Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Epstein Barr virus Nuclear Antigen 3C regulated genes in Lymphoblastoid Cell Lines

ABSTRACT: Epstein Barr virus (EBV) nuclear antigen 3C (EBNA3C) is an essential transcription factor for initiating and maintaining human B lymphocyte transformation to lymphoblastoid cell lines (LCLs). To comprehensively identify EBNA3C regulated cell genes in LCLs, oligonucleotide arrays were used to compare RNA abundances in 3 different LCLs transformed by an EBV that conditionally expresses EBNA3C. Cell RNA levels were assessed in actively growing LCLs, under non-permissive or permissive conditions or under non-permissive conditions after transcomplementation with wild type EBNA3C. A two-way ANOVA model with covariates including the 3 different clone effects and the 3 EBNA3C expression levels, identified 550 EBNA3C regulated genes, with False Discovery Rate <0.01 and >1.5 fold change. A seeded Bayesian network analysis of the 80 most significantly EBNA3C regulated genes that changed >1.5 fold, positioned RAC1, LYN and TNF upstream of other EBNA3C regulated genes. Further, Gene Set Enrichment Assay (GSEA) identified EBNA3C regulated genes to be enriched for MAP kinase signaling, cytokine-cytokine receptor interactions, JAK-STAT signaling, and cell adhesion molecule effects, implicating these pathways in LCL growth or survival. Moreover, 106 EBNA3C regulated genes could be placed in protein interaction networks. Since CXCL12 and CXCR4 signaling are implicated in LCL growth and were EBNA3C up-regulated, up-regulation of CXCL12 was validated by qRT-PCR and effects on induced LCL migration were confirmed. EBNA3C regulated genes significantly overlapped with EBNA2 and EBNA3A regulated genes, consistent with a central role for RBP/CSL in these effects. RNAs from three different Lymphoblastoid Cell Lines(LCLs) expressing conditional EBNA3C grown under permissive or non-permissive conditions for 7 days; the same LCLs transcompletemented with EBNA3C expressed in trans at full wild-type level were used to identify EBNA3C regualted cellular genes. Total cell RNAs were hybrdized to Affymetrix U-133 Plus 2.0 microarrays. A two way ANOVA model was developed with covariates including the 3 different clone effects and the 3 EBNA3C expression levels and identified 550 EBNA3C regulated genes.

ORGANISM(S): Homo sapiens

SUBMITTER: Bo Zhao

PROVIDER: E-GEOD-24362 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

Epstein-Barr virus nuclear antigen 3C regulated genes in lymphoblastoid cell lines.

Zhao Bo B Mar Jessica C JC Maruo Seiji S Lee Sungwook S Gewurz Benjamin E BE Johannsen Eric E Holton Kristina K Rubio Renee R Takada Kenzo K Quackenbush John J Kieff Elliott E

Proceedings of the National Academy of Sciences of the United States of America 20101220 1

EBV nuclear antigen 3C (EBNA3C) is an essential transcription factor for EBV transformed lymphoblast cell line (LCL) growth. To identify EBNA3C-regulated genes in LCLs, microarrays were used to measure RNA abundances in each of three different LCLs that conditionally express EBNA3C fused to a 4-OH-Tamoxifen-dependent estrogen receptor hormone binding domain (EBNA3CHT). At least three RNAs were assayed for each EBNA3CHT LCL under nonpermissive conditions, permissive conditions, and nonpermissive ...[more]

