The Role of Gene Expression Noise in Phenotypic Heterogeneity

描述(申请人提供)：人们普遍观察到生物系统对遗传和环境扰动都表现出很强的耐受性。然而，也观察到克隆的细胞群体总是表现出不同的表型。这种异质性是几个人类健康问题的根源，包括病原体的抗生素耐药性、衰老、癌症进展以及癌细胞对药物治疗的反应。这项提案中描述的工作将试图阐明表型异质性背后的机制。有人提出，基因表达中的噪声是表型变异的一个重要来源，但很难直接检验这一假说。我们实验室的工作已经确定了一组酵母突变株，它们在形态表型上表现出高度的可变性。这组基因丰富了与染色质调节有关的基因，如SNF6。有趣的是，SNF6突变体也显示出转录噪声增加。这似乎支持这样的假设，即基因表达中的噪声与表型变异有关；然而，已知的几个与SNF6在染色质重塑中合作的基因在突变时不会增加形态可变性。为了阐明转录噪声和表型变异之间的关系，我们必须在不同的遗传背景下一起测量这些现象。这个项目的主要目标是阐明导致同基因细胞群体表型异质性的机制。我们假设基因表达中的噪音是表型变异的许多方面的基础。为了验证这一假设，我们将使用SWI/SNF染色质重塑复合体作为案例研究。我们将首先测量SWI/SNF复合体12个亚基中每个突变体的表型可变性。我们将通过测量细胞形态和生长速率的异质性来扩展以前的研究。此外，我们将测量荧光报告蛋白的表达，以确定每个突变体中转录噪音的数量和类型。许多拟议的实验将利用我们实验室开发的高通量显微镜和自动图像分析系统。使用这个系统，我们将对成千上万的细胞进行成像，这些细胞被固定、染色并沉积在玻璃底96孔板中。然后我们将量化每个突变背景中存在的形态可变性。此外，我们还将利用这个系统来监控 研究个体微集落的生长情况，并决定每种基因的生长率分布。最后，我们将使用由各种启动子驱动的荧光报告蛋白来确定每个SWI/SNF突变体对基因表达噪声的影响。这些实验中产生的数据将使我们能够确定随机基因表达是否是表型异质性的必要条件，或者表型的可变性是否可以通过其他方式产生。 公共卫生相关性：人们普遍认为，生物表型完全由遗传学、环境和基因-环境相互作用决定。然而，即使在高度同质的环境中，同基因的细胞群体也总是表现出一系列的表型。我们将检验这一假设，即基因表达的随机波动有助于表型可变性。

DESCRIPTION (provided by applicant): It is widely observed that biological systems are phenotypically robust to both genetic and environmental perturbation. However, it is also observed that a clonal population of cells will invariably display heterogeneous phenotypes. Such heterogeneity is at the root of several human health issues including antibiotic resistance in pathogens, aging, cancer progression, and the response of cancer cells to drug therapies. The work described in this proposal will seek to elucidate the mechanisms that underlie phenotypic heterogeneity. It has been proposed that noise in gene expression is an important source of phenotypic variability, but it has been difficult to test this hypothesis directly. Work n our lab has identified a set of yeast mutants that display a high degree of variability in morphological phenotypes. This set is enriched for genes with roles in chromatin regulation, such as SNF6. Interestingly, SNF6 mutants have also been shown to exhibit increased transcriptional noise. This seems to support the hypothesis that noise in gene expression is associated with phenotypic variation; however, several genes that are known cooperate with SNF6 in chromatin remodeling do not increase morphological variability when mutated. To clarify the relationship between transcriptional noise and phenotypic variation, we must measure these phenomena together, in a variety of genetic backgrounds. The primary objective of this project is to elucidate the mechanisms that cause phenotypic heterogeneity in a population of isogenic cells. We hypothesize that noise in gene expression underpins many aspects of phenotypic variability. To test this hypothesis, we will use the SWI/SNF chromatin remodeling complex as a case study. We will first measure the phenotypic variability of mutants in each of the 12 subunits of the SWI/SNF complex. We will expand upon previous investigations by measuring heterogeneity in cell morphology and growth rate. Additionally, we will measure the expression of fluorescent reporter proteins to determine the amounts, and types, of transcriptional noise in each of these mutants. Many of the proposed experiments will utilize a system of high-throughput microscopy and automated image analysis that has been developed in our lab. Using this system, we will image hundreds of thousands of cells which are fixed, stained, and deposited in glass-bