-1 expression on T cells in the chimeric mice, the extravasation of the inflammatory cells in these mice was significantly diminished, compared to control mice. Finally, the altered number and composition of infiltrating leukocytes in Thy-1-deficient mice modified the chemokine/cytokine and protease expression at the site of inflammation. In conclusion, Thy-1 is involved in the control of inflammatory cell recruitment and, thus, also in conditioning the inflammatory microenvironment.Key words: Extravasation . Inflammation . Thy-1 Supporting Information available online IntroductionThe recruitment of inflammatory cells to sites of inflammation plays an important role in the pathogenesis of several inflammatory diseases. Leukocyte adhesion to endothelial cells (ECs) follows a multistep process, including the capture of free leukocytes out of the blood stream, rolling, firm adhesion, and transendothelial diapedesis. The importance of several adhesion molecules in this series of events has been described previously [1]. In ICAM-1-deficient mice, neutrophil recruitment was significantly reduced, but it was not completely blocked in a chemical peritonitis model or in a lipopolysaccharide 645(LPS)-induced airway inflammation model, indicating the involvement of additional adhesion molecules [2,3]. Furthermore, leukocyte recruitment in experimental colitis was not affected by blocking ICAM-1 or MadCAM, whereas the blocking of VCAM-1 resulted in a significant attenuation of colitis [4]. Thus, under specific inflammatory conditions, certain adhesion molecules mediate adhesion and transmigration of leukocytes into the perivascular tissue.Recently, human Thy-1 expressed on ECs was identified as an adhesion molecule mediating the binding of neutrophils and monocytes to activated microvascular ECs [5]. Thy-1 is a highly glycosylated GPI-anchored surface protein and a member of the immunoglobulin superfamily [6,7,8]. In humans, Thy-1 is expressed on ECs at sites of inflammation or in tumours whereas ECs do not express Thy-1 in healthy tissue [5,9]. Thy-1 is also expressed on fibroblasts, neurons, and a su...
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