Imaging and manipulating oncoprotein phase separation and compartmentalization

摘要 包括c-Myc和MYCN在内的MYC癌蛋白是人类肿瘤发生的主要驱动因素。MYC的表达 在许多类型的人类癌症中是失调和增强的。例如，MYCN是 儿童癌症虽然上调的MYC表达在许多组织中诱导肿瘤发展，但缺失MYC表达可诱导肿瘤发生。 在各种肿瘤模型中，MYC的表达消除了肿瘤发生并导致肿瘤消退。尽管取得了重大 尽管在理解MYC上调与肿瘤发生之间的联系方面取得了进展，但仍存在许多关键问题 关于MYC表达上调如何导致肿瘤发生。MYC之间的关系 上调及其转化活性尚未以定量方式研究，这是重要的 因为表达升高的一个潜在后果是当蛋白质在细胞中的表达增加时发生蛋白质相分离， 蛋白质浓度高于临界浓度。最近，许多转录因子， 已经报道了本征无序区（IDR）经历浓度依赖性液-液 相分离（LLPS），形成生物分子缩合物（也称为无膜区室， 液滴）。MYC癌蛋白是转录因子，含有IDR，我们的初步研究表明， 在Kelly MYCN扩增的神经母细胞瘤细胞的细胞核中存在MYCN的点状结构，这表明 MYCN在MYCN高度表达的Kelly细胞中形成缩合物。生物相的功能- 分离的缩合物包括区室化的信号传导。转录因子的浓缩物已经被 提出划分转录机制和重塑基因转录。MYC致癌 信号传导依赖于其与包括MAX和TRRAP在内的关键蛋白的相互作用来实现转录活性 和转化，抑制这些相互作用导致MYC失活。为了理解上调的 MYC癌蛋白及其与肿瘤发生的联系，需要解决几个基本的分子生物学问题。 讨论了MYC缩合物的生物物理/化学机制和功能作用，包括：1） MYC在生物化学和细胞生物学中是否以及如何经历相分离并形成凝聚物 系统; 2）MYC浓缩物是否将转录机器区室化; 3）MYC阶段是否 分离重塑下游基因表达，以及MYC的缩合物是否是下游基因表达所必需的。 转化活动。由于LLPS依赖于蛋白质浓度， 分离经常与表达水平变化的影响纠缠在一起。解剖MYC LLPS， 因此，需要能够操纵MYC LLPS而不影响其功能的化学遗传学工具。 改变蛋白质水平，理想情况下，这种操纵可以在两个相反的方向上进行： 冷凝物形成和溶解存在的冷凝物。在这里，我们建议开发这样的化学遗传 工具，并将其与荧光蛋白标记技术和荧光成像相结合， 剖析MYCN缩合物的生化机制和功能作用。

Abstract MYC oncoproteins including c-Myc and MYCN are major drivers of human tumorigenesis. Expression of MYC is deregulated and enhanced in many types of human cancer. For instance, MYCN is a major driver of childhood cancers. While upregulated MYC expression induces tumor development in many tissues, depletion of MYC abolishes tumorigenesis and results in tumor regression in various tumor models. Despite significant progress in understanding the link between MYC upregulation and tumorigenesis, many key questions remain regarding how upregulated expression of MYC leads to tumorigenesis. The relationship between MYC upregulation and its transforming activity has not been investigated in a quantitative manner, which is important because one potential consequence of elevated expression is protein phase separation that occurs when protein concentration is above critical concentration. Recently, many transcription factors that contain intrinsically disordered region (IDR) have been reported to undergo concentration-dependent liquid-liquid phase separation (LLPS), forming biomolecular condensates (also known as membraneless compartments, liquid droplets). MYC oncoproteins are transcription factors and contain IDR and our preliminary study reveals punctate structures of MYCN in the nucleus of Kelly MYCN-amplified neuroblastoma cells, suggesting that MYCN forms condensates in the Kelly cells where MYCN is highly expressed. Functions of biological phase- separated condensates include compartmentalized signaling. Condensates of transcriptional factors have been proposed to compartmentalize transcriptional machineries and remodel gene transcription. MYC oncogenic signaling relies on its interaction with the key proteins including MAX and TRRAP for the transcriptional activity and transforming, and inhibition of these interactions results in MYC inactivation. To understand upregulated MYC oncoproteins and their link to tumorigenesis, several basic molecular biological questions need to be addressed regarding biophysical/chemical mechanisms and functional roles of MYC condensates, including: 1) Whether and how MYC undergoes phase separation and forms condensates in biochemical and cellular systems; 2) Whether MYC condensates compartmentalize transcriptional machineries; 3) Whether MYC phase separation remodels downstream gene expression and whether condensates of MYC is necessary for the transforming activity. Because LLPS is dependent on protein concentration, biological functions of phase separation are often entangled with effects from changes of expression levels. To dissect MYC LLPS and functional consequences, there is a need for chemogenetic tools that can manipulate MYC LLPS without changing protein levels, and ideally such manipulation can be performed in two opposite directions: driving condensate formation and dissolving existing condensates. Here we propose to develop such chemogenetic tools and combine them with fluorescent protein labeling technology and multicolor fluorescence imaging to dissect biochemical mechanisms and functional roles of MYCN condensates.