REL Domain of NFATc2 Binding to Five Types of DNA Using Protein Binding Microarrays.

NFATc2 is a DNA binding protein in the Rel family transcription factors, which binds a CGGAA motif better when both cytosines in the CG dinucleotide are methylated. Using protein binding microarrays (PBMs), we examined the DNA binding of NFATc2 to three additional types of DNA: single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) with either 5-methylcytosine (5mC, M) or 5-hydroxymethylcytosine (5hmC, H) in one strand and a cytosine in the second strand. ATTTCCAC, the complement of the core GGAA motif, is better bound as ssDNA compared to dsDNA. dsDNA containing the 5-mer CGGAA with either 5mC or 5hmC in one DNA strand is bound stronger than CGGAA. In contrast, the reverse complement TTCCG is bound weaker when it contains 5mC. Analysis of the available NFATc2:dsDNA complexes rationalizes these PBM data.

Impact of cytosine methylation on DNA binding specificities of human transcription factors.

The majority of CpG dinucleotides in the human genome are methylated at cytosine bases. However, active gene regulatory elements are generally hypomethylated relative to their flanking regions, and the binding of some transcription factors (TFs) is diminished by methylation of their target sequences. By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment), we found that there are also many TFs that prefer CpG-methylated sequences. Most of these are in the extended homeodomain family. Structural analysis showed that homeodomain specificity for methylcytosine depends on direct hydrophobic interactions with the methylcytosine 5-methyl group. This study provides a systematic examination of the effect of an epigenetic DNA modification on human TF binding specificity and reveals that many developmentally important proteins display preference for mCpG-containing sequences.

5-Hydroxymethylcytosine in E-box motifs ACAT|GTG and ACAC|GTG increases DNA-binding of the B-HLH transcription factor TCF4.

We evaluated DNA binding of the B-HLH family members TCF4 and USF1 using protein binding microarrays (PBMs) containing double-stranded DNA probes with cytosine on both strands or 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) on one DNA strand and cytosine on the second strand. TCF4 preferentially bound the E-box motif (CAN|NTG) with strongest binding to the 8-mer CAG|GTGGT. 5mC uniformly decreases DNA binding of both TCF4 and USF1. The bulkier 5hmC also inhibited USF1 binding to DNA. In contrast, 5hmC dramatically enhanced TCF4 binding to E-box motifs ACAT|GTG and ACAC|GTG, being better bound than any 8-mer containing cytosine. Examination of X-ray structures of the closely related TCF3 and USF1 bound to DNA suggests TCF3 can undergo a conformational shift to preferentially bind to 5hmC while the USF1 basic region is bulkier and rigid precluding a conformation shift to bind 5hmC. These results greatly expand the regulatory DNA sequence landscape bound by TCF4.