True equilibrium measurement of transcription factor-DNA binding affinities using automated polarization microscopy.
True equilibrium measurement of transcription factor-DNA binding affinities using automated polarization microscopy.
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DOI：
10.1038/s41467-018-03977-4
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发表时间：
2018-04-23
影响因子：
16.6
通讯作者：
Gaul U
中科院分区：
文献类型：
--
作者：
Jung C;Bandilla P;von Reutern M;Schnepf M;Rieder S;Unnerstall U;Gaul U
在后生动物中，复杂的基因表达模式是由转录因子（TFs）与调控DNA的选择性结合控制的。为了提高对这一过程的定量理解，我们开发了一种新的方法，在受控递送系统中使用荧光各向异性测量来测定溶液中的TF-DNA结合能，具有高灵敏度和高通量。由于其大的动态范围，该方法被称为高性能荧光各向异性（HiP-FA），可以可靠地定量弱和强结合；结合特异性是基于平衡常数测量的突变DNA变体计算。我们确定了26个tf的结合偏好景观，并测量了高绝对亲和力，但大多数结合特异性低于其他方法报道的特异性。修订后的结合偏好提高了对体内TF占用和增强子表达的预测。我们的方法为基因调控的系统生物学分析提供了一个强大的新工具。测量选择性转录因子- dna结合的方法往往缺乏敏感性，而且不能在溶液中进行。在这里，作者开发了一种具有高灵敏度和高通量的转录因子- dna结合能荧光各向异性测量方法。
The complex patterns of gene expression in metazoans are controlled by selective binding of transcription factors (TFs) to regulatory DNA. To improve the quantitative understanding of this process, we have developed a novel method that uses fluorescence anisotropy measurements in a controlled delivery system to determine TF-DNA binding energies in solution with high sensitivity and throughput. Owing to its large dynamic range, the method, named high performance fluorescence anisotropy (HiP-FA), allows for reliable quantification of both weak and strong binding; binding specificities are calculated on the basis of equilibrium constant measurements for mutational DNA variants. We determine the binding preference landscapes for 26 TFs and measure high absolute affinities, but mostly lower binding specificities than reported by other methods. The revised binding preferences give rise to improved predictions of in vivo TF occupancy and enhancer expression. Our approach provides a powerful new tool for the systems-biological analysis of gene regulation. Methods to measure selective transcription factor-DNA binding often lack sensitivity and are not performed in solution. Here the authors develop a method to perform fluorescence anisotropy measurements of transcription factor-DNA binding energies with high sensitivity and throughput.
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