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

Abstract

The Epstein-Barr virus (EBV) B-ZIP transcription factor (TF) 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 where 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 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 and (where =5mC) were preferentially bound. 5mC inhibits Zta binding to both TRE motif half sites and . Like the CREB1 homodimer, the Zta homodimer and the cJun|cFos heterodimer bind the C/EBP half site tetranucleotide stronger when it contains 5mC. Zta also binds dsDNA sequences containing 5hmC in one strand, although the effect is less dramatic than 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.

Replacing C189 in the bZIP domain of Zta with S, T, V, or A changes DNA binding specificity to four types of double-stranded DNA

“…Zta, Zta(C189S), and Zta(C189T) bind many features containing DNA(5mC∣C), and median and average fluorescent intensity for all features increases relative to DNA(C∣C) (Table S2). For Zta and Zta(C189S), many well-bound DNA(5mC∣C) 8-mers contain the C/EBP half-site GM 2 AA (Fig 2A,S4) as observed previously for Zta [7, 8] and CREB1 [9]. While most GM 2 AA -containing 8-mers are better bound by Zta compared to Zta(C189S), several 8-mers are strongly bound by both proteins (e.g.…”

“…Unlike previous findings [7], our data indicates that Zta does not bind meTRE ( M −4 G −3 ) as strongly as the TRE ( C −4 G −3 ) or meZRE2 ( M 2 G 3 ) sequences. We and others have also shown Zta can bind DNA sequences with 5mC outside of CG dinucleotides, in the case of the methylated C/EBP half-site GMAA [7, 8], similar to that observed for another bZIP domain, CREB1 [9]. In addition, the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC, H ) can increase Zta binding to hydroxymethylated C/EBP half-sites ( GHAA )[8].…”

“…Single-stranded DNA 60-mers on microarrays were double stranded with: 1) cytosine, producing dsDNA with cytosine in both strands (DNA(C∣C)), 2) 5mC ( M ), producing dsDNA with 5mC in one strand and cytosine in the second strand (DNA(5mC∣C)), or 3) 5hmC ( H ), producing DNA(5hmC∣C) [8, 18, 20]. A fourth type of dsDNA was generated by enzymatic methylation of the two cytosines in all CG dinucleotides (one cytosine on each strand) [17].…”

“…For each PBM, image quantification and calculation of 8-mer Z-scores were performed as described previously [8, 18]. All proteins were assayed with at least two replicates (representing individual IVT reactions and arrays), with good agreement between replicates and previously published PBM data for Zta [8] (R≥0.8, Table S1, Fig S2).…”

“…Extending this work, we recently examined the bZIP domain of Zta binding to four types of double-stranded DNA (dsDNA) using protein binding microarrays (PBMs) [8]. We found that many TRE variants containing methylated cytosines at the aforementioned two positions ( M −4 G −3 and M 2 G 3 ) were well bound by Zta, with motifs containing a methylated CG dinucleotide at M 2 G 3 being among the strongest bound sequences.…”