Quantitative Characterization of the Interactions among c-myc Transcriptional Regulators FUSE, FBP, and FIR
Quantitative Characterization of the Interactions among c-myc Transcriptional Regulators FUSE, FBP, and FIR
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DOI：
10.1021/bi9021445
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发表时间：
2010-06-08
期刊：
影响因子：
2.9
通讯作者：
Braddock, Demetrios T.
中科院分区：
文献类型：
--
作者：
Hsiao, Hsin-hao;Nath, Abhinav;Braddock, Demetrios T.
人类c-myc基因对细胞的稳态和生长至关重要，但如果调控不当，它也是一种强有力的致癌因子。c-myc远端上游元件（FUSE）在转录活跃时融合成单链DNA，非编码链FUSE招募激活子[FUSE结合蛋白（FBP）]和阻遏子[FBP相互作用阻遏子（FIR）]，以实时方式微调c-myc转录。尽管详细的生物学实验描述了这种独特的转录调控模式，但仍然缺乏对调节蛋白质-DNA相互作用的物理常数的定量测量。在这里，我们首先证明了两个融合链采用不同的构象后融化，与非编码链DNA在一个扩展的，线性的形式。FBP与线性非编码FUSE结合，解离常数在纳摩尔范围内。FIR与FUSE的结合更弱，其解离常数在低微摩尔范围内。FIR在接近生理条件下是单体的，但在与FUSE结合后，由RRM介导二聚成2：1的FIR 2-FUSE复合物。在三方相互作用中，我们的分析表明FIR逐步添加到激活FBP-FUSE复合物上以形成四元FIR 2-FBP-FUSE抑制复合物。我们的定量表征增强了对DNA链偏好和FUSE-FBP-FIR调节系统中逐步形成复合物的机制的理解。
Human c-myc is critical for cell homeostasis and growth but is a potent oncogenic factor if improperly regulated. The c-myc far-upstream element (FUSE) melts into single-stranded DNA upon active transcription, and the noncoding strand FUSE recruits an activator [the FUSE-binding protein (FBP)] and a repressor [the FBP-interacting repressor (FIR)] to fine-tune c-myc transcription in a real-time manner. Despite detailed biological experiments describing this unique mode of transcriptional regulation, quantitative measurements of the physical constants regulating the protein-DNA interactions remain lacking. Here, we first demonstrate that the two FUSE strands adopt different conformations upon melting, with the noncoding strand DNA in an extended, linear form. FBP binds to the linear noncoding FUSE with a dissociation constant in the nanomolar range. FIR binds to FUSE more weakly, having its modest dissociation constants in the low micromolar range. FIR is monomeric under near-physiological conditions but upon binding of FUSE dimerizes into a 2:1 FIR2-FUSE complex mediated by the RRMs. In the tripartite interaction, our analysis suggests a stepwise addition of FIR onto an activating FBP-FUSE complex to form a quaternary FIR2-FBP-FUSE inhibitory complex. Our quantitative characterization enhances understanding of DNA strand preference and the mechanism of the stepwise complex formation in the FUSE-FBP-FIR regulatory system.