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

Abstract

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. Cytosine methylation outside CG dinucleotide has recently been identified in stem cells and the brain. To explore potential changes in sequence-specific DNA binding of transcription factors, we use Agilent DNA microarrays and did the double stranding reaction with 5mC or 5hmC. Using this technical innovation we explored DNA binding specificity of two helix-loop-helix proteins. For USF1, these modifications inhibited binding, while for TCF4, new sequences were bound. This innovation of DNA microarray slides opens up new possibilities to explore how modification of DNA alters transcription factor binding to mediate changes of biological importance.

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

“…T7 DNA polymerase can efficiently incorporate 5mC and 5hmC when double-stranding single-stranded DNA 27 . We exploited this property to double-strand single-stranded DNA 60-mers on an Agilent microarray using cytosine, 5mC or 5hmC, creating an asymmetric distribution of 5mC and 5hmC 15, 17 . To create the 4 th type of DNA, we enzymatically methylated the two cytosines in all CG dinucleotides (one cytosine in each strand) 20 , producing DNA that Zta is known to bind 106 .…”

“…To determine the effect of individual methylated cytosines on Zta binding to DNA, we double-stranded the single-stranded DNA on the PBMs with 5mC 17 . For these analyses, we compute the Z-scores for all 65,536 8-mers, as complements are structurally different, unlike the CG dinucleotide methylation data where complements are identical.…”

“…Enzymatic methylation of CG dinucleotides on the HK microarray was performed as previously described 20 . Double-stranding of array probes with 5mC or 5hmC was also performed as described 17 using either 5-methylcytosine (5mC, NEB) or 5-hydroxymethylcytosine (5hmC, Zymo Research). Protein binding reactions were performed as described previously 15, 20 .…”

“…For each PBM, image quantification and calculation of Z-scores were performed as described previously 17 . Microarray images were analyzed using ImaGene (BioDiscovery Inc.), and the extracted data (probe intensity values) were used for further analysis.…”

TET proteins in natural and induced differentiation

“…5caC has been observed to augment the interaction between basic helix-loop-helix (bHLH) family members and E-box sites. In other cases, the presence of 5caC at positions just flanking an E-box motif without a CpG substantially increased affinity of bHLH family members for this sequence [32,33], perhaps by altering DNA shape [34]. In other cases, modification of bases within the recognition sequence could promote binding or alter binding preference compared to unmodified DNA.…”