ABSTRACT: We report the profiling of small RNAs from Nanoarchaeum equitans by high-throughput sequencing. Over 16 million Illumina HiSeq 2000 reads were mapped to the 490,885-nucleotide small genome of Nanoarchaeum equitans. The mapping allowed the identification of highly abundant C/D box sRNAs and crRNAs in an organism that has to import its nucleotides from Ignicoccus hospitalis. The precursor tRNA halves of trans-spliced tRNAs were identified. Analysis of small RNA from one sample of Nanoarchaeum equitans.
Project description:We report the profiling of small RNAs from Methanopyrus kandleri by high throughput sequencing. Over 83 million Illumina Hi Seq2000 reads were obtained for six independent RNA libraries. The reads were mapped to the M. kandleri AV19 genome (Genbank: NC_003551, 1694969 bp). The small RNome of M. kandleri was analyzed. Analysis of small RNome from six Methanopyrus kandleri RNA samples
Project description:We aimed to determine the binding sites of the putative transcriptional regulator PafBC under DNA damage stress induced by mitomycin C or under oxidative stress induced by hydrogen peroxide. Therefore, we chose a ChIP-seq approach, crosslinking cells before PafBC-DNA complexes were immunoprecipitated with a PafBC-specific antibody.
Project description:The scope of the project is to find DNA binding sites of the DNA binding protein AtDEK3. For the experiments AtDEK3 overexpressor (OE) plants have been obtained. The AtDEK3 was tagged with a CFP-tag. With ChIP the protein was pulled down and eluted DNA was send for sequencing. Gene ontology (GO) term classification of genes enriched for AtDEK3 binding indicated that AtDEK3 target genes are involved in diverse biological processes. ChIP Seq of three indenpendendt AtDEK3 OE and their respective inputs were used.
Project description:Transcriptional changes during early infection of macrophage-like THP-1 cell line with pathogenic bacterium Mycobacterium tuberculosis. RNAseq samples were taken at 0h (THP-1 cells growing in the RPMI medium), and after 4h, 24h and 48h post infection. Bacterial enrichment was performed to increase the amount of bacterial mRNA in the samples. Non-enriched samples were used to map THP-1 cells transcripts; enriched samples were used to map M. tuberculosis transcripts the corresponding genomes.
Project description:The goal of this study was to elucidate the effects of inflammation on bone metabolism. As we found IL-17A is induced immediately after bone injury and Il17aâ/â mice showed delayed healing, we analyzed the effects of IL-17A on mesenchymal cells in the repair tissue. Most of the IL-17RA+ cells were PαS cells. We collected these cells and analyzed their response to IL-17A by RNA sequencing. This analysis will provide a mechanistic insight into the mechanism of how IL-17A promote bone formation in the context of bone fracture healing. PαS cells were harvested from the injury tissue of wild-type mice and cultured with or without IL-17A or BMP-2. RNAs were harvested at day 7.
Project description:The goal of the experiment is to obtain a differential gene expression profile for Mycobacterium smegmatis mc2-155 SMR5 (Msm) wild-type in comparison to a pafBC deletion strain (MSMEG_3888 and MSMEG_3889). In addition, the gene expression is to be analyzed with respect to different growth/stress conditions. Four groups were etablished: (1) Msm wild-type under standard conditions (2) Msm delta-pafBC under standard conditions (3) Msm wild-type treated with mitomycin C (4) Msm delta-pafBC treated with mitomycin C.
