Project description:ChIP-seq experiments with the antibody BZ1 directed against the EBV protein BZLF1 prior to and 15 hours post induction with doxycycline (100 ng/ml). The doxycycline regulated conditional BZLF1 allele is encoded by an inducible oriP plasmid vector stably introduced into DG75 cells. Experiments were performed as duplicates.
Project description:(i) Transcripts of DG75 iBZLF1 cells were analyzed comparing non-induced cells and cells induced with doxycycline (100 ng/ ml) for 6 h. Doxycycline induces the expression of the full length viral transcription factor BZLF1. (ii) For control purposes, the transcripts of DG75 cells equipped with a doxycycline-regulated truncated BZLF1 allele lacking its transactivation domain (DG75 iBZLF1 AD-truncated) were analyzed prior to and after induction with doxycycline for 6 h. (iii) For an additional control, the transcripts of parental, unmodified DG75 cells were analyzed prior to and after adding doxycycline for 6 h. All experiments were performed as triplicates. The analysis is based on Homo sapiens (human) genome assembly GRCh37 (hg19) from Genome Reference Consortium. Artificial ERCC Spike-in RNAs (Thermo Scientific) were added as external controls.
Project description:The parental DG75 cell line is EBV negative and cannot express the viral BZLF1 protein, thus, this set of experiments served as a negative control. After ChIP, peaks were identified in both the human and viral (EBV (Human Herpesvirus 4) genome KF717093.1) genome. Only few peaks were identified. Experiments were performed as duplicates.
Project description:Identification of specific chromatin interactions of 49 selected genes with the Capture-C technique in DG75 iBZLF1 cells prior to and 15 h after expression of EBV's protein BZLF1. The experiments were performed as triplicates.
Project description:The aim of the experiment was to identify accessible (open) chromatin regions in the genome prior to and post induction of EBV's BZLF1 protein. Two versions of BZLF1 were employed: full-length and activation-domain (AD)-truncated BZLF1 non-induced and induced for 15 h. The experiments were performed as triplicates.
Project description:ChIP-seq experiments with the antibody BZ1 directed against the EBV protein BZLF1 prior to and 15 hours post induction with doxycycline (100 ng/ml). The doxycycline regulated conditional BZLF1 allele is encoded by an inducible plasmid stably introduced into Raji cells. Experiments were performed as duplicates.
Project description:Epstein-Barr virus (EBV) infection converts resting human B cells into permanently growing lymphoblastoid cell lines (LCLs). The viral Epstein-Barr virus nuclear antigen 2 (EBNA2) plays key role in this process. It preferentially binds to B cell enhancers and establishes a specific viral and cellular gene expression program in LCLs. The cellular DNA binding factor CBF1/CSL serves as a sequence specific chromatin anchor for EBNA2. The ubiquitous expression of this highly conserved protein raises the question whether additional cellular factors might determine EBNA2 chromatin binding selectively in B cells. Here we used CBF1 deficient B cells to identify cellular genes up or downregulated by EBNA2 as well as CBF1 independent EBNA2 chromatin binding sites. Both, CBF1 independent EBNA2 target genes and chromatin binding sites are less frequent than CBF1 dependent EBNA2 functions. CBF1 independent EBNA2 binding sites are highly enriched for EBF1 binding motifs. We show that EBNA2 binds to EBF1 in CBF1 proficient and deficient B cells and requires EBF1 to bind to CBF1 independent binding sites. Our results identify EBF1 as a co-factor of EBNA2 which conveys B cell specificity to EBNA2. In order to test, if EBNA2 can exert any functions in the absence of its DNA adaptor CBF1, a microarray based genome wide screen for EBNA2 target genes in DG75 B cells that are either proficient (wt) or deficient (ko) for CBF1 was performed. CBF1 deficient DG75 cells (SM224.9) cells had been generated by gene targeting using homologous recombination in the somatic B cell line DG75. Both cell lines, the CBF1 proficient DG75 parental cell line and the CBF1 deficient somatic knock-out cell line constitutively express an estrogen receptor (ER) hormone binding domain EBNA2 fusion protein (ER/EBNA2). ER/EBNA2 is retained in the cytoplasm of the cell but is rapidly activated and translocated to the nucleus in response to estrogen. For expression profiling, DG75, DG75 CBF1 ko (SM224.9), DG75 ER/EBNA2 CBF1 wt (SM295 D6) and DG75 ER/EBNA2 CBF1 ko (SM296 D3) cells were cultured in estrogen supplemented media for 24 h, total cellular RNAs were harvested and processed for the hybridization of gene arrays. The cellular system used for this study has been published: Maier S, Santak M, Mantik A, Grabusic K, Kremmer E, Hammerschmidt W, et al. A somatic knockout of CBF1 in a human B-cell line reveals that induction of CD21 and CCR7 by EBNA-2 is strictly CBF1 dependent and that downregulation of immunoglobulin M is partially CBF1 independent. Journal of Virology. 2005 Jul;79(14):8784-92. PubMed PMID: 15994772.
Project description:Lytic infection by the Epstein-Barr virus (EBV) poses numerous health risks, such as infectious mononucleosis and lymphoproliferative disorder. We demonstrate that JQ1 and other BET inhibitors block two different steps in the sequential cascade of the EBV life cycle: expression of the immediate-early gene BZLF1 and lytic genome replication. This represents a novel mode of action for antiviral drugs that may increase efficacy and decrease emergence of resistance. The sequenced total DNA data in this series show that JQ1 causes a decrease in EBV genome replication upon antibody induction.