Genome wide map of H3K4me3 histone modification in C6 rat glioma cells [SOLiD]
ABSTRACT: We report a map of H3K4me3 - an activiting expression histone modification in C6 rat glioma cells. The data was obtained using whole genome high throughput technology. The sequencing was performed on Solid 5500xl platform. Examination of H3K4me3 histone modification in C6 rat glioma cell line
Project description:We report maps of H3K4me3 and H3ac - activiting expression histone modifications in C6 rat glioma cells. The data was obtained using whole genome high throughput technology. The sequencing was performed on HiSeq Ilumina platform. Examination of H3K4me3 histone modification and H3ac histone modification in C6 rat glioma cell line
Project description:We report a map of Stat3 binding sites in C6 rat glioma cells. The data was obtained using whole genome technology using NimbleGen microarrays. Examination of Stat3 binding sites in C6 rat glioma cell line
Project description:Extracellular matrix remodeling, degradation and glioma cell motility are critical aspects of glioblastoma multiforme (GBM). Despite being a rich source of potential biomarkers and targets for therapeutic advance, the dynamic changes within the extracellular environment that are specific to GBM cell motility have yet to be fully resolved. The gap junction protein connexin43 (Cx43) increases glioma migration and invasion in a variety of in vitro and in vivo models. In this study, the conditioned media of Cx43-expressing C6 rat glioma cells was found to increase the motility of the parental line. Demonstrating the selective engagement of ECM-remodelling pathways, secretome analysis revealed the near-binary expression of osteopontin and matrix metalloproteinase-3 (MMP3).
Project description:We report a map of H3K4me3 - an activiting expression histone modification in C6 rat glioma cells. The data was obtained using whole genome high throughput technology. The sequencing was performed on Solid 5500xl platform. Overall design: Examination of H3K4me3 histone modification in C6 rat glioma cell line
Project description:Several independent microglia preparations performed on different days were plated onto wells of 24-well plates, and either left untreated (control), treated with the C6 glioma-conditioned medium (GCM). RNA samples were prepared at 6, 24 and 48 hours from the start of the treatment, as well as from the control untreated microglia, with each sample then separately labeled and hybridized. These RNA samples were prepared from from at three independent microglia preparations, so they constitute biological replicates.
Project description:(R,R’)-4-methoxy-1-naphthylfenoterol (MNF) inhibits in vitro proliferation of several types of cancer cell lines. In this study, the in vivo antitumor effects of MNF were evaluated using rat C6 glioma cells implanted subcutaneously into the lower flank of 5 week-old NMRI/Nude female Swiss mice. Three days after the inoculation, the mice were subjected to intraperitoneal injections of saline or MNF (2mg/kg) for five days per week for16 days. Tumor volumes were measured everyday using slide calipers. Significant reductions in mean tumor volumes were observed in mice receiving MNF when compared with the saline-treated group (p<0.001, n=17-19). At the end of the study, animals were sacrificed and tumors were collected for cDNA microarray, quantitative RT-PCR and immunoblot analyses. Significant decrease in expression of genes involved in cellular proliferation, including mitotic checkpoint kinase MAD3L (Bub1b), cyclin-dependent kinase inhibitor 3 (Cdkn3) and cyclin A2 (Ccna2), as well as molecular markers for glioblastoma, such as oligodendrocyte transcription factor 1 (Olig1) and SRY-box 4 (Sox4) was observed in tumors of MNF-treated mice as compared to saline-injected controls. The efficacy of MNF against C6 glioma cancer in vivo was accompanied by marked reduction in the expression of cell cycle regulator proteins, including cyclin A and cyclin D1, in tumor extracts. This study is the first demonstration of MNF-dependent chemoprevention in a glioblastoma xenograft model in the mouse and may offer a potential mechanism for its anticancer action in vivo. Keywords: fenoterol derivative; C6 glioma; tumor xenografts; microarray analysis; Total RNA was isolated from rat C6 glioma xenografts harvested from vehicle and MNF-treated nude mice (n = 3 per group, cohort 1). This analysis was repeated in a second cohort of animals (n = 3 per group, cohort 2). Total cellular RNA was extracted using an RNeasy plus mini kit (QIAGEN, Valencia, CA), and its quality was assessed using an Agilent BioAnalyzer using RNA 6000 Nano Chips (Agilent Technologies, Santa Clara, CA). Transcriptional profiling was determined using Illumina Sentrix BeadChips (Illumina, San Diego, CA). Total RNA was used to generate biotin-labeled cRNA with the Illumina TotalPrep RNA Amplification Kit. In short, 0.5ug of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double-stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated incorporating biotin-16-UTP. A total of 0.75µg of biotin-labeled cRNA was hybridized at 58 °C for 16 h to Illumina's Sentrix Rat Ref-12 Expression BeadChips. The arrays were washed, blocked and the labeled cRNA was detected by staining with streptavidin-Cy3. Hybridized arrays were scanned using an Illumina BeadStation 500X Genetic Analysis Systems scanner and the image data extracted using Illumina’s GenomeStudio software, version 1.6.1. For statistical analysis, the expression data were filtered to include only probes with a consistent signal on each chip and an Illumina detection p value < 0.02.
