No abstract

We analyzed the levels of acetylated histones and histone H3 dimethylated on lysine 4 (H3K4me2) at the LMP2A promoter (LMP2Ap) of Epstein-Barr virus in well-characterized type I and type III lymphoid cell line pairs and additionally in the nasopharyngeal carcinoma cell line C666-1 by using chromatin immunoprecipitation. We found that enhanced levels of acetylated histones marked the upregulated LMP2Ap in lymphoid cells. In contrast, in C666-1 cells, the highly DNA-methylated, inactive LMP2Ap was also enriched in acetylated histones and H3K4me2. Our results suggest that the combinatorial effects of DNA methylation, histone acetylation, and H3K4me2 modulate the activity of LMP2Ap.Epstein-Barr virus (EBV) is closely associated with a variety of neoplasms. In latently infected cells, depending on the gene expression pattern, three main classes of latency have been described (17). Latent membrane protein 2A (LMP2A) is transcribed from the LMP2A promoter (LMP2Ap) in large quantities in type III latency, at variable levels in type II latency, and at low levels or not at all in type I latency (15,20,32).Previous in vitro binding and reporter gene experiments charted two CBF1 sites and other elements in the regulatory region of LMP2Ap (10,15,18,[30][31][32]. Our previous in vivo analysis of LMP2Ap showed that, in lymphoid cell lines, the characteristic footprints on two CBF1 and further binding sites, together with the overall hypomethylation of CpG dinucleotides, correlate well with promoter activity. In contrast, the absence of several genomic footprints, as well as the presence of patches of highly methylated CpG dinucleotides, is characteristic of silent LMP2Ap's in lymphoid cells (20). However, in addition to DNA methylation and protein-DNA interactions, the acetylation of histone H3 and H4 and methylation on the lysine 4 residue of histone H3 may play an important role in the regulation of LMP2Ap, as they lead to chromatin relaxation and subsequent modulation of gene expression (4,7,16,24,26). It was also shown previously that the binding of EBNA2 to CBF1 directs the p300, CBP, and P/CAF histone acetyltransferase coactivators to the LMP1 promoter (28). On the other hand, CBF1 in the absence of EBNA2 or activated NotchIC represses transcription, in part by tethering a histone deacetylase corepressor complex (11)(12)(13)29). Furthermore, previous studies have shown good correlation between the levels of acetylated histones and histone H3 dimethylated at the lysine 4 residue (H3K4me2) and the activity of the CBF1-regulated C-promoter of EBV (1, 5; G. Fejer et al., submitted for publication). Therefore, we wished to analyze the levels of acetylated histone H3 (AcH3), acetylated histone H4 (AcH4), and H3K4me2 at LMP2Ap in well-characterized type I (Mutu-BL-I-Cl-216 and Rael) and type III (Mutu-BL-III-Cl-99 and CB-M1-Ral-STO) lymphoid cell line pairs carrying the same viral strains (20, 21) and additionally in the only available EBV-positive nasopharyngeal carcinoma (NPC) cell line, C666-1, representative of latency type...

In this study, the binding of the insulator protein CCCTC-binding factor (CTCF) to the region located between Rep* and the C promoter (Cp) of Epstein–Barr virus (EBV) was analysed using chromatin immunoprecipitation and in vivo footprinting. CTCF binding was found to be independent of Cp usage in cell lines corresponding to the major EBV latency types. Bisulfite sequencing and an electrophoretic mobility-shift assay (using methylated and unmethylated probes) revealed that CTCF binding was insufficient to induce local CpG demethylation in certain cell lines and was unaffected by CpG methylation in the region between Rep* and Cp. In addition, CTCF binding to the latency promoter, Qp, did not correlate with Qp activity.