Binding of CCCTC-binding factor in vivo to the region located between Rep and the C promoter of Epstein-Barr virus is unaffected by CpG methylation and does not correlate with Cp activity

1 Microbiological Research Group, National Center for Epidemiology, Pihenö u. 1, H-1529 Budapest, Hungary 2 Department of Microbiology and Hygiene, University of Regensburg, Franz-Josef-Strauss Allee 11, D-93053 Regensburg, Germany Correspondence Daniel Salamon saladili{at}yahoo.com 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. These authors contributed equally to this work. The GenBank/EMBL/DDBJ accession numbers for the sequences of the region between Rep* and Cp are FM178371–FM178378 (cell lines C666-1, CB-M1-Ral-STO, Daudi, Mutu-BL-I-Cl-216, Mutu-BL-III-Cl-99, NPC-C15, NPC-C18 and Rael, respectively). In the Raji, LCL-721, IARC-171 and KR4 cell lines, the sequences between nt 10046 and 10891 did not show any deviation from the B95-8 sequence (Baer et al. , 1984). The GenBank/EMBL/DDBJ accession number for the sequence of the EBER region for C666-1 is AM397661.