Project description:We present results of RNA-Seq, Ribo-Seq, and RIP-Seq (YB-1, YB-3) experiments performed in HEK293T cells, as well as in HEK293T cells with YB-1 knockout and overexpression. The data shows YB-1 function as a global translation inhibitor and YB-3 ability to substitute YB-1 in its function in YB-1 knockout mutant.
Project description:EBV encodes 44 mature miRNAs, a number of which have been found to promote carcinogenesis by targeting host genes. To determine the biological functions of the BART cluster miRNAs, we singly transfected these miRNAs into HEK293T cells. Interestingly, we found that overexpression of EBV-miR-BART6-3p in HEK293T cells dramatically altered HEK293T cell shape. To explore the mechanism of EBV-miR-BART6-3p-mediated inhibition of cell migration and invasion, we attempted to identify its downstream host cellular genes by whole genome microarray, which contains probes to all known human protein coding genes (mRNAs) and long non-coding RNA genes (lncRNAs).
Project description:To identify the directly bound transcripts of Flag antibody (the backgroud control for our METTL16 RIP-seq), RNA immunoprecipitation sequencing (RIP-seq) was conducted HEK293T. Briefly, HEK293T cells were infected with pmiRNA1-empty vector. Only the GFP-positive cells were used for study and expanded in DMEM medium.
Project description:EBNA1 is the EBV-encoded nuclear antigen required for viral episome maintenance during latency. EBNA1 is a sequence specific DNA binding protein with high affinity binding sites for the viral genome, especially OriP. EBNA1 can also bind sequence specifically to a large number of sites in the host cellular genome, but the function of these binding sites has remained elusive. EBNA1 is also known to provide a host cell survival function, but the molecular mechanisms accounting for this function are not completely understood. Here, we show by integrating ChIP-Seq and RNA-Seq with experimental validation that MEF2B, IL6R, and EBF1 are high confidence target genes of EBNA1 that are essential for viability of B-lymphocytes latently infected with EBV. We show that EBNA1 binds to ~1000 sites with many, but not all, universally bound in different cell types, including Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC). We find that a large subset of EBNA1 binding sites are located proximal to transcription start sites and correlate genome-wide with transcription activity. EBNA1 bound to genes of high significance for B-cell growth and function, including MEF2B, IL6R, EBF1, RNF145, POU2F1, KDM4C, FGR, EGFR, LAIR, CDC7, CD44, and IL17A. EBNA1 depletion from latently infected LCLs results in the loss of cell proliferation, and the loss of gene expression for some EBNA1-bound genes, including MEF2B, EBF1, and IL6R. Depletion of MEF2B, EBF1, or IL6R partially phenocopies EBNA1-depletion by decreasing EBV-positive cell growth and viability. These findings indicate that EBNA1 binds to a large cohort of cellular genes important for cell viability, and implicates EBNA1 as a master coordinator of host cell gene expression important for enhanced survival of latently infected cells. Examination of EBNA1 binding in Raji, MutuI, LCL and C666-1 cells and EBNA1 knockdown effect on mRNA gene expression in LCL
Project description:EBNA1 is the EBV-encoded nuclear antigen required for viral episome maintenance during latency. EBNA1 is a sequence specific DNA binding protein with high affinity binding sites for the viral genome, especially OriP. EBNA1 can also bind sequence specifically to a large number of sites in the host cellular genome, but the function of these binding sites has remained elusive. EBNA1 is also known to provide a host cell survival function, but the molecular mechanisms accounting for this function are not completely understood. Here, we show by integrating ChIP-Seq and RNA-Seq with experimental validation that MEF2B, IL6R, and EBF1 are high confidence target genes of EBNA1 that are essential for viability of B-lymphocytes latently infected with EBV. We show that EBNA1 binds to ~1000 sites with many, but not all, universally bound in different cell types, including Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC). We find that a large subset of EBNA1 binding sites are located proximal to transcription start sites and correlate genome-wide with transcription activity. EBNA1 bound to genes of high significance for B-cell growth and function, including MEF2B, IL6R, EBF1, RNF145, POU2F1, KDM4C, FGR, EGFR, LAIR, CDC7, CD44, and IL17A. EBNA1 depletion from latently infected LCLs results in the loss of cell proliferation, and the loss of gene expression for some EBNA1-bound genes, including MEF2B, EBF1, and IL6R. Depletion of MEF2B, EBF1, or IL6R partially phenocopies EBNA1-depletion by decreasing EBV-positive cell growth and viability. These findings indicate that EBNA1 binds to a large cohort of cellular genes important for cell viability, and implicates EBNA1 as a master coordinator of host cell gene expression important for enhanced survival of latently infected cells.
