Epstein-Barr virus (EBV) nuclear antigens EBNALP (LP) and EBNA2 (E2) are coexpressed in EBV-infected B lymphocytes and are critical for lymphoblastoid cell line outgrowth. LP removes NCOR and RBPJ repressive complexes from promoters, enhancers, and matrix-associated deacetylase bodies, whereas E2 activates transcription from distal enhancers. LP ChIP-seq analyses identified 19,224 LP sites of which ∼50% were ±2 kb of a transcriptional start site. LP sites were enriched for B-cell transcription factors (TFs), YY1, SP1, PAX5, BATF, IRF4, ETS1, RAD21, PU.1, CTCF, RBPJ, ZNF143, SMC3, NFκB, TBLR, and EBF. E2 sites were also highly enriched for LP-associated cell TFs and were more highly occupied by RBPJ and EBF. LP sites were highly marked by H3K4me3, H3K27ac, H2Az, H3K9ac, RNAPII, and P300, indicative of activated transcription. LP sites were 29% colocalized with E2 (LP/E2). LP/E2 sites were more similar to LP than to E2 sites in associated cell TFs, RNAPII, P300, and histone H3K4me3, H3K9ac, H3K27ac, and H2Az occupancy, and were more highly transcribed than LP or E2 sites. Gene affected by CTCF and LP cooccupancy were more highly expressed than genes affected by CTCF alone. LP was at myc enhancers and promoters and of MYC regulated ccnd2, 23 med complex components, and MYC regulated cell survival genes, igf2r and bcl2. These data implicate LP and associated TFs and DNA looping factors CTCF, RAD21, SMC3, and YY1/INO80 chromatinremodeling complexes in repressor depletion and gene activation necessary for lymphoblastoid cell line growth and survival.genome-wide ChIP-seq analysis | gene expression E pstein-Barr virus (EBV) nuclear antigens EBNALP (LP) and EBNA2 (E2) are EBV-encoded transcription factors (TFs) that are coordinately expressed within hours after EBV infection of resting B lymphocytes (RBLs) and are important for B-lymphocyte conversion to lymphoblastoid cell lines (LCLs) (1-7). However, the biochemical mechanisms by which LP and E2 coordinately affect RBL transformation to LCLs are largely unknown. LP coactivates transcription by heterodimerizing with HA95 and Hsp70/72 to relocate HDAC4 from the nucleus to the cytoplasm, displaces Sp100 and Hp1α from ND10 bodies, and disrupts matrix-associated deacetylase (MAD) bodies, broadly affecting repressor localization in cell nuclei (8-16). LP also decreases repressive NCOR and RBPJ occupancy at E2 sites, without altering E2 occupancy (9). E2 enhances gene expression by localizing to cell TF sites through . E2/RBPJ sites localize in six clusters of EBF, ETS1, ZNF143, PU.1, NFκB, and RUNX1 sites. Encyclopedia of DNA elements (ENCODE) ChIP-sequencing experiments (ChIP-seqs) indicated high-level cell TF cooccupancy at E2 sites, consistent with these sites being open to cell or virus TF occupancy. Indeed, E2 chromatin sites in LCLs are open chromatin sites in RBLs, before EBV infection, consistent with EBF and RBPJ as pioneering factors that displace nucleosomes (17). E2 increases H3K4me1 signals allowing E2 and cell TF occupancy and transcription activation. The E2 a...
