A unique DNA binding domain converts T-cell factors into strong Wnt effectors
A unique DNA binding domain converts T-cell factors into strong Wnt effectors
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DOI：
10.1128/mcb.02132-06
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发表时间：
2007-12-01
影响因子：
5.3
通讯作者：
Waterman, Marian L.
中科院分区：
文献类型：
--
作者：
Atcha, Fawzia A.;Syed, Adeela;Waterman, Marian L.
基因表达的Wnt调控需要LEF/T细胞因子（LEF/TCF）转录因子与Wnt应答元件（WRE）的结合以及激活剂β-连环蛋白的募集。LEF/TCF家族成员调节Wnt靶基因的能力存在显著差异。例如，具有C-末端"E"尾的TCF-1和TCF-4的可变剪接同种型在它们激活LEF 1和CDX 1方面是独特有效的。在这里，我们报告说，负责这种独特的活动的机制是一个辅助的30个氨基酸的DNA相互作用基序，在这里被称为“半胱氨酸钳"（或C-钳）。C-夹含有不变的半胱氨酸、芳香族和碱性残基，重组C-夹蛋白的表面等离子体共振（SPR）研究表明，它结合双链DNA，但不结合单链DNA或RNA（平衡解离常数= 16 nM）。CASTing（循环扩增和目标选择）实验用于测试该基序是否影响WRE识别。全长LEF-1、TCF-1 E和具有突变C-夹的TCF-1 E都结合几乎相同的WRE（TYYCTTTGATSTT），表明C-夹不改变WRE特异性。然而，WRE下游的GC元件（RCCG）富含野生型TCF-1 E结合位点，但不富含突变型TCF-1 E结合位点。我们的结论是，C-钳是一个序列特异性的DNA结合基序。C-夹突变破坏了β-连环蛋白调节LEF 1启动子的能力，并且它们严重损害了TCF-1调节结肠癌细胞生长的能力。因此，TCF的E-尾同种型利用两种DNA结合活性来接近对细胞生长重要的Wnt靶标的子集。
Wnt regulation of gene expression requires binding of LEF/T-cell factor (LEF/TCF) transcription factors to Wnt response elements (WREs) and recruitment of the activator beta-catenin. There are significant differences in the abilities of LEF/TCF family members to regulate Wnt target genes. For example, alternatively spliced isoforms of TCF-1 and TCF-4 with a C-terminal '' E '' tail are uniquely potent in their activation of LEF1 and CDX1. Here we report that the mechanism responsible for this unique activity is an auxiliary 30-amino-acid DNA interaction motif referred to here as the '' cysteine clamp '' (or C-clamp). The C-clamp contains invariant cysteine, aromatic, and basic residues, and surface plasmon resonance (SPR) studies with recombinant C-clamp protein showed that it binds double-stranded DNA but not single-stranded DNA or RNA (equilibrium dissociation constant = 16 nM). CASTing (Cyclic Amplification and Selection of Targets) experiments were used to test whether this motif influences WRE recognition. Full-length LEF-1, TCF-1E, and TCF-1E with a mutated C-clamp all bind nearly identical WREs (TYYCTTTGATSTT), showing that the C-clamp does not alter WRE specificity. However, a GC element downstream of the WRE (RCCG) is enriched in wild-type TCF-1E binding sites but not in mutant TCF-1E binding sites. We conclude that the C-clamp is a sequence-specific DNA binding motif. C-clamp mutations destroy the ability of beta-catenin to regulate the LEF1 promoter, and they severely impair the ability of TCF-1 to regulate growth in colon cancer cells. Thus, E-tail isoforms of TCFs utilize two DNA binding activities to access a subset of Wnt targets important for cell growth.