Screen for Small Molecule Inhibitors of ATP Dependent Chromatin Remodeling

描述（由申请人提供）：尽管人类基因组编码大约30种不同的atp依赖性染色质重塑复合物，但它们的机制在很大程度上是未知的。在某种程度上，这是由于缺乏快速作用的小分子抑制剂，这种抑制剂可以允许进行机制研究所需的精确时间分析。其中一个基于Brg和Brm atp酶的复合物家族（BAF或mSWI/SNF复合物）是诱导多能状态所必需的，在某些情况下也足以诱导多能状态。BAF复合物亚基也是肿瘤抑制因子，在某些人类恶性肿瘤中发生杂合性缺失。最后，它们已被证明是显性衰老所必需的，它们的缺失增加了人类成纤维细胞的增殖寿命。蛋白质组学分析表明，BAF复合物的不同功能是来自编码亚基的基因家族的复合物组合组装的结果。对胚胎干细胞中BAF复合物占用的全基因组分析表明，它们通常从远处抑制其靶基因，这表明了一种意想不到的作用机制。为了了解SWI/ snf样BAF复合物促进多能性和抑制肿瘤的不明确机制，该项目旨在通过敲入ESC报告系确定一套全面的快速作用小分子抑制剂。该细胞系含有插入Bmi1位点的荧光素酶，并在BAF复合物的atp酶亚基基因缺失时快速激活。2 MDa复合物包含13个亚基，预计表面积为50至250万E2。该复合物的超大表面被假设可以进行一系列连续的反应，这些反应可以用小分子进行排序和探测。一旦确定，我们将使用在该复合物亚基中具有零突变的已建立的胚胎干细胞系来确定小分子抑制剂的结合位点。由于抑制剂也可以阻断影响BAF复合物的调节机制，我们将描述它们对复合物翻译后修饰的影响。每种抑制剂在抑制Polycomb基因和激活重要多能性靶点（如Fgf4和Bmp4）中的作用时间将有助于进一步确定每种快速作用抑制剂的功能水平。具体来说，它们的作用将取决于高阶染色质结构、远程相互作用、染色质可及性、核小体定位和转录因子占用。这项全面而系统的研究将有助于我们更深入地了解这种染色质重塑复合物的作用，它在多能性、人类肿瘤抑制和细胞衰老中发挥着重要作用。此外，这个有价值的小分子探针工具箱对于其他研究染色质调控的研究人员来说将是无价的。

DESCRIPTION (provided by applicant): Although the human genome encodes about 30 different ATP-dependent chromatin remodeling complexes, their mechanism[s] are largely unknown. In part, this is due to the lack of rapidly acting small molecule inhibitors that could permit precise temporal analysis needed for mechanistic studies. One family of these complexes based on the Brg and Brm ATPases (BAF or mSWI/SNF complexes) are both necessary and in some contexts sufficient to induce the pluripotent state. BAF complex subunits are also tumor suppressors and undergo loss of heterozygosity in certain human malignancies. Finally, they have been shown to be necessary for explicative senescence and their deletion increases the proliferative life of human fibroblasts. Proteomic analysis indicates that the diverse functions of BAF complexes are the result of combinatorial assembly of the complexes from gene families encoding the subunits. Genome wide analysis of the occupancy of BAF complexes in ES cells has revealed that they most commonly suppress their target genes from a distance, suggesting an unanticipated mechanism of action. To understand the poorly defined mechanisms used by SWI/SNF-like BAF complexes to promote pluripotency and suppress tumors, this project aims to identify a comprehensive set of rapidly acting small- molecule inhibitors using a knock-in ESC reporter line. This line contains luciferase inserted into the Bmi1 locus and undergoes rapid activation upon genetic deletion of the ATPase subunit of the BAF complex. The 2 MDa complex contains 13 subunits and has an expected surface area of 0.5 to 2.5 million E2. The extraordinarily large surface of the complex is hypothesized to carry out a sequential series of reactions that can be ordered and probed with small molecules. Once identified, we will define the binding sites of the small molecule inhibitors using established embryonic stem cell lines with null mutations in subunits of the complex. Because the inhibitors could also block regulatory mechanisms impinging upon BAF complexes, we will characterize their effect upon defined post-translational modifications of the complex. The time-of-action of each of the inhibitors in both the repression of Polycomb genes and the activation of important pluripotency targets such as Fgf4 and Bmp4 will help to further define the level of function of each of the rapidly acting inhibitors. Specifically, their effects will be determined on higher-order chromatin structures, long-range interactions, chromatin accessibility, nucleosome positioning, and transcription factor occupancy. This comprehensive and systematic investigation should lead to deeper understanding of the actions of this chromatin-remodeling complex, which plays an essential role in pluripotency, human tumor suppression and cellular senescence. In addition, this valuable toolbox of small molecule probes will be invaluable for other researchers studying chromatin regulation. PUBLIC HEALTH RELEVANCE: We intend to find small molecules that inhibit the activities of a complex of proteins that control the packaging of DNA into the nucleus of a cell. This protein complex plays a critical role in human embryonic stem cells and human tumor suppression. Inhibitors of this complex could lead to improved stem cell therapies and new cancer treatments.