Replacing C189 in the bZIP domain of Zta to S, T, V, and A changes DNA binding specificity to four types of double-stranded DNA (mC)

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 (TGAG/CTCA) 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. Protein binding microarray (PBM) experiments were performed for the DNA binding domain of the Epstein Barr Virus Zta protein and for 4 mutant constructs of Zta in which the cysteine at position 189 of wild type Zta was changed to serine (C189S), threonine (C189T), alanine (C189A) or valine (C189V). Briefly, the PBMs involved binding GST-tagged DNA-binding proteins to double-stranded 44K Agilent microarrays (Berger et al., Nature Biotechnology 2006) in order to determine their binding preferences to sequences containing modified cytosines. We modified the double stranding step of the PBM protocol in which the nucleotide mixture contained 5-methylcytosine or 5-hydroxymethylcytosine. Details of the modified cytosine PBM protocol are described in Khund-Sayeed S et al., Integrative Biology, 2016.