The Epstein-Barr Virus B‑ZIP Protein Zta Recognizes Specific DNA Sequences Containing 5‑Methylcytosine and 5‑Hydroxymethylcytosine

The
Epstein-Barr virus (EBV) B-ZIP transcription factor Zta binds
to many DNA sequences containing methylated CG dinucleotides. Using
protein binding microarrays (PBMs), we analyzed the sequence specific
DNA binding of Zta to four kinds of double-stranded DNA (dsDNA): (1)
DNA containing cytosine in both strands, (2) DNA with 5-methylcytosine
(5mC) in one strand and cytosine in the second strand, (3) DNA with
5-hydroxymethylcytosine (5hmC) in one strand and cytosine in the second
strand, and (4) DNA in which both cytosines in all CG dinucleotides
contain 5mC. We compared these data to PBM data for three additional
B-ZIP proteins (CREB1 and CEBPB homodimers and cJun|cFos heterodimers).
With cytosine, Zta binds the TRE motif TGAC/GTCA as previously reported. With CG dinucleotides containing 5mC on
both strands, many TRE motif variants containing a methylated CG dinucleotide
at two positions in the motif, such as MGAGTCA and TGAGMGA (where M = 5mC), were preferentially
bound. 5mC inhibits binding of Zta to both TRE motif half-sites GTCA and CTCA. Like the CREB1 homodimer, the
Zta homodimer and the cJun|cFos heterodimer more strongly bind the
C/EBP half-site tetranucleotide GCAA when it contains
5mC. Zta also binds dsDNA sequences containing 5hmC in one strand,
although the effect is less dramatic than that observed for 5mC. Our
results identify new DNA sequences that are well-bound by the viral
B-ZIP protein Zta only when they contain 5mC or 5hmC, uncovering the
potential for discovery of new viral and host regulatory programs
controlled by EBV.