Project description:Experimental design Using Circular chromosome conformation capture (4C) assay to uncovers epigenetically regulated intra- and inter-chromosomal interaction network To examine restrictions imposed by the nuclear structure on such cis/trans-chromosomal networks, we developed a high-throughput screening assay 4C and identified 114 different sequences from all autosomes in mouse liver cells. To check the relative frequencies of interactions, the 114 sequences were PCR amplified and spotted on glass to make dedicated microarray. The microarray and 3C validation showed that most of the sequences interact with primarily the maternally inherited H19 imprinting control region (ICR). The epigenetic feature of these interactions was highlighted by the observation that 19% of 4C library sequences are derived from 11 different imprinted domains. In some of these instances, differentially methylated regions (DMRs) interact both in cis and in trans. Moreover, the patterns of chromosomal interactions undergo reprogramming during in vitro maturation of embryonic stem cells. We propose that the nuclear organization of mammalian cells displays considerable plasticity to potentially throw new light on development, cancer epigenetics and evolution of imprinting. Key words: Chromosome conformation capture, CTCF, ICR(Imprinting Control Region) Experimental factors: Genetic variation Experimental design: The 4C samples from both neonatal liver cell SD7 x 142* and 142* x SD7; differentiated embryonic cells and undifferentiated embryonic cells were amplified, fluorescently labeled, and hybridized to 4C dedicated microarray. Quality control steps taken: using pooled samples and gDNA as input, about half of most frequently interactions were confirmed by replicates and dyes swaps. Processed data and a data transformation protocol can be download from ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/MEXP/E-MEXP-870/

Project description:Comparison of polysomal profiles of murine adult olig2 cortical progenitors, murine tumor olig2 cells derived from hPDGF-B-driven glioblastomas, and murine olig2 proliferative recruited glioma cells contributing to the tumor mass but not derived from the cell of origin An unbiased way to define a cell population lies in identification of its gene expression signature and subsequent comparison to gene expression signatures of known normal or cancer cells to define its position on the axis of tumorigenesis. To quantify similarities and differences in the expression profiles of recruited cells and tumor cells using microarray analysis, we used the bacTRAP technology that allows immunoprecipitation of polysomes from specific cell types in vivo (Heiman et al 2008, Doyle et al 2008). We compared polysomal expression profiles of hPDGFb-driven Ntv-a Ink4a/Arf+/- olig2 mouse glioma cells representing a histologically defined glioma population, recruited olig2 cells derived from Ink4a/Arf+/- olig2 RP-eGFP mice transplanted with non-fluorescent glioma cells, and normal adult cortical olig2 mouse progenitors, using Affymetrix 430 mouse 2.0 chips and Genespring GX10 software (Doyle et al 2008, Heiman et al 2008). Translational profiles of recruited olig2 cells and glioma olig2 cells were similar to each other and clearly distinct from the normal olig2 progenitors, majority of the differences accounted for by the “statistically significantly changed” set of mRNAs (ANOVA, p<0.05), and sample clustering reduced upon removal of the ANOVA-tested mRNA set. Unsupervised hierarchical mRNA clustering likewise indicated that recruited olig2 cells clustered more closely to glioma olig2 cells. The caveat is that olig2 expression in the adult normal brain of bacTRAP olig2 RP-eGFP mice labels both progenitors and mature oligodendrocytes, while recruited and tumor olig2 cells are almost entirely progenitors; therefore, some of the differences seen in the comparative analysis of normal, tumor and recruited olig2 cells may in part result from the shift in relative abundance of different progenitor and mature olig2 cell populations. Normal olig2 progenitors were collected from three replicates of pooled cortices of three olig2 RP-eGFP bacTRAP reporter mice. Mouse gliomas induced by injection of a non-fluorescent RCAS-hPDGFb (Shih et al 2004) or by transplantation of muring glioma cells derived from Ntv-a gliomas induced by the non-fluorescent RCAS-hPDGFb were collected from Ntv-a olig2 RP-eGFP bacTRAP reporter mice and immunoprecipitated; each tumor was processed as a separate sample. Briefly, mouse tissues were collected into ice-cold cyclohexamide-containing buffer, homogenized, cells were lysed in NP-40 and DHPC-containing buffer, centrifuged at 20,000g for 15 min, supernatant incubated with anti-eGFP-conjugated protein G beads 30 min at 4C, washed and RNA collected using Trizol reagent as per manufacturer’s instructions (Doyle et al., 2008; Heiman et al., 2008). RNA was purified, concentrated using Qiagen RNeasy kit, its quality confirmed by Agilent Bioanalyzer, 15ng per sample amplified using Affymetrix Two-Cycle Amplification kit and hybridized to Affymetrix 430 mouse 2.0 chips as described in Doyle et al., 2008; Heiman et al., 2008. Data was analyzed using Genespring GX10 software, as described in the text and in Doyle et al., 2008; Heiman et al., 2008. Briefly, samples were normalized using GCRMA, filtered to remove probe sets with low intensity, and analyzed using ANOVA (p<0.05) with SNK post-hoc and Benjamini and Hochberg FDR multiple testing correction. Probe quality, Pearson’s correlation, hierarchical clustering, heatmaps, gene profile plots were generated using Genespring GX10 software. Pathway analysis was performed using Ingenuity as per manufacturer’s instructions.

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