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.

中文翻译：

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E-box基序ACAT | GTG和ACAC | GTG中的5-羟甲基胞嘧啶增加B-HLH转录因子TCF4的DNA结合。

我们使用蛋白质结合微阵列（PBM）评估B-HLH家族成员TCF4和USF1的DNA结合，该蛋白质结合微阵列（PBM）包含双链DNA探针，两条链上都带有胞嘧啶，一条DNA链上带有5-甲基胞嘧啶（5mC）或5-羟甲基胞嘧啶（5hmC）第二条是胞嘧啶。TCF4优先结合最牢固的E-box基序（CAN | NTG）与8-mer CAG | GTGGT。5mC均匀降低TCF4和USF1的DNA结合。更大的5hmC也抑制了USF1与DNA的结合。相反，5hmC显着增强了TCF4与E-box基序ACAT | GTG和ACAC | GTG的结合，比任何含8个胞嘧啶的胞嘧啶都更好地结合。对与DNA紧密相关的TCF3和USF1的X射线结构的研究表明，TCF3可以经历构象转变，优先与5hmC结合，而USF1基本区域更大且刚性强，排除了构象转变以与5hmC结合。这些结果大大扩展了由TCF4结合的调控DNA序列的格局。