A new super-resolution proximity assay to probe RNA transcription condensates

尽管每个细胞都以基因组DNA的形式在细胞核中具有相同的遗传信息，但这些信息的哪一部分在特定细胞中被用来（“转录”）制造蛋白质是受到严格调控的，从而使细胞能够实现大量不同的功能。尽管这种转录调控的机制对每个生物体都很重要，但它们的机制还不完全清楚。最近已经发现，在细胞核内有专门的微观区域，参与遗传转录的分子集中在那里。为了确定这些微观区域或“凝聚体”的作用，我们将研究哪些分子在其中相互作用，以及当细胞暴露于压力时这些相互作用是如何变化的。由于微观凝析物的多样性和非常小的尺寸，使用传统方法很难检测和量化微观凝析物内部的相互作用。因此，我们将开发并应用一种新的显微镜技术，以非常高的空间分辨率来解剖分子相互作用，这样我们就可以直接看到(i)哪些分子相互作用，（ii）这些分子在哪里相互作用，（iii）有多少分子以这种方式相互作用，所有这些都在微观凝聚体中。为了达到这些严格的要求，我们将使用纳米技术的方法，利用合成DNA序列来测试两个分子是否彼此接近（比如在10纳米内），并通过一系列被称为“光学超分辨率显微镜”的显微镜技术提供的非常高的分辨率对其进行成像，这些显微镜技术允许在~ 10纳米内检测和定位这些蛋白质对。该项目将专注于利用DNA纳米技术和超分辨率成像的原理，适应和增强新技术，称为ePD-PAINT（增强邻近依赖性PAINT）。ePD-PAINT将首先用DNA本身制成的合成测试样品进行验证，并将其称为“DNA折纸”，类似于折纸艺术，将DNA分子折叠成定义的分子模式。改进的ePD-PAINT方法随后将在生物细胞中进行验证，使用试验，可以操纵蛋白质以诱导分子相互作用，以响应小化学雷帕霉素的类似物，雷帕霉素也被用作免疫抑制剂。为了完成验证，使用ePD-PAINT技术的测量将与经典的生化蛋白质相互作用分析进行比较。我们将使用ePD-PAINT直接在微观凝聚物中研究参与转录的几个关键蛋白之间的相互作用，并测试当转录受损时这些相互作用是如何变化的，无论是通过干预转录过程中的步骤，还是通过将细胞暴露于已知的外部压力下，在细胞响应和适应外部约束时改变转录。这些发现将对基础细胞生物学产生重大影响，并使人们能够更深入地了解基因转录机制集中区域的蛋白质-蛋白质相互作用。此外，它们可能会支持基因表达调控的主要新机制。这不仅对所有细胞生物学都很重要，而且还可能为在疾病期间发生错误时操纵过程的新策略提供信息。值得注意的是，许多已知积聚在凝聚体中的蛋白质（包括BRD4，这里将进行研究）与癌症和神经变性有关。

Although each cell has the same genetic information in the cell nucleus in the form of genomic DNA, which parts of this information are used ("transcribed") in a given cell to make proteins is closely regulated, allowing cells to fulfil a large number of different functions. The mechanisms that underlie the regulation of this transcription are incompletely understood despite their fundamental importance for every organism. Recently it has been discovered that there are specialised microscopic regions within the cell nucleus where molecules involved in genetic transcription are concentrated. To establish the roles of these microscopic regions or "condensates" we will study which molecules interact in them and how these interactions change when the cell is exposed to stress.Detecting and quantifying interactions within the microscopic condensates is difficult using traditional methods due to their multiplicity and very small size. We will therefore develop and apply a new microscopy technology to dissect the molecular interactions with very high spatial resolution so that we can directly see (i) which molecules interact, (ii) where these molecules interact and (iii) how many molecules interact in this way, all within the microscopic condensates. To achieve these stringent requirements we will use methods of nanotechnology that employ synthetic DNA sequences to test if two molecules are close to each other (say within 10 nm) and image this with the very high resolution provided by a range of microscopy techniques termed "optical super-resolution microscopy" which allow detection and localisation of such protein pairs to within ~ 10 nm.This project will focus on adapting and enhancing the new technology, termed ePD-PAINT (enhanced proximity-dependent PAINT), by using the principles of DNA nanotechnology and super-resolution imaging. ePD-PAINT will be first validated with synthetic test samples, made of DNA itself, and termed "DNA origami" in analogy with the art of paper folding, folding DNA molecules to defined molecular patterns. The improved ePD-PAINT approach will then be validated in biological cells using assays where proteins can be manipulated to induce molecular interactions in response to an analogue of the small chemical rapamycin, which is also used as an immune-suppressant. To complete validation, the measurements with the ePD-PAINT technology will be compared to classical biochemical protein interaction assays.We will investigate the interactions between several key proteins involved in transcription using ePD-PAINT directly in the microscopic condensates and test how these interactions change when transcription is impaired, either by interventions in the steps performed during transcription or by exposing the cell to external stress which is known to change transcription as the cell responds and adapts to the external constraints.These findings will have significant impact on basic cell biology and enable a deeper understanding of protein-protein interactions in regions where the gene transcription machinery is concentrated. Furthermore, they may come to underpin a major new mechanism underlying the regulation of gene expression. Not only is this important for all of cell biology, but it may also inform novel strategies to manipulate the process when errors occur during disease. Of note, many of the proteins known to accumulate in condensates (including BRD4 as will be studied here) are implicated in cancer and neurodegeneration.