Multivalency regulates activity in an intrinsically disordered transcription factor.
Multivalency regulates activity in an intrinsically disordered transcription factor.
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DOI：
10.7554/elife.36258
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发表时间：
2018-05-01
期刊：
影响因子：
7.7
通讯作者：
Barbar EJ
中科院分区：
文献类型：
--
作者：
Clark S;Myers JB;King A;Fiala R;Novacek J;Pearce G;Heierhorst J;Reichow SL;Barbar EJ
转录因子ASCIZ（ATMIN，ZNF 822）对其主要靶基因的产物，中枢蛋白LC 8（DYNLL 1）具有异常高的识别基序数量。使用生物物理方法，结构分析，NMR和电子显微镜，和细胞转录测定的组合，我们开发了一个模型，提出了内在的障碍和多个LC 8结合事件在调节LC 8转录的协调作用。我们证明，长期内在无序的C-末端结构域的ASCIZ结合LC 8形成一个动态的整体的复合物与梯度的转录活性，这是成反比的LC 8占用。在饱和LC 8浓度下与人和果蝇ASCIZ的低占有率复合物的偏好表明负协同性是ASCIZ-LC 8相互作用的重要特征。转录因子的内在紊乱和多价性的普遍存在表明，异质性、动态复合物的形成是调节转录调控的一种普遍机制。蛋白质有助于调节身体中的几乎每一个过程，并以各种形式，大小和目的出现。细胞含有数千种不同的蛋白质，但并不是每种蛋白质都是必需的。为了创造新的蛋白质，基因上的信息首先需要在称为转录的过程中转录成RNA（DNA的模板分子）。然后，细胞内的一个复杂机制将拷贝作为模板组装蛋白质。所谓的转录因子（也称为蛋白质）可以通过与基因的起始点结合来开启或关闭复制过程。它们可以单独或与其他蛋白质复合起作用。例如，称为ASCIZ的转录因子有助于调节称为LC 8的蛋白质的产生。LC 8附着于100多种不同的蛋白质，在许多细胞过程中发挥重要作用。因此，对其生产进行微调至关重要。蛋白质的形状对其用途至关重要。像大多数蛋白质一样，转录因子由氨基酸链组成，这些氨基酸链折叠成特定的三维（3D）结构，具有识别并结合特定DNA序列的区域。但许多转录因子也包含柔性的、“无序”的区域，这些区域不会折叠成刚性的3D形状。这些可能有助于控制基因的活性，但它们的确切作用尚不清楚。ASCIZ包含一个非常长的无序区域，该区域沿着其链具有多个结合LC 8的位置。先前的研究表明，ASCIZ与LC 8基因结合，并增加转录以产生更多的LC 8蛋白。一旦蛋白质水平足够高，LC 8被认为与ASCIZ的无序区域结合并关闭转录。人类ASCIZ蛋白有11个LC 8分子结合位点，而果蝇有7个。到目前为止，还不清楚为什么存在如此多的不同结合位点。为了解决这个问题，Clark等人结合了生物物理、结构和分子生物学技术来分析人类和果蝇的蛋白质，并测试它们在人类细胞中的作用。这表明LC 8和ASCIZ形成复合物的动态混合物，而不是单个完全占据的复合物。随着与ASCIZ结合的LC 8分子数量的增加，转录速率下降。然而，所有的结合位点很少被完全占据。相反，三到四个附着的LC 8分子似乎足以确保LC 8水平保持平衡。当LC 8分子的数量超过该值时，额外LC 8的附着速率减慢。因此，即使存在过量的LC 8，大多数人ASCIZ结合位点也仅部分被填充。通过这种方式，LC 8蛋白质的生产被减缓，而不是完全关闭。因此，细胞能够微调LC 8的转录速率，并保持这些蛋白质的稳定和平衡。这项工作表明，转录因子上的无序区域可以帮助细胞系统在不断变化的条件下保持稳定。在未来，这里使用的方法的组合可以揭示关于其他具有无序区域的蛋白质的新信息。
The transcription factor ASCIZ (ATMIN, ZNF822) has an unusually high number of recognition motifs for the product of its main target gene, the hub protein LC8 (DYNLL1). Using a combination of biophysical methods, structural analysis by NMR and electron microscopy, and cellular transcription assays, we developed a model that proposes a concerted role of intrinsic disorder and multiple LC8 binding events in regulating LC8 transcription. We demonstrate that the long intrinsically disordered C-terminal domain of ASCIZ binds LC8 to form a dynamic ensemble of complexes with a gradient of transcriptional activity that is inversely proportional to LC8 occupancy. The preference for low occupancy complexes at saturating LC8 concentrations with both human and Drosophila ASCIZ indicates that negative cooperativity is an important feature of ASCIZ-LC8 interactions. The prevalence of intrinsic disorder and multivalency among transcription factors suggests that formation of heterogeneous, dynamic complexes is a widespread mechanism for tuning transcriptional regulation. Proteins help to regulate almost every process in the body, and come in various forms, sizes and purposes. Cells contain thousands of different proteins, but not every protein is needed at all times. To create new proteins, the information on a gene first needs to be transcribed into RNA (template molecules of the DNA) in a process known as transcription. A complex machinery inside the cell then uses the copy as a template to assemble the protein. So-called transcription factors (also proteins) can switch the copying process on or off by binding to the start point of a gene. They can act alone or in complex with other proteins. The transcription factor called ASCIZ, for example, helps to regulate the production of a protein called LC8. LC8 attaches to more than 100 different proteins and plays an important role in many cell processes. Therefore, fine-tuning its production is essential. The shape of a protein is critical to its purpose. Like most proteins, transcription factors are made up of chains of amino acids that fold into a specific three-dimensional (3D) structurewith a region that recognizes and binds to a specific DNA sequence. But many transcription factors also contain flexible, ‘disordered’ regions that do not fold into a rigid 3D shape. These may help to control the activity of genes, but their exact role is unclear. ASCIZ contains an exceptionally long, disordered region that has multiple positions for binding LC8 along its chain. Previous research has shown that ASCIZ binds to the LC8 gene and increases transcription to produce more LC8 proteins. Once the protein levels are high enough, LC8 is thought to bind to the disordered region of ASCIZ and switch off transcription. Human ASCIZ proteins have 11 binding sites for LC8 molecules, while fruit flies have seven. Until now it was not clear why so many different binding sites exist. To address this question, Clark et al. combined biophysical, structural and molecular biology techniques to analyze proteins from humans and fruit flies and to test their role in human cells. This revealed that LC8 and ASCIZ form a dynamic mixture of complexes, instead of a single fully-occupied complex. As the number of LC8 molecules bound to ASCIZ increased, the rate of transcription dropped. However, all of the binding sites were rarely fully occupied. Instead, three to four attached LC8 molecules seemed to be sufficient to ensure that LC8 levels remain balanced. When the number of LC8 molecules exceeded this value, the attachment rate for additional LC8 slowed down. So, even when there was an excess of LC8, most of the human ASCIZ binding sites were only partially filled. This way, the production of LC8 proteins was slowed, rather than fully shut down. As a result, the cells were able to fine-tune the transcription rate of LC8 and maintain a stable and balanced pool of these proteins. This work suggests that disordered regions on transcription factors could help to keep cellular systems steady in the face of changing conditions. In the future, the combination of methods used here could reveal new information about other proteins with disordered regions.