168 research outputs found
Dissection of gene regulatory networks in embryonic stem cells by means of high-throughput sequencing
Transcription factor regulation of gene expression and chromatin-controlled epigenetic memory systems are closely cooperating in establishing the pluripotent state of embryonic stem (ES) cells and maintaining cell fate decisions throughout development of an organism. A thorough understanding of the regulatory transcriptional circuitry that rules the underlying plastic yet heritable gene expression programs in ES cells is of great importance. With the advent of next-generation sequencing technologies facilitating the quantitative assessment of functional genomics assays it is now feasible to interrogate transcription networks at a genome-wide scale. Here, we discuss the application of next-generation sequencing in elucidating the molecular mechanisms underlying ES cell functio
Identifizierung und Charakterisierung von Histonmethyltransferasen in Drosophila melanogaster
In eukaryotischen Zellen ist die DNA mit Histonen und anderen chromosomalen Proteinen zu Chromatin komplexiert. Das sich wiederholende Grundelement des Chromatins, das Nukleoso-m, besteht aus je zwei Kopien der Histone H2A, H2B, H3 und H4, um die 146 Ba-senpaare DNA gewunden sind. Durch Kondensierung der Nukleosomenreihen bilden sich übergeordnete Chromatinstrukturen, die noch wenig verstanden sind. Zur Ausführung DNA-abhängiger Prozesse muss die Verpackung der DNA teilweise gelockert werden, um zu ge-währleisten, dass die ausführenden Proteine Zugang zu ihren Zielsequenzen haben. Daher existieren verschiedene biochemische Systeme, die die grundlegende Struktur des Chromatins dynamisch kontrollieren. Das Zusammenspiel verschiedener Histonmodifizierungen, wie Acetylierung, Phosphorylierung, Ubiquitinierung und Methylierung setzt sowohl synergisti-sche als auch antagonistische Signale, die mit der Häufigkeit, mit der ein Gen transkribiert wird, korrelieren. Dieser „Histoncode“ erzeugt eine Architektur des Chromatins, die von Nichthistonproteinen erkannt wird und dann den Übergang zwischen transkriptionsaktiven und –inaktiven Chromatindomänen bewirken. Untersuchungen in den 1970er und 1980er Jahren zeigten für die Histonmethylierung, die sowohl an Argininen als auch an Lysinen erfolgen kann, eine Beteiligung an der eu- und hete-rochromatischen Transkriptionsregulation. Enzyme, die Histone methylieren können, waren jedoch zu Beginn dieser Arbeit nicht bekannt. Durch die chromatographische Fraktionierung von embryonalem Drosophila-Kernex-trakt konnten hier drei argininspezifische Histonmethyltransferasen identifiziert werden, die den Säugerproteinen CARM1, PRMT1 und PRMT3 homolog sind. Die cDNAs wurden klo-niert und die rekombinanten Proteine biochemisch charakterisiert. Während dieser Arbeit wurden von anderen Gruppen mehrere lysinspezifische Histon-methyltransferasen mit dem katalytisch aktiven Cys-SET(-Domäne)-Cys-Sequenzmotiv in Säugern und Hefe identifiziert. Außerdem wurde eine zentrale Rolle der lysinspezifischen Histonmethylierung in der epigenetischen Genrepression gezeigt. Im Verlauf der vorliegenden Arbeit wurden sieben in Drosophila vorkommende Cys-SET-Cys-Proteine identifiziert und kloniert. Für alle Proteine wurde Histonmethyltransferase-Aktivität mit teilweise unterschiedlicher Substratspezifität gezeigt. Darunter befindet sich auch ASH1 (Absent, Small or Homeotic discs), ein bekannter epigenetischer Aktivator der Trithorax-Gruppe. ASH1 methyliert in Histon H3 Lysin 4 und 9 und in Histon H4 Lysin 20. Die ASH1-abhängige Transkriptionsaktivierung konnte mit der Methylierung der drei Lysin-reste in der Promotorregion von ASH1-Zielgenen korreliert werden. Mittels in vitro Interakti-onsexperimenten und Chromatin-Immunpräzipitationen konnte gezeigt werden, dass durch ASH1-vermittelte Histonmethylierung die Assoziation von Repressoren, wie Polycomb und Heterochromatin-Protein 1 (HP1), verhindert wird. Außerdem konnte eine Korrelation zwi-schen der epigenetischen ASH1-abhängigen Aktivierung der Ultrabithorax-Transkription in Drosophila mit ASH1-vermittelter Histonmethylierung und Rekrutierung oder Stabilisierung der Bindung des aktivierenden Chromatin-Remodelling-Komplexes BRAHMA, dem Dro-sophila SWI/SNF-Homolog, nachgewiesen werden. Die Histonmethyltransferase-Aktivität von ASH1 könnte daher spezifische Signale für die Etablierung epigenetisch aktiver Transkriptionsmuster setzen. Diese Arbeit legt die Grundlagen für die Einbeziehung des Modellsystems Drosophila melanogaster in die Erforschung der Funktionen der Histonmethylierung, für ein besseres Verständnis der epigenetischen Aktivierung und erweitert die Komplexität des „Histoncodes“
Tracking and characterization of partial and full epithelial-mesenchymal transition cells in a mouse model of metastatic breast cancer
