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Abstract
The Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1) protein is required for the establishment of EBV latent infection in proliferating B-lymphocytes. EBNA1 is a multifunctional DNA-binding protein that stimulates DNA replication at the viral origin of plasmid replication (OriP), regulates transcription of viral and cellular genes, and tethers the viral episome to the cellular chromosome. EBNA1 also provides a survival function to B-lymphocytes, potentially through its ability to alter cellular gene expression. To better understand these various functions of EBNA1, we performed a genome-wide analysis of the viral and cellular DNA sites associated with EBNA1 protein in a latently infected Burkitt lymphoma B-cell line. Chromatin-immunoprecipitation (ChIP) combined with massively parallel deep-sequencing (ChIP-Seq) was used to identify cellular sites bound by EBNA1. Sites identified by ChIP-Seq were validated by conventional real-time PCR, and ChIP-Seq provided quantitative, high-resolution detection of the known EBNA1 binding sites on the EBV genome at OriP and Qp. We identified at least one cluster of unusually high-affinity EBNA1 binding sites on chromosome 11, between the divergent FAM55 D and FAM55B genes. A consensus for all cellular EBNA1 binding sites is distinct from those derived from the known viral binding sites, suggesting that some of these sites are indirectly bound by EBNA1. EBNA1 also bound close to the transcriptional start sites of a large number of cellular genes, including HDAC3, CDC7, and MAP3K1, which we show are positively regulated by EBNA1. EBNA1 binding sites were enriched in some repetitive elements, especially LINE 1 retrotransposons, and had weak correlations with histone modifications and ORC binding. We conclude that EBNA1 can interact with a large number of cellular genes and chromosomal loci in latently infected cells, but that these sites are likely to represent a complex ensemble of direct and indirect EBNA1 binding sites.
Highlights
Epstein-Barr virus (EBV) is a human lymphotropic gammaherpesvirus associated with a spectrum of lymphoid and epithelial cell malignancies, including Burkitt’s lymphoma, Hodgkin’s disease, nasopharyngeal carcinoma, and post-transplant lymphoproliferative disease
ChIP-Seq Analysis of EBV and human genomes Raji Burkitt lymphoma cells were selected for EBNA1ChIP-Seq experiments because they maintain a stable copy number of EBV episomes, and because the genomes are incapable of lytic replication, which might complicate ChIP analysis
We found three major peaks for EBNA1 mapping to the family of repeats (FR), dyad symmetry (DS) and Q promoter (Qp) region, as were predicted from earlier genetic and biochemical studies of EBNA1 binding to EBV DNA
Summary
Epstein-Barr virus (EBV) is a human lymphotropic gammaherpesvirus associated with a spectrum of lymphoid and epithelial cell malignancies, including Burkitt’s lymphoma, Hodgkin’s disease, nasopharyngeal carcinoma, and post-transplant lymphoproliferative disease (reviewed in [1,2]). EBNA1 is a nuclear phosphoprotein that binds with high-affinity to three major DNA sites within the EBV genome [5](reviewed in [6]). EBNA1 binding to OriP is essential for plasmid DNA replication and episome maintenance, and can function as a transcriptional enhancer of the C promoter (Cp) [7,8]. At the Q promoter (Qp), EBNA1 binds to two 18 bp sequences immediately downstream of the transcriptional start site, and functions as an inhibitor of transcription initiation and mRNA accumulation [9]. In addition to direct DNA binding through the C-terminal domain, EBNA1 tethers the EBV genome to metaphase chromosomes through its amino terminal domain [13,14]. The precise chromosomal sites, proteins, or structures through which EBNA1 attaches during metaphase are not completely understood [14,15,16]
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