title: 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.
authors.affiliation: Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Room 3128, Building 37, Bethesda, MD 20892, USA. vinsonc@mail.nih.gov.
authors.forename: Syed
authors.initials: S
authors.lastname: Khund-Sayeed
authors.forename: Ximiao
authors.initials: X
authors.lastname: He
authors.forename: Timothy
authors.initials: T
authors.lastname: Holzberg
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.forename: Divya
authors.initials: D
authors.lastname: Rajagopal
authors.forename: Shriyash
authors.initials: S
authors.lastname: Upadhyay
authors.forename: Stewart R
authors.initials: SR
authors.lastname: Durell
authors.forename: Sanjit
authors.initials: S
authors.lastname: Mukherjee
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Weirauch
authors.forename: Robert
authors.initials: R
authors.lastname: Rose
authors.forename: Charles
authors.initials: C
authors.lastname: Vinson
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives* / Q000737:chemistry; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000737:chemistry* / Q000648:ultrastructure; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000648:ultrastructure; D024721:E-Box Elements; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D000073940:Transcription Factor 4 / Q000737:chemistry* / Q000648:ultrastructure
chemical_list: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C532096:TCF4 protein, human; D000073940:Transcription Factor 4; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D004247:DNA