Zta is a bZIP transcription factor (TF) in the Epstein-Barr virus that binds unmethylated and methylated DNA sequences. Substitution of cysteine 189 of Zta to serine (Zta(C189S)) results in a virus that is unable to execute the lytic cycle, which was attributed to a change in binding to methylated DNA sequences. To learn more about the role of this position in defining sequence-specific DNA binding, we mutated cysteine 189 to four other amino acids, producing Zta(C189S), Zta(C189T), Zta(C189A), and Zta(C189V) mutants. Zta and mutants were used in protein binding microarray (PBM) experiments to evaluate sequence-specific DNA binding to four types of double-stranded DNA (dsDNA): 1) with cytosine in both strands (DNA(C|C)), 2) with 5-methylcytosine (5mC) in one strand and cytosine in the second strand (DNA(5mC|C)), 3) with 5-hydroxymethylcytosine (5hmC) in one strand and cytosine in the second strand (DNA(5hmC|C)), and 4) with both cytosines in all CG dinucleotides containing 5mC (DNA(5mCG)). Zta(C189S) and Zta(C189T) bound the TRE (AP-1) motif (TGA/TCA) more strongly than wild-type Zta, while binding to other sequences, including the C/EBP half site GCAA was reduced. Binding of Zta(C189S) and Zta(C189T) to DNA containing modified cytosines (DNA(5mC|C), DNA(5hmC|C), and DNA(5mCG)) was reduced compared to Zta. Zta(C189A) and Zta(C189V) had higher non-specific binding to all four types of DNA. Our data suggests that position C189 in Zta impacts sequence-specific binding to DNA containing modified and unmodified cytosine.

No abstract

In mammalian cells, 5-methylcytosine (5mC) occurs in genomic double-stranded DNA (dsDNA) and is enzymatically oxidized to 5-hydroxymethylcytosine (5hmC), then to 5-formylcytosine (5fC), and finally to 5-carboxylcytosine (5caC). These cytosine modifications are enriched in regulatory regions of the genome. The effect of these oxidative products on five bZIP dimers (CREB1, ATF2, Zta, ATF3|cJun, and cFos|cJun) binding to five types of dsDNA was measured using protein binding microarrays. The five dsDNAs contain either cytosine in both DNA strands or cytosine in one strand and either 5mC, 5hmC, 5fC, or 5caC in the second strand. Some sequences containing the CEBP half-site GCAA are bound more strongly by all five bZIP domains when dsDNA contains 5mC, 5hmC, or 5fC. dsDNA containing 5caC in some TRE (AP-1)-like sequences, e.g., TGACTAA, is better bound by Zta, ATF3|cJun, and cFos|cJun.