Project description:The piRNA-interacting Piwi protein is involved in transcriptional silencing of transposable elements in ovaries of D. melanogaster. Here we characterized the genome-wide effect of nuclear Piwi elimination on the presence of the heterochromatic H3K9me3 mark and HP1a, as well as on the transcription-associated mark H3K4me2. Our results demonstrate that a significant increase in the H3K4me2 level upon nuclear Piwi loss is not accompanied by the alterations in H3K9me3 and HP1a levels for several germline-expressed transposons, suggesting that in this case Piwi prevents transcription by a mechanism distinct from H3K9 methylation. We found that the targets of Piwi-dependent chromatin repression are mainly related to the elements that display a higher level of H3K4me2 modification in the absence of silencing, i.e. most actively transcribed elements. We also show that Piwi-guided silencing does not significantly influence the chromatin state of dual-strand piRNA-producing clusters. In addition, host protein-coding gene expression is essentially not affected due to the nuclear Piwi elimination, but we noted an increase in small nuclear spliceosomal RNAs abundance and propose Piwi involvement in their posttranscriptional regulation. Our work reveals new aspects of transposon silencing in Drosophila, indicating that transcription of transposons can underpin their Piwi dependent silencing, while canonical heterochromatin marks are not obligatory for their repression. Examination of histone modifications in ovaries from two different fly lines- piwiNt/piwi2 (mutant) and piwi/+ (wildtype)
Project description:Evolutionary alterations to cis-regulatory sequences are likely to cause adaptive phenotypic complexity, through orchestrating changes in cellular proliferation, identity and communication. For non-model organisms with adaptive key-innovations, patterns of regulatory evolution have been predominantly limited to targeted sequence-based analyses. Chromatin-immunoprecipitation with high-throughput sequencing (ChIP-seq) is a technology that has only been used in genetic model systems and is a powerful experimental tool to screen for active cis-regulatory elements. Here, we show that it can also be used in ecological model systems and permits genome-wide functional exploration of cis-regulatory elements. As a proof of concept, we use ChIP-seq technology in adult fin tissue of the cichlid fish Oreochromis niloticus to map active promoter elements, as indicated by occupancy of trimethylated Histone H3 Lysine 4 (H3K4me3). The fact that cichlids are one of the most phenotypically diverse and species-rich families of vertebrates could make them a perfect model system for the further in-depth analysis of the evolution of transcriptional regulation. examination of H3K4me3 in adult fin tissue of the Nile tilapia (Oreochromis niloticus)
Project description:TCF7L2 is one of the strongest type 2 diabetes (T2DM) candidate genes to emerge from GWAS studies, but the mechanisms by which it regulates the pathways which are important in the pathogenesis of type 2 diabetes are unknown. Previous in vitro and in vivo studies have focused on the link between TCF7L2 and insulin secretion as an explanation for the association between TCF7L2 and T2DM. However, TCF7L2 and the Wnt/β-catenin pathway are important for metabolic zonation in the liver. This raises the interesting possibility that TCF7L2 may influence glucose homeostasis by regulating hepatic glucose production (HGP). To examine this question, we utilized the H4IIE cell as a model of HGP. Inhibition of HGP in H4IIE cells from lactate and pyruvate was highly sensitive to physiological concentrations of insulin and metformin. Silencing of TCF7L2 protein expression induced a 5-fold increase in basal HGP (P<0.0001), and this was accompanied by marked increase in the expression of several key gluconeogenic genes. FBPase, PEPCK and G6Pase mRNA were up-regulated 2.5-fold (P<0.0001), 1.4-fold (P<0.01) and 2.3-fold (P<0.0001), respectively, compared to scramble siRNA. Compared to their respective baseline values, insulin and metformin suppressed HGP equally in the scramble and TCF7L2 siRNA cells, but HGP remained elevated in TCF7L2 silenced cells due to the increased baseline HGP. Using chromatin immunoprecipitation sequencing (ChIP-Seq), we investigated the direct transcriptional targets of TCF7L2 in hepatocytes. A total of 2119 ChIP peaks were detected, of which 36% were located inside gene boundaries and, overall, a total of 65% of all binding events were within 50 Kb of a gene. De novo motif analysis revealed remarkable conservation of the long and short TCF7L2 consensus binding sites in the rat hepatocytes. Pathway analysis showed that the top two disease categories over-represented in our dataset were “non-insulin dependent diabetes” (155 genes; P = 1.63 x 10-10) and “diabetes mellitus” (245 genes; P = 7.4 x 10-12). Inspection of genes in these categories revealed that TCF7L2 directly binds to multiple genes important in the regulation of glucose metabolism in the liver, including PEPCK, FBP1, IRS1, IRS2, AKT2 ADIPOR1, PDK4 and CPT1A. Our findings suggest a novel mechanism for the regulation of HGP by TCF7L2, and provide a possible explanation for the association of TCF7L2 polymorphisms with the incidence of T2DM. two samples: TCF7L2 ChIP-Seq and Input DNA
