CpG-Methylation Regulates a Class of Epstein-Barr Virus Promoters
Figure 7
Zta binds preferentially to CpG-methylated promoters.
In EMSAs, Zta bound preferentially to the CpG methylated promoters of BRLF1, BALF2, BSLF2/BMLF1, BALF5 and BMRF1 and BSLF1 but it bound only the CpG-methylated promoters of BBLF4 and BBLF2/3. CpG methylation did not influence Zta binding to the promoters of BZLF1, BHLF1, and BHRF1. EMSAs were performed with affinity-purified Strep/FLAG-tagged BZLF1 fusion protein transiently expressed in HEK293 cells. Eleven PCR fragments encompassing EBV promoters with BZLF1 binding sites identified in Figures 2 to 5 served as radioactive probes. PCR fragments (Table S3 in Text S1) were used either unmethylated or fully CpG-methylated in vitro with M.SssI. Supershifts with a FLAG-antibody confirmed the identity of the protein-DNA complexes. The signals were scanned and the percent ratios of bound DNAs versus total input DNAs are shown for each sample.