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

尽管每个细胞都以基因组DNA的形式在细胞核中拥有相同的遗传信息，但这些信息的哪些部分在给定的细胞中被使用(“转录”)来制造蛋白质是受到严格调控的，使细胞能够履行大量不同的功能。这种转录调控的基础机制还不完全清楚，尽管它们对每个生物体都很重要。最近，人们发现在细胞核内有一些特殊的微观区，参与基因转录的分子集中在那里。为了确定这些微观区域或“凝聚物”的作用，我们将研究哪些分子在其中相互作用，以及当细胞暴露在压力下时，这些相互作用如何变化。由于微观凝聚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这些微观凝聚物中的相互作用。因此，我们将开发和应用一种新的显微技术，以非常高的空间分辨率剖析分子相互作用，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物中。为了达到这些严格的要求，我们将使用纳米技术的方法，利用合成的DNA序列来测试两个分子是否彼此接近(比如在10 nm以内)，并用一系列被称为“光学超分辨率显微镜”的显微技术提供的非常高的分辨率对其成像，该技术允许检测和定位这种蛋白质对到大约10 nm以内。这个项目将专注于通过使用DNA纳米技术和超分辨率成像的原理来适应和增强这项名为EPD-Paint(增强型邻近相关涂料)的新技术。EPD-Paint将首先用合成的测试样本进行验证，这些样本由DNA本身制成，并被称为“DNA折纸”，类似于折叠纸张的艺术，将DNA分子折叠成定义的分子模式。然后，改进的EPD-PAINT方法将在生物细胞中得到验证，在生物细胞中，蛋白质可以被操纵，以诱导分子相互作用，以响应一种小的化学物质雷帕霉素的类似物，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。我们将直接在微观凝集物中使用EPD-PAINT来研究参与转录的几个关键蛋白质之间的相互作用，并测试当转录受到损害时，这些相互作用如何变化，无论是通过对转录过程中执行的步骤进行干预，还是通过将细胞暴露在外部压力下，当细胞对外部约束做出反应并适应时，外部压力会改变转录。这些发现将对基础细胞生物学产生重大影响，并使我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，它们可能成为基因表达调控的主要新机制的基础。这不仅对所有细胞生物学都很重要，而且还可能提供新的策略，当疾病期间发生错误时操纵这一过程。值得注意的是，许多已知在凝集物中积累的蛋白质(包括这里将要研究的BRD4)都与癌症和神经退化有关。

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.