Project description:Glycosylation is an abundant post-translational modification of both intracellular and extracellular proteins [1]. The majority of glycans are classified as N-linked chains, where the carbohydrate moiety is attached to asparagine residues, or O-linked chains, most commonly linked to a serine or threonine. N-linked glycosylation is initiated by the oligosaccharyltransferase complex with only two paralogs of the catalytic subunit, whereas O-glycan initiation is more complex. There are several types of O-linked glycosylation, but among the most diverse is the mucin or GalNAc type (hereafter referred to as O-glycosylation). O-glycosylation is initiated by 20 evolutionarily conserved polypeptide GalNAc-transferases (GalNAc-Ts), which catalyze the first step in the O-glycosylation of proteins by adding GalNAc residues to threonine, serine, and tyrosine amino acids (Fig 1A). Each of the GalNAc-Ts are differentially expressed in various tissues and have both distinct and overlapping peptide substrate specificities [2-12]. Thus, the repertoire of GalNAc-Ts expressed in a given cell determines the subset and O-glycosite pattern of glycosylated proteins [13]. Substantial efforts have been made to characterize and predict the substrate specificities of GalNAc-Ts in vitro, but understanding of the in vivo specificities of the individual GalNAc-Ts or their biological functions is limited [13-15]. This lack of insight prevents an understanding of how site-specific O-linked glycosylation affects diseases, such as metabolic disorders, cardiovascular disease, and various malignancies, that have been associated with GalNAc-Ts through genome-wide association studies and other linkage studies [16-26]. Therefore, it is imperative that we establish how O-glycosylation at specific sites in proteins affects protein function. A major task in achieving this goal is to identify the non-redundant biological functions of site-specific O-glycosylation. We and others recently developed new strategies for identifying specific sites on proteins that undergo O-glycosylation in different cell types and tissues [27-31]. Characterization of the O-glycoproteomic landscape in isolated human cells and multiple human cell lines suggests that more than 80 % of all proteins that traffic through the secretory pathway are O-glycoproteins [28, 30]. Probing the non-redundant contributions of individual GalNAc-Ts in cells with and without specific GalNAc-Ts [32-34] has revealed broad substrate specificities for some of the individual isoforms, whereas others seem to have very restricted substrate specificities [33-35]. Assessing all of the mapped O-glycosylation sites to identify associations between O-glycosites and protein annotations, we recently found that O-glycans are over-represented close to tandem repeat regions, protease cleavage sites, within propeptides, and on a select group of protein domains [28, 30, 36]. Although such general associations between the location of O-glycans and protein functions may direct future investigations, the strategy does not define the function of site-specific glycosylation. Further progress in discovering and defining novel functions of site-specific glycosylation events requires direct quantitative analysis of potential biological responses induced by the loss of distinct GalNAc-T isoforms, and such biological responses are not easily observed in single cell culture systems. Instead, more complex model systems can be used to examine and dissect the molecular mechanisms underlying the important biological functions of site-specific glycosylation. We previously used an organotypic tissue model equipped with genetically engineered cells to decipher the function of elongated O-glycans [29]. In the present study, we use the model combined with quantitative O-glycoproteomics and phosphoproteomics to perform open-ended discovery of the biological functions of site-specific glycosylation governed by GalNAc-Ts (Fig 1B). With this combinatorial strategy, we demonstrate that loss of individual GalNAc-T isoforms has distinct phenotypic consequences through their effect on distinct biological pathways, suggesting specific roles during epithelial formation.