Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling by array of human LB31 cells infected with a series of EBNA3-knockout Epstein-Barr Viruses

ABSTRACT: Epstein-Barr virus (EBV) is able to drive the transformation of B-cells, resulting in the generation of lymphocyte cell lines (LCLs) in vitro. EBV nuclear proteins EBNA3A and EBNA3C are necessary for efficient transformation, while EBNA3B is dispensable. We describe a transcriptome analysis of BL31 cells infected with a series of EBNA3-knockout EBVs, including one deleted for all three EBNA3 genes. BL31 cells were infected with a variety of EBV mutants and their revertants, generated in the EBV-BAC system developed by Henri-Jacques Delecluse and Wolfgang Hammerschmidt. These are mutants of the EBV EBNA3 genes - specifically individual knockouts of EBNA3A, EBNA3B and EBNA3C (in this case the first exon of EBNA3C was retained) and a deletion of the total EBNA3 locus (ie all three EBNA3 genes missing).
RNA from cultures of cell lines carrying these mutant EBVs (3-4 independent cell lines for each mutant; 2-3 for each revertant, plus three each for uninfected BL31 and parental B95-8 BAC-infected lines) was isolated using RNeasy midi kits (qiagen) and then ribominus-treated and then labelled and hybridised to Affymetrix Human Exon 1.0ST arrays. Array analysis was performed using the MMBGX algorithm developed by Ernest Turro and Alex Lewin.

ORGANISM(S): Homo sapiens

SUBMITTER: Rob White

PROVIDER: E-MEXP-2767 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Human aging implies many phenotypic modifications and an increased susceptibility to many common diseases, phenomena that cannot be fully explained by the constrained genetic setting. An alternative pathway that could explain the age-associated alterations is epigenetic drifts. To address this issue, we performed Whole Genome Bisulfite Sequencing (WGBS) of a newborn and of a centenarian. The centenarian DNA exhibited a significant loss in DNA methylation and a poor correlation in the methylation status of neighboring CpGs throughout the genome. From a gene-regulatory region standpoint, we observed that the DNA hypomethylation events occurred mainly at CpG-poor promoters and in tissue-specific genes, whereas a greater level of DNA methylation was observed in CpG island promoters. Most importantly, we extended the study to a larger cohort of newborn and nonagenarian samples using a 450,000 CpG-site DNA methylation microarray. The 450K DNA methylation approach enabled validation of the WGBS data and revealed additional age-related DNA methylation events. Interestingly, we also observed the DNA methylation fingerprint characteristic of a healthy aged cell in samples from patients with premature-aging disorders such as Hutchinson-Gilford progeria and Werner syndrome. Overall, we suggest that defects in the physiological DNA methylation profile contribute to the process of human aging. As the disease associated cells were immortalized B cells, the effect of EBV immortalization was determined in advance to the study using immortalized and naive B cells. DNA was quantified by Quant-iT PicoGreen dsDNA Reagent (Invitrogen) and the integrity was analyzed in a 1.3% agarose gel. Bisulfite conversion of 600 ng of each sample was perform according to the manufacturer's recommendation for Illumina Infinium Assay. Effective bisulphite conversion was checked for three controls that were converted simultaneously with the samples. 4 ul of bisulfite converted DNA were used to hybridize on Infinium HumanMethylation 450 BeadChip, following Illumina Infinium HD Methylation protocol. Chip analysis was performed using Illumina HiScan SQ fluorescent scanner. The intensities of the images are extracted using GenomeStudio (2010.3) Methylation module (1.8.5) software. Methylation score of each CpG is represented as beta value.

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Transcription profiling by array of human LB31 cells infected with a series of EBNA3-knockout Epstein-Barr Viruses

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