PMID: 21173222

Similar Datasets

Project description:Epstein-Barr Virus (EBV) Latent Membrane Protein 1 (LMP1) transforms rodent fibroblasts and is expressed in most EBV-associated malignancies. LMP1 Transformation Effector Site 2 (TES2)/C-Terminal Activation Region 2 (CTAR2) activates NF-kappaB, p38, JNK, ERK and IRF7 pathways. We have investigated LMP1 TES2 genome-wide RNA effects at 4 time points after LMP1 TES2 expression in HEK 293 cells. Using a False Discovery Rate (FDR) of < 0.001 after correction for multiple hypotheses, LMP1 TES2 caused > 2-fold changes in 1916 mRNAs; 1479 RNAs were up-regulated and 437 down-regulated. In contrast to TNFalpha stimulation, which transiently up-regulates many target genes, LMP1 TES2 maintained most RNA effects through the time course, despite robust and sustained induction of negative feedback regulators, such as IkappaBalpha and A20. LMP1 TES2 regulated RNAs encode many NF-kappaB signaling proteins and secondary interacting proteins. Consequently, many LMP1 TES2-regulated RNAs encode proteins that form an extensive interactome. Gene Set Enrichment Analyses found LMP1 TES2 up-regulated genes to be significantly enriched for Pathways in Cancer, B-and T-cell receptor signaling, and Toll-like receptor signaling. Surprisingly, LMP1 TES2 and IkappaBalpha super-repressor co-expression decreased LMP1 TES2 RNA effects to only 5 RNAs with FDR<0.001 and >2 fold change. Thus, canonical NF-kappaB activation is critical for almost all LMP1 TES2 RNA effects in HEK-293 cells and a more significant therapeutic target than previously appreciated. An LMP1 double point mutant (P204A, Q206A) was used to construct a HEK-293 TET-On LMP1 TES2 cell line. Stable cell clones were selected that carry an inducible system for LMP1 TES2 expression. The Tet-system is composed of three parts: 1) the LMP1 TES2 cDNA cloned into the tetracycline-regulated pJEF vector; 2) a tetracycline suppressor (tTS) that binds Tet-operator sites in the absence of tetracyclines and silences expression; 3) a reverse-tetracycline transactivator fused to the 4-hydroxy tamoxifen (4HT) ligand binding domain (rTTA M2). LMP1 expression was induced by addition of doxycycline (1 ug/ml) and 4HT (100 nM). For simultaneous inducible expression of LMP1 TES2 and an IkBa super-repressor, a stable cell line was derived. A pJEF vector encoding IkBa residues 37-317 was introduced into the inducible LMP1 TES2 cell line. Cell lines were cultured with DMEM (GIBCO) supplemented with 10% tetracycline-free serum (Clontech). LMP1 expression was induced by addition of tetracycline (1 ug/ml). RNA samples were collected from triplicate samples using RNABee (Qiagen) according to the manufacturer’s instructions from 293 cells induced for LMP1 TES2 (and IkBa super-repressor, where indicated) expression at 0, 6, 9, 12 and 24 hours of induction. Cells were in the log-phase of growth. Gene expression profiles were assayed using the Affymetrix HU-133 plus2 GeneChip according to the manufacturer's instructions. Real-time RT-PCR was performed with the Power SYBR Green RNA-to-CT™ 1-Step Kit (Applied Biosystems, Foster City, CA). Fold changes were determined using delta delta Ct method and normalized by GAPDH expression levels. RNA expression data were normalized using RMA and array quality assessed by NUSE and RLE scores. The data was collected according to methods that fall under the MIAME standards.

Project description:Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs) are a widely used renewable resource for functional genomic studies in humans. The ability to accumulate multidimensional data pertaining to the same individual cell lines, from complete genomic sequences to detailed gene regulatory profiles, further enhances the utility of LCLs as a model system. However, the extent to which LCLs are a faithful model system is relatively unknown. We have previously shown that gene expression profiles of newly established LCLs maintain a strong individual component. Here, we extend our study to investigate the effect of freeze-thaw cycles on gene expression patterns in mature LCLs, especially in the context of inter-individual variation in gene regulation. We found a profound difference in the gene expression profiles of newly established and mature LCLs. Once newly established LCLs undergo a freeze-thaw cycle, the individual specific gene expression signatures become much less pronounced as the gene regulatory programs in LCLs from different individuals converge to a more uniform profile, which reflects a mature transformed B cell phenotype. As expected, previously identified eQTLs are enriched among the relatively few genes whose regulations in mature LCLs maintain marked individual signatures. We thus conclude that findings and insight drawn from gene regulatory studies in mature LCLs are generally not affected by artificial nature of the LCL model system and are likely to faithfully reflect regulatory interactions in primary tissues. However, our data indicate that many aspects of primary B cell biology cannot be observed and studied in mature LCL cultures. We obtained unpurified buffy coats (of a unit of blood) from six unrelated healthy individuals of Caucasian ethnicity (age range: 20-45). We isolated CD20+ B cells and established six independent cultures of LCLs between October 2009 and January 2010. From each of these samples, we obtained genome wide gene expression data using Illumina HumanHT-12 v3 Expression BeadChip arrays. We refer to data from these experiments as M-bM-^@M-^\cycle 0M-bM-^@M-^] to acknowledge the fact that the LCLs from this cycle were newly established and therefore not frozen/thawed. Between February 2011 and October 2012, we thawed each of these LCL cultures every 3 months and cultured them until obtaining ~10 million cells (February 2011=cycle 1, June 2011=cycle 2, October 2011=cycle 3, February 2012=cycle 4, June 2012=cycle 5, and October 2012=cycle 6). We used Illumina HumanHT-12 v4 Expression BeadChip arrays and obtained genome wide gene expression data on cycle 2, cycle 4 and cycle 6 LCLs. At each hybridization batch we included a subset of RNA samples that were also hybridized in a previous batch. This allowed us to effectively consider the batch effect on gene expression profiles. Our final dataset included genome wide gene expression data from 187 samples collected at 4 different time points.

Dataset Information

Epstein Barr virus Nuclear Antigen 3C regulated genes in Lymphoblastoid Cell Lines

Publications

Epstein-Barr virus nuclear antigen 3C regulated genes in lymphoblastoid cell lines.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets