bZIP dimers CREB1, ATF2, Zta, ATF3|cJun, and cFos|cJun prefer binding some DNA sequences containing 5-formylcytosine and 5-carboxylcytosine

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. Protein binding microarray (PBM) experiments were performed for a set of bZIP transcription factors: Atf2, Creb1, and Zta homodimers, and cJun-cFos and Atf3-cJun heterodimers. 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, 5-hydroxymethylcytosine, 5-formylcytosine, or 5-carboxymethylcytosine. Details of the modified cytosine PBM protocol are described in Khund-Sayeed S et al., Integrative Biology, 2016.