通过超高分辨率荧光成像技术研究细胞基因转录噪声组合调控的分子机制

Gene transcriptions are inherently random due to the stochastic activities of the molecules. Random fluctuations in gene expression can potentially affect the functioning of genetic circuits and downstream signaling pathways, thus may lead to phenotypic heterogeneity in a population of cells. An appealing view on transcriptional noise is that it may not be a kind of dysregulation of the central dogma, instead, it could be utilized by cells to achieve certain functions. A number of recent studies have addressed this fascinating point, suggesting that gene fluctuations may be under the direct control of cells in responding to intracellular and extracellular signals. ..Despite great details of the spatial and temporal patterns have been revealed by direct observations on gene transcriptions in living cells, the mechanism that cells can utilize to control the gene expression noise and phenotypic heterogeneity is still poorly understood. Recently, an experiment on single cells suggested that cells can control the diverse patterns of gene expression through combinatorial gene regulation by modulating the relative timing of two TFs. Another single-cell imaging study found that two TFs can form dynamic logic gates in regulating their target genes. These studies inspired us the role of combinatorial gene regulation in controlling phenotypic heterogeneity...In this project, we plan to examine the role and mechanism of combinatorial gene regulation in modulating transcriptional noise. We started by formulating a single-TF model that can describe transcriptional bursting in single cells. Next, we extended the single-TF model to the two-TF model by introducing combinatorial logic to represent the interactions between TFs. Then, based on the models and recent experimental findings, we generated three progressive hypotheses to interrogate the relationships between combinatorial gene regulation and gene expression noise. First, we hypothesize that combinatorial gene regulation can be utilized by cells to generate larger noise of gene expression comparing to single-TF regulation. Testing on this hypothesis will help to answer whether combinatorial regulation is responsible for the additional noise. Then, we plan to examine whether the noises are due to the adoption of various regulatory logics by testing the second hypothesis. In order to conduct this test, we develop a model of combinatorial gene regulation using experimental data. Identification of the model can obtain the predicted regulatory logic. Then, by aggregating the cells with the same predicted logic in the same group, we are able to compare the means among different logic groups, to investigate whether the noises are different in different logic groups. Further, we ask whether there is extra layer of noise control beyond the regulatory logic. We found that variations in the initial phases of TF binding might alter the bursting patterns only when the gene is regulated by multiple TFs. And, there is no mechanism for two cells to coordinate TF binding. As such, we hypothesize that, the lacking of coordination mechanism on TF binding between cells, can be leveraged by combinatorial regulation to generate extra variations in gene expression. ..To test these hypotheses, we plan to construct a single-lac system, a single-tet system, and several hybrid tet-lac systems in E. coli cells, using lacZ as the target gene. The fluorescence signals of gene expression and TF binding will be measured using super-resolution microscopy. Using E. coli cells rather than eukaryotic cells as the validation platform, can bring additional advantages to avoid potential confounding effects of nucleosome occupancy and TATA box. Moreover, using bacteria cells in this study make it convenient for model identification.

即使在具有共同遗传背景和生存环境的细胞群体内，基因表达也往往存在差异。这种差异称作转录噪声。一方面，细胞通过适当表达转录噪声产生群体多样性，从而提高其环境适应性。另一方面，细胞也可以通过压制转录噪声水平来实现对表型的精确控制。本项目旨在研究细胞控制转录噪声的分子机制。为此，我们构造了单细胞转录脉冲的理论模型，并据此提出研究假设以回答以下科学问题：1.多转录因子组合调控与单因子调控相比，是否会产生不同的转录噪声？2.多因子调控中不同的逻辑组合是否将导致不同的噪声水平？3.多因子调控中，转录因子结合DNA的时间相位是否可以调控噪声水平？我们将构造多套不同的大肠杆菌系统，分别模拟单转录因子调控及不同的双转录因子组合调控模式。我们拟使用超高分辨显微镜对目标基因的转录活动及转录因子的结合状态进行动态观测。结合理论模型以及观测数据，我们将揭示多因子调控在细胞控制转录噪声中的作用机制。

基因转录本质上是随机的，分子随机活动会导致基因表达的随机波动，这可能会影响基因调控和下游信号通路的功能。目前流行的观点是认为转录噪声的产生可能不是中心法则失调。相反，转录噪声可能被细胞利用来实现某些特定的功能。研究发现基因转录活动的波动可能在细胞对内外信号的响应中进行了直接控制，细胞可以通过调节两个转录因子的相对时间和组合基因来控制基因表达的多种模式。两个转录因子可以形成动态逻辑门调控以调节靶基因的转录活性。但是，如何通过组合基因调节来控制转录噪声的机制仍然不清楚。.我们研究了组合基因调节在调节细胞转录噪声中的作用和机制。我们首先制定了一个单转录因子模型用于描述单个细菌细胞中的转录波动。我们通过引入组合逻辑来表示转录因子之间的相互作用，将单转录因子模型扩展为双转录因子模型。然后，基于模型和最近的实验发现，我们提出了三个假设，以探究组合基因调节对基因表达噪声的影响。我们假设双转录因子调节会导致比单转录因子调节更大的基因表达噪声。实验中发现由于组合基因调节引起的大振幅振荡信号可能产生更高水平的噪声。我们通过第二个假设来检查不同调节逻辑的噪声是否不同。为了进行该验证，我们开发了组合基因调控的理论框架。求解计算模型可以确定一个细胞中主导的调控逻辑。通过将具有相同逻辑的细胞聚类到同一组中，我们可以研究不同逻辑组中的噪音是否不同。我们尝试探索了是否有超出调控逻辑的噪音控制机制。通过模拟研究我们发现TF结合的相位变化可以改变由两个或多个TF调控的基因波动模式。因此，我们假设在细胞中TF结合的不同步可以被基因调控逻辑利用以产生额外的基因表达变异。.这个项目的主要创新包括：（i）通过直接观察揭示组合基因调控逻辑对单一细菌细胞中的转录噪音的影响；（ii）描述TF结合不同步调控基因表达噪音的机制；（iii）开发了量化框架，对单个细胞中的组合基因调控进行建模，可以表征基因转录中波动模式的动态行为。