Single-molecule dynamics of human transcription regulation

我的实验室对理解细胞如何“决定”什么基因以及何时启动很感兴趣。例如，了解基因是如何被调控的，可以为临床医生提供治疗遗传疾病的新药，为科学家提供将正常细胞转化为干细胞的新工具。以前，科学家们不得不从遗传学（通过跟踪遗传特征）或生化实验（通过粉碎细胞并分析其化学成分）中推断基因是如何工作的。然而，还没有人“看到”人类基因分子开启，这限制了我们对基因调控的理解。最近，我们的实验室开发了一种成像技术，可以直接“看到”基因分子被单个酶分子打开。在这个提议中，我们将利用我们的技术在单分子分辨率下重建基因激活的最小电路，这将给我们关于基因如何被调节的基本新想法。从物理上讲，基因是细胞核中的DNA分子；大多数细胞每个基因只包含两个分子。开启一个基因意味着根据DNA编码的信息制造一种蛋白质。细胞通过向基因分子招募一种叫做Pol II的酶来决定开启一个基因。然后Pol II沿着基因运行，同时制造（转录）基因的“活性副本”，称为RNA，然后将其用作制造蛋白质的模板。这项提议的重点是找出Pol II是如何“决定”首先与基因结合的。这是一个重要的问题，因为许多遗传疾病（如癌症）可以追溯到Pol II结合和复制一组错误的基因。Pol II与基因的结合需要其他几个分子，称为转录因子。虽然大多数转录因子是已知的，但它们相互作用将Pol II带入基因的顺序尚不清楚。通过类比，要理解足球，仅仅识别球场上的每个球员是不够的：人们必须了解球员如何相互作用，才能在门柱之间把球（Pol II）拿到（基因）。我们建议通过观察整个分子游戏来阐明转录因子如何将Pol II带到基因中。为此，我们将从细胞中分离出一组最小的参与者——Pol II和五种转录因子，并将它们混合在一滴生理溶液中。然后我们把一个基因分子放在显微镜载玻片上，用液滴覆盖，然后在暗室里的显微镜下观察基因发生了什么。为了看到Pol II分子和转录因子，我们将用不同颜色的染料（如蓝色、绿色和红色）标记它们，这将使分子在黑暗的背景下像星星一样发光。通过观察蓝色、绿色和红色星形与基因结合的顺序，我们将确定转录因子如何将Pol II带到基因中。在我们阐明了由五个因子组成的最小团队是如何进行游戏的之后，我们将增加一个参与者——转录“激活因子”Sp1，它在快速分裂和癌细胞中含量很高——并确定Sp1如何改变游戏（例如，与哪些转录因子相互作用），使Pol II产生更多的基因RNA拷贝。我们对单分子如何开启基因的分析可能会从根本上改变科学家对基因调控的看法。在教科书中，打开一个基因通常被描述为打开一个开关。然而，由于细胞中的每个基因仅由两个分子表示，因此，由于微观世界中分子的布朗运动，打开一个基因可能是一个随机（“草率”）事件。因此，细胞做出的所有决定（例如，变成癌细胞的决定）都可能受到Pol II、转录因子和基因之间的随机碰撞的影响——这可能解释了为什么一些细胞行为难以控制。

My lab is interested in understanding how cells 'decide' what genes, and when, to switch on. Knowing how genes are regulated could, for example, provide clinicians with new drugs to cure genetic diseases, and scientists with new tools to turn regular cells into stem cells. Previously, scientists had to infer how genes work from genetics (by following inherited traits), or from biochemical experiments (by grinding up cells and analyzing their chemical composition). However, no one has ever 'seen' a human gene molecule switch on, which limited our understanding of gene regulation. Recently, our lab has developed an imaging technology to directly 'see' gene molecules being switched on by single enzyme molecules. In this proposal, we will use our technology to reconstruct a minimal circuit of gene activation at single-molecule resolution, which will give us fundamentally new ideas on how genes are regulated.Physically, genes are molecules of DNA located in the cell nucleus; most cells contain only two molecules of each gene. To switch a gene on means to make a protein based on the information encoded in the DNA. The cell decides to switch on a gene by recruiting an enzyme, called Pol II, to the gene molecule. Pol II then runs along the gene while making (transcribing) an 'active copy' of the gene, called RNA, which is then used as a template for making protein. The focus of this proposal is to find out how Pol II 'decides' to bind to a gene in the first place. This is an important question, because many genetic diseases (e.g. cancer) can be traced back to Pol II binding and copying a wrong set of genes.Binding of Pol II to genes requires several other molecules, called transcription factors. Although most of transcription factors are known, the order in which they interact with each other to bring Pol II to a gene is not clear. By analogy, to understand football, it is not enough to identify each player in the field: one has to understand how the players interact with each other to get the ball (Pol II) between the goalposts (to a gene). We propose to elucidate how transcription factors bring Pol II to a gene, literally, by watching the entire molecular game live. To do that, we will isolate a minimal team of players -- Pol II and five transcription factors -- from cells and mix them together in a drop of physiological solution. We will then put a single gene molecule on a microscope slide, cover it with the drop, and watch what is going on at the gene under a microscope in a dark room. To see the molecules of Pol II and transcription factors, we will label them with dyes of different colors (e.g. blue, green, and red), which will make the molecules glow against dark background like stars. By watching in what order the blue, green, and red stars bind to the gene, we will determine how the transcription factors bring Pol II to the gene.After we elucidate how the minimal team of five factors plays the game, we will add one more player -- transcription 'activator' Sp1, which is present at high levels in rapidly dividing and cancerous cells -- and determine how Sp1 changes the game (e.g. with which transcription factors it interacts) to make Pol II produce more RNA copies of a gene. Our analysis of how single molecules switch genes on may fundamentally change the way scientists think about gene regulation. In textbooks, switching a gene on is often shown as flipping on a switch. However, because each gene in a cell is represented by only two molecules, switching a gene on could be a stochastic ('sloppy') event, due to the Brownian motion of molecules in the microscopic world. Therefore, all decisions made by the cell (for instance, a decision to turn into a cancer cell) could be affected by stochastic collisions between Pol II, transcription factors and genes -- which may explain why some cell behaviors are difficult to control.