Project description:RIP-Chip was performed on DG75-eGFP, DG75-10/12, BCBL-1, BL41, BL41 B95.8 and Jijoye using anti-human Ago2 (11A9) antibodies. Anti-BrdU antibodies were used as controls for DG75-eGFP, DG75-10/12 and BCBL-1. Total RNA was used as control for BL41, BL41 B95.8 and Jijoye. Samples were analyzed on Affymetrix Gene ST 1.0 Arrays (2 independent biological replicates / sample) KSHV, EBV and cellular miRNA targets were determined by RIP-Chip using monoclonal antibodies to human Ago2
Project description:Subpopulations of B-lymphocytes traffic to different sites and organs to provide diverse and tissue-specific functions. Here, we provide evidence that epigenetic differences confer a neuroinvasive phenotype. An EBV+ B cell lymphoma cell line (M14) with low frequency trafficking to the CNS was neuroadapted to generate a highly neuroinvasive B-cell population (MUN14). MUN14 B cells efficiently infiltrated the CNS within one week and produced neurological pathologies. We compared the gene expression profiles of viral and cellular genes using RNA-Seq and identified one viral (EBNA1) and several cellular gene candidates, including secreted phosphoprotein 1/osteopontin (SPP1/OPN), neuron navigator 3 (NAV3), CXCR4, and germinal center-associated signaling and motility protein (GCSAM)) that were selectively upregulated in MUN14. ATAC-Seq and ChIP-qPCR revealed that these gene expression changes correlated with epigenetic changes at gene regulatory elements. The neuroinvasive phenotype could be attenuated with a neutralizing antibody to OPN, confirming the functional role of this protein in trafficking EBV+ B cells to the CNS. These studies indicate that B-cell trafficking to the CNS can be acquired by epigenetic adaptations and provide a new model to study B-cell neuroinvasion associated CNS lymphoma and autoimmune disease of the CNS, including multiple sclerosis (MS).cell infection to investigate the underlying mechanisms that control EBV-induced B-cell immortalization. ATAC-seq revealed that over a third of accessible chromatin is altered with the most perturbed sites overlapping Ets-family, including PU.1 and RUNX1 transcription factors. EBV nuclear antigens (EBNAs) clustered with different gene categories and RNA-seq identified the transcriptional response of these gene. Focusing on EBNA1 revealed a selection of gene targets involved in nucleotide metabolism. Metabolomics indicated that adenosine and purine metabolism are significantly altered by EBV immortalization, and we validated that adenosine deaminase (ADA) is a direct and critical target of EBNA1 and the EBV-directed immortalization process. These findings reveal that purine metabolism and ADA inhibitors may be a useful therapeutic for EBV-driven lymphoid cancers
Project description:Subpopulations of B-lymphocytes traffic to different sites and organs to provide diverse and tissue-specific functions. Here, we provide evidence that epigenetic differences confer a neuroinvasive phenotype. An EBV+ B cell lymphoma cell line (M14) with low frequency trafficking to the CNS was neuroadapted to generate a highly neuroinvasive B-cell population (MUN14). MUN14 B cells efficiently infiltrated the CNS within one week and produced neurological pathologies. We compared the gene expression profiles of viral and cellular genes using RNA-Seq and identified one viral (EBNA1) and several cellular gene candidates, including secreted phosphoprotein 1/osteopontin (SPP1/OPN), neuron navigator 3 (NAV3), CXCR4, and germinal center-associated signaling and motility protein (GCSAM)) that were selectively upregulated in MUN14. ATAC-Seq and ChIP-qPCR revealed that these gene expression changes correlated with epigenetic changes at gene regulatory elements. The neuroinvasive phenotype could be attenuated with a neutralizing antibody to OPN, confirming the functional role of this protein in trafficking EBV+ B cells to the CNS. These studies indicate that B-cell trafficking to the CNS can be acquired by epigenetic adaptations and provide a new model to study B-cell neuroinvasion associated CNS lymphoma and autoimmune disease of the CNS, including multiple sclerosis (MS).cell infection to investigate the underlying mechanisms that control EBV-induced B-cell immortalization. ATAC-seq revealed that over a third of accessible chromatin is altered with the most perturbed sites overlapping Ets-family, including PU.1 and RUNX1 transcription factors. EBV nuclear antigens (EBNAs) clustered with different gene categories and RNA-seq identified the transcriptional response of these gene. Focusing on EBNA1 revealed a selection of gene targets involved in nucleotide metabolism. Metabolomics indicated that adenosine and purine metabolism are significantly altered by EBV immortalization, and we validated that adenosine deaminase (ADA) is a direct and critical target of EBNA1 and the EBV-directed immortalization process. These findings reveal that purine metabolism and ADA inhibitors may be a useful therapeutic for EBV-driven lymphoid cancers