5-hydroxymethylcytosine in E-Box motifs ACAT∣GTG and ACAC∣GTG increases DNA-binding of the B-HLH transcription factor TCF4.

By Syed Khund-Sayeed, Ximiao He, Timothy R Holzberg, J. Wang, Divya Rajagopal, Shriyash Upadhyay, S. Durell, S. Mukherjee, Matthew T. Weirauch, R. Rose and 1 more. 2016

21 citations recorded by the source corpus.

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.

Subfields: Molecular Biology

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In Molecular Biology

1. Dirk Schübeler
2. Jussi Taipale
3. Paul Adrian Ginno
4. P. Beachy
5. Benjamin L. Ebert
6. Siddhartha Jaiswal

Citations in the corpus

• Impact of cytosine methylation on DNA binding specificities of human transcription factors (2017, background): “In addition to increased methylation, the Tet-TKO cell line also lacks 5hydroxymethylcytosine, which has been shown to specifically affect TF binding (52).”