Project description:<p>In this study, lymphoblastoid cell line cultures (LCLs) from women with premenstrual dysphoric disorder (PMDD) and asymptomatic controls were compared via whole-transcriptome sequencing (RNA-seq) during untreated (ovarian steroid-free) conditions and after estradiol and progesterone treatment. In untreated LCLs, pathway analysis of the LCL transcriptome revealed, among others, over-expression of ESC/E(Z) complex genes (an ovarian steroid-regulated gene silencing complex) with significant overexpression of MTF2, PHF19, and SIRT1 (P<0.05) in cells from women with PMDD compared with controls. Finally, mRNA expression of several ESC/E(Z) complex genes were increased by progesterone in controls only, and decreased by estradiol in PMDD LCLs. </p>
Project description: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.
Project description: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.
Project description:To identify gene expression changes and pathways induced by interferon-β (IFN-β) in B cells, we studied the in vitro response of EBV-transformed B cells (lymphoblast cell lines-LCLs). LCLs were derived from an MS patient repository. Whole genome expression analysis identified 115 genes that were more than two-fold differentially up-regulated following IFN-β exposure, with over 50 novel IFN-β response genes. Pathways analysis demonstrated that IFN-β affected LCLs in a similar manner to other cell types by activating known IFN-β canonical pathways. Additionally, IFN-β increased the expression of innate immune response genes, while down-regulating many B cell receptor pathway genes and genes involved in adaptive immune responses. Several of the novel response genes were tested and validated as IFN-β response genes in human primary B cells.
Project description:To identify gene expression changes and pathways induced by interferon-? (IFN-?) in B cells, we studied the in vitro response of EBV-transformed B cells (lymphoblast cell lines-LCLs). LCLs were derived from an MS patient repository. Whole genome expression analysis identified 115 genes that were more than two-fold differentially up-regulated following IFN-? exposure, with over 50 novel IFN-? response genes. Pathways analysis demonstrated that IFN-? affected LCLs in a similar manner to other cell types by activating known IFN-? canonical pathways. Additionally, IFN-? increased the expression of innate immune response genes, while down-regulating many B cell receptor pathway genes and genes involved in adaptive immune responses. Several of the novel response genes were tested and validated as IFN-? response genes in human primary B cells. Total RNA from LCL samples treated for four hours with interferon beta (100u/ml) was compared to RNA from untreated LCLs (paired analysis). LCL samples were from patients with MS.
Project description:Statin-induced gene expression differences observed in LCLs may be influenced by their transformation, and thus differ from those observed in native B-cells. To assess this possibility, we prepared LCLs and purified B-cells from the same donors, and compared mRNA profiles after 24hr incubation with simvastatin (2M-BM-5M) or sham buffer. We prepared LCLs and purified B-cells from the same donors, and compared mRNA profiles after 24hr incubation with simvastatin (2M-BM-5M) or sham buffer. Genes involved in cholesterol metabolism were similarly regulated between the two cell types under both the statin and sham treated conditions, and the statin-induced changes were significantly correlated.
Project description:In this study, we investigated PPA-induced changes in gene expression profiles of lymphoblastoid cell lines (LCLs) from unaffected individuals, compared with gene expression profiles of LCLs from sex-matched siblings with ASD. Global gene expression profiling analysis revealed that 96 genes in LCLs from the unaffected individuals were significantly altered after PPA exposure, exhibiting expression levels similar to those of their respective siblings with autism. Biological pathway analyses of these PPA-responsive genes suggested significant association with many neurological functions associated with autism, including synaptic transmission, neuronal cell differentiation and apoptosis. Moreover, we demonstrated that PPA also deregulated several of the responsive genes, including APOE, LIFR, NR3C1, and PTK2, in the neuroblastoma cell line SH-SY5Y. Functional analyses further showed that PPA exposure negatively impacted neurite outgrowth and promoted neurodegeneration in the human neuronal cell model. This study indicates that PPA exposure induces global changes in gene expression profiles of LCLs from non-autistic individuals that reflect expression patterns of LCLs from affected individuals.
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.
Project description:We performed an evolutionary comparison of the binding of the TF CTCF in human, chimpanzee, gorilla, orang-utan, macaque, baboon and marmoset using lymphoblastoid cell lines (LCLs). We also probes YY1 binding in human, chimpanzee, orang-utan and baboon LCLs as well as human and mouse liver.