The various stages of epithelial-mesenchymal transition (EMT) generate phenotypically heterogeneous populations of cells. Here, we detail a dual recombinase lineage tracing system using a transgenic mouse model of metastatic breast cancer to trace and characterize breast cancer cells at different EMT stages. We describe analytical steps to label cancer cells at an early partial or a late full EMT state, followed by tracking their behavior in tumor slice cultures. We then characterize their transcriptome by five-cell RNA sequencing. For complete details on the use and execution of this protocol, please refer to Luond et al. (2021)
Quantitative in vivo analysis of chromatin binding of Polycomb and Trithorax group proteins reveals retention of ASH1 on mitotic chromatin
The Polycomb (PcG) and Trithorax (TrxG) group proteins work antagonistically on several hundred developmentally important target genes, giving stable mitotic memory, but also allowing flexibility of gene expression states. How this is achieved in quantitative terms is poorly understood. Here, we present a quantitative kinetic analysis in living Drosophila of the PcG proteins Enhancer of Zeste, (E(Z)), Pleiohomeotic (PHO) and Polycomb (PC) and the TrxG protein absent, small or homeotic discs 1 (ASH1). Fluorescence recovery after photobleaching and fluorescence correlation spectroscopy reveal highly dynamic chromatin binding behaviour for all proteins, with exchange occurring within seconds. We show that although the PcG proteins substantially dissociate from mitotic chromatin, ASH1 remains robustly associated with chromatin throughout mitosis. Finally, we show that chromatin binding by ASH1 and PC switches from an antagonistic relationship in interphase, to a cooperative one during mitosis. These results provide quantitative insights into PcG and TrxG chromatin-binding dynamics and have implications for our understanding of the molecular nature of epigenetic memor
The BET protein FSH functionally interacts with ASH1 to orchestrate global gene activity in Drosophila
BACKGROUND: The question of how cells re-establish gene expression states after cell division is still poorly understood. Genetic and molecular analyses have indicated that Trithorax group (TrxG) proteins are critical for the long-term maintenance of active gene expression states in many organisms. A generally accepted model suggests that TrxG proteins contribute to maintenance of transcription by protecting genes from inappropriate Polycomb group (PcG)-mediated silencing, instead of directly promoting transcription. RESULTS AND DISCUSSION: Here we report a physical and functional interaction in Drosophila between two members of the TrxG, the histone methyltransferase ASH1 and the bromodomain and extraterminal family protein FSH. We investigated this interface at the genome level, uncovering a widespread co-localization of both proteins at promoters and PcG-bound intergenic elements. Our integrative analysis of chromatin maps and gene expression profiles revealed that the observed ASH1-FSH binding pattern at promoters is a hallmark of active genes. Inhibition of FSH-binding to chromatin resulted in global down-regulation of transcription. In addition, we found that genes displaying marks of robust PcG-mediated repression also have ASH1 and FSH bound to their promoters. CONCLUSIONS: Our data strongly favor a global coactivator function of ASH1 and FSH during transcription, as opposed to the notion that TrxG proteins impede inappropriate PcG-mediated silencing, but are dispensable elsewhere. Instead, our results suggest that PcG repression needs to overcome the transcription-promoting function of ASH1 and FSH in order to silence genes
Methods for analyzing deep sequencing expression data: constructing the human and mouse promoterome with deepCAGE data
A set of methods is presented for normalization, quantification of noise and co-expression analysis for gene expression studies using deep sequencing
Organ specific gene expression in the regenerating tail of Macrostomum lignano
Temporal and spatial characterization of gene expression is a prerequisite for the understanding of cell-, tissue-, and organ-differentiation. In a multifaceted approach to investigate gene expression in the tail plate of the free-living marine flatworm Macrostomum lignano, we performed a posterior-region-specific in situ hybridization screen, RNA sequencing (RNA-seq) of regenerating animals, and functional analyses of selected tail-specific genes. The in situ screen revealed transcripts expressed in the antrum, cement glands, adhesive organs, prostate glands, rhabdite glands, and other tissues. Next we used RNA-seq to characterize temporal expression in the regenerating tail plate revealing a time restricted onset of both adhesive organs and copulatory apparatus regeneration. In addition, we identified three novel previously unannotated genes solely expressed in the regenerating stylet. RNA interference showed that these genes are required for the formation of not only the stylet but the whole male copulatory apparatus. RNAi treated animals lacked the stylet, vesicula granulorum, seminal vesicle, false seminal vesicle, and prostate glands, while the other tissues of the tail plate, such as adhesive organs regenerated normally. In summary, our findings provide a large resource of expression data during homeostasis and regeneration of the morphologically complex tail regeneration and pave the way for a better understanding of organogenesis in M. lignano
Detection of isoforms and genomic alterations by high-throughput full-length single-cell RNA sequencing in ovarian cancer
Understanding the complex background of cancer requires genotype-phenotype information in single-cell resolution. Here, we perform long-read single-cell RNA sequencing (scRNA-seq) on clinical samples from three ovarian cancer patients presenting with omental metastasis and increase the PacBio sequencing depth to 12,000 reads per cell. Our approach captures 152,000 isoforms, of which over 52,000 were not previously reported. Isoform-level analysis accounting for non-coding isoforms reveals 20% overestimation of protein-coding gene expression on average. We also detect cell type-specific isoform and poly-adenylation site usage in tumor and mesothelial cells, and find that mesothelial cells transition into cancer-associated fibroblasts in the metastasis, partly through the TGF-β/miR-29/Collagen axis. Furthermore, we identify gene fusions, including an experimentally validated IGF2BP2::TESPA1 fusion, which is misclassified as high TESPA1 expression in matched short-read data, and call mutations confirmed by targeted NGS cancer gene panel results. With these findings, we envision long-read scRNA-seq to become increasingly relevant in oncology and personalized medicine
Statistical tests for intra-tumour clonal co-occurrence and exclusivity
Tumour progression is an evolutionary process in which different clones evolve over time, leading to intra-tumour heterogeneity. Interactions between clones can affect tumour evolution and hence disease progression and treatment outcome. Intra-tumoural pairs of mutations that are overrepresented in a co-occurring or clonally exclusive fashion over a cohort of patient samples may be suggestive of a synergistic effect between the different clones carrying these mutations. We therefore developed a novel statistical testing framework, called GeneAccord, to identify such gene pairs that are altered in distinct subclones of the same tumour. We analysed our framework for calibration and power. By comparing its performance to baseline methods, we demonstrate that to control type I errors, it is essential to account for the evolutionary dependencies among clones. In applying GeneAccord to the single-cell sequencing of a cohort of 123 acute myeloid leukaemia patients, we find 1 clonally co-occurring and 8 clonally exclusive gene pairs. The clonally exclusive pairs mostly involve genes of the key signalling pathways
Effect of Bacterial Infection on Ghrelin Receptor Regulation in Periodontal Cells and Tissues.
The effect of bacterial infection on the expression of growth hormone secretagogue receptor (GHS-R) was investigated in periodontal cells and tissues, and the actions of ghrelin were evaluated. GHS-R was assessed in periodontal tissues of rats with and without periodontitis. Human gingival fibroblasts (HGFs) were exposed to Fusobacterium nucleatum in the presence and absence of ghrelin. GHS-R expression was determined by real-time PCR and immunocytochemistry. Furthermore, wound healing, cell viability, proliferation, and migration were evaluated. GHS-R expression was significantly higher at periodontitis sites as compared to healthy sites in rat tissues. F. nucleatum significantly increased the GHS-R expression and protein level in HGFs. Moreover, ghrelin significantly abrogated the stimulatory effects of F. nucleatum on CCL2 and IL-6 expressions in HGFs and did not affect cell viability and proliferation significantly. Ghrelin stimulated while F. nucleatum decreased wound closure, probably due to reduced cell migration. Our results show original evidence that bacterial infection upregulates GHS-R in rat periodontal tissues and HGFs. Moreover, our study shows that ghrelin inhibited the proinflammatory actions of F. nucleatum on HGFs without interfering with cell viability and proliferation, suggesting that ghrelin and its receptor may act as a protective molecule during bacterial infection on periodontal cells
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