EBV nuclear antigen 2 (EBNA2) and EBV nuclear antigen LP (EBNALP) are critical for B-lymphocyte transformation to lymphoblastoid cell lines (LCLs). EBNA2 activates transcription through recombination signal-binding immunoglobulin κJ region (RBPJ), a transcription factor associated with NCoR repressive complexes, and EBNALP is implicated in repressor relocalization. EBNALP coactivation with EBNA2 was found to dominate over NCoR repression. EBNALP associated with NCoR and dismissed NCoR, NCoR and RBPJ, or NCoR, RBPJ, and EBNA2 from matrix-associated deacetylase (MAD) bodies. In non-EBV-infected BJAB B lymphoma cells that stably express EBNA2, EBNALP, or EBNA2 and EBNALP, EBNALP was associated with hairy and enhancer of split 1 (hes1), cd21, cd23, and arginine and glutamate-rich 1 (arglu1) enhancer or promoter DNA and was associated minimally with coding DNA. With the exception of RBPJ at the arglu1 enhancer, NCoR and RBPJ were significantly decreased at enhancer and promoter sites in EBNALP or EBNA2 and EBNALP BJAB cells. EBNA2 DNA association was unaffected by EBNALP, and EBNALP was unaffected by EBNA2. EBNA2 markedly increased RBPJ at enhancer sites without increasing NCoR. EBNALP further increased hes1 and arglu1 RNA levels with EBNA2 but did not further increase cd21 or cd23 RNA levels. EBNALP in which the 45 C-terminal residues critical for transformation and transcriptional activation were deleted associated with NCoR but was deficient in dismissing NCoR from MAD bodies and from enhancer and promoter sites. These data strongly support a model in which EBNA2 association with NCoR-deficient RBPJ enhances transcription and EBNALP dismisses NCoR and RBPJ repressive complexes from enhancers to coactivate hes1 and arglu1 but not cd21 or cd23.lymphoma | notch E BV causes posttransplantation and AIDS-related lymphomas and is a cofactor in African Burkitt lymphoma (BL), and Hodgkin disease. In vitro, EBV latency III infection converts B lymphocytes to immortal lymphoblast cell lines (LCLs) (1).EBV nuclear antigen 2 (EBNA2) and EBNALP are the first proteins expressed in latency III infection (2, 3). EBNA2 is essential for B-cell transformation (4, 5) and for up-regulation of EBV latency III and cell RNA expression through CSL/CBF1/ recombination signal-binding protein for immunoglobulin κJ region (RBPJ), a DNA sequence-specific cell transcription factor (6-8). EBNA2 up-regulated cell genes include hairy and enhancer of split 1 (hes1), myc, cd21, and cd23 (2, 9, 10). The EBNA2 acidic activation domain recruits basal and activated transcription factors (TF) including TFIIB, TFIIE, TFIIH, histone acetyl transferases p300, CBP, and PCAF, and RNA polymerase II (11-16).RBPJ is in repressive complexes containing NCoR1 and -2, SKIP, and SHARP (17-23). NCoR recruits class I and II histone deacetylases (HDAC), including HDAC3, which repress transcription and associate with matrix-associated deacetylase (MAD) bodies, subnuclear sites of repressed transcription (24, 25). NCoR overexpression represses EBNA2 up-regulation of promot...
The Epstein-Barr virus (EBV) lytic transactivator Rta activates promoters through direct binding to cognate DNA sites termed Rta response elements (RREs). Rta also activates promoters that apparently lack Rta binding sites, notably Zp and Rp. Chromatin immunoprecipitation (ChIP) of endogenous Rta expressed during early replication in B95-8 cells was performed to identify Rta binding sites in the EBV genome. Quantitative PCR (qPCR) analysis showed strong enrichment for known RREs but little or no enrichment for Rp or Zp, suggesting that the Rta ChIP approach enriches for direct Rta binding sites. Rta ChIP combined with deep sequencing (ChIP-seq) identified most known RREs and several novel Rta binding sites. Rta ChIP-seq peaks were frequently upstream of Rta-responsive genes, indicating that these Rta binding sites are likely functioning as RREs. Unexpectedly, the BALF5 promoter contained an Rta binding peak. To assess whether BALF5 might be activated by an RRE-dependent mechanism, an Rta mutant (Rta K156A), deficient for DNA binding and RRE activation but competent for Zp/Rp activation, was used. Rta K156A failed to activate BALF5p, suggesting this promoter can be activated by an RRE-dependent mechanism. Rta binding to late gene promoters was not seen at early time points but was specifically detected at later times within the Rta-responsive BLRF2 and BFRF3 promoters, even when DNA replication was inhibited. Our results represent the first characterization of Rta binding to the EBV genome during replication, identify previously unknown RREs, such as one in BALF5p, and highlight the complexity of EBV late gene promoter activation by Rta.
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