Super-resolution microscopy for dynamic analysis of focal enhancer amplifications in cancer

项目摘要 癌症通常被认为是一种遗传病。例如，特定的蛋白质编码突变可以 激活致癌基因。然而，致癌基因也可以通过改变到 非编码DNA。具体地说，增强子是可以激活基因表达的非编码DNA元件 跨越了遥远的基因组距离。在许多癌症中，基因改变导致增强剂过度激活 致癌基因。这也被称为增强子劫持。 在这里，我们关注的是上皮癌，激活myc癌基因的增强子被复制。 并进行了聚焦放大。这与更高的MYC表达和更高的致瘤性有关。但是 分子机制还不是很清楚。实现机械性的理解很重要，因为它可能 确定分子‘阿喀琉斯高跟鞋’，可以作为治疗的靶点。 实现对增强子作用和增强子劫持的一般机制的理解 癌症一般需要在高时空精度下测量增强子-基因通讯的能力 在活细胞中。建议的项目将建立和验证这样一个基于超分辨率的平台 显微镜，它使跟踪单个增强剂和基因成为可能，精度为10纳米 在活细胞中，以及基因活动的实时。将致力于验证超级- 通过广泛的对照实验，分辨率显微镜方法。我们还将作出重大努力 将技术创新和技术诀窍传播给更广泛的社区。 综上所述，我们建议建立和验证一个创新的、综合的实验和 用于研究焦点增强子放大的计算超分辨率显微镜平台。虽然我们最初的 重点是MYC增强剂在上皮性癌症中的放大作用，拟议的平台可能会发现 在MYC之外的广泛使用。

Project Summary Cancer is often thought of as a genetic disease. For example, specific protein-coding mutations can activate cancer-causing oncogenes. However, cancer-causing oncogene can also be activated by alterations to non-coding DNA. Specifically, enhancers are non-coding DNA elements that can activate gene expression across vast genomic distances. In numerous cancers, genetic alterations cause enhancers to over-activate cancer-causing oncogenes. This is also referred to as enhancer hijacking. Here we focus on epithelial cancers, where enhancers that activate the MYC oncogene get duplicated and focally amplified. This correlates with higher MYC expression and increased tumorigenicity. But the molecular mechanisms are not well understood. Achieving a mechanistic understanding is important since it may identify molecular ‘Achilles Heels’ that can be targeted therapeutically. Achieving an understanding of the mechanism of enhancer action in general and enhancer hijacking in cancer in general requires an ability to measure at high spatiotemporal precision enhancer-gene communication in living cells. The proposed project will establish and validate such a platform based on super-resolution microscopy, which make it possible to track individual enhancers and genes with a precision of ten nanometers in living cells as well as gene activity in real time. Significant efforts will be devoted to validating the super- resolution microscopy approach through extensive control experiments. Significant efforts will also be devoted to disseminating the technological innovations and know-how to the greater community. In summary, we propose to establish and validate an innovative and integrative experimental and computational super-resolution microscopy platform for studying focal enhancer amplifications. While our initial focus is on amplifications of MYC enhancers in epithelial cancers, the proposed platform is likely to find widespread use beyond MYC.