Project description:The aim of the experiment was to identify differences in gene expression in the microglia due to the presence of a brain tumor, at 14 days after a stereotactic injection of 5 x 10E4 rat C6 glioma cells into the right striatum of the rat. The control (naive) animals were left untreated. The brain hemispheres from the control animals, or the tumor-bearing hemispheres were dissected, gently dissociated into cells and the microglia (CD11b+CD45low cells) were sorted out.
Project description:Analysis of Hoechst 33342 dye-effluxing side population cells (SP cells defined as glioma stem cells, GSCs) and dye-retaining main population cells (MP cells defined as non-GSCs) that were FACS-sorted from the C6 glioma cell line stably expressing EGFP (C6-eGFP). ECM-related genes, such as Col4a1 and Col4a2, and the iron carrier gene Tf are upregulated in MP cells. Results provide the insight into molecular basis underlying the maintenance of GSCs by non-GSCs. Gene expression profiles were compared between SP and MP cells just after FACS-sorting from the whole C6-eGFP cells based on their Hoechst-effluxing abilities.
Project description:The aim of the experiment was to identify differences in gene expression in the whole brain hemisphere due to the presence of a brain tumor. At 21 days after a stereotactic injection of 5 x 10E4 rat C6 glioma cells into the right striatum of the rat the animals were sacrificed and the tumor-bearing brain hemispheres were isolated. The control animals were left untreated. The focus of the experiment was on the changes in gene expression due to the immune cells infiltrating the tumor and the surrounding brain parenchyma.
Project description:TET proteins convert 5-methylcytosine to 5-hydroxymethylcytosine, an emerging dynamic epigenetic state of DNA that can influence transcription. Evidence has linked TET1 function to epigenetic repression complexes, yet mechanistic information, especially for the TET2 and TET3 proteins, remains limited. Here, we show a direct interaction of TET2 and TET3 with O-GlcNAc transferase (OGT). OGT does not appear to influence hmC activity, rather TET2 and TET3 promote OGT activity. TET2/3-OGT co-localize on chromatin at active promoters enriched for H3K4me3 and reduction of either TET2/3 or OGT activity results in a direct decrease in H3K4me3 and concomitant decreased transcription. Further, we show that Host Cell Factor 1 (HCF1), a component of the H3K4 methyltransferase SET1/COMPASS complex, is a specific GlcNAcylation target of TET2/3-OGT, and modification of HCF1 is important for the integrity of SET1/COMPASS. Additionally, we find both TET proteins and OGT activity promote binding of the SET1/COMPASS H3K4 methyltransferase, SETD1A, to chromatin. Finally, studies in Tet2 knockout mouse bone marrow tissue extend and support the data as decreases are observed of global GlcNAcylation and also of H3K4me3, notably at several key regulators of haematopoiesis. Together, our results unveil a step-wise model, involving TET-OGT interactions, promotion of GlcNAcylation, and influence on H3K4me3 via SET1/COMPASS, highlighting a novel means by which TETs may induce transcriptional activation. ChIP-Seq experiments were performed on Illumina HiScanSQ sequencer in wild-type HEK293T cells for H3K4me3 histone marks, O-GlcNAc and HCF1, for HT-TET2, HT-TET3 and HT-OGT in HEK293T cells overexpressing those three fusion proteins and in TET2 Kd HEK293T cells for H3K4me3 histone marks. ChIP-Seqs were also performed in mouse bone marrow tissues for H3K4me3 histone marks, O-GlcNAc, endogenous Tet2 and in Tet2 Ko bone marrow tissues for H3K4me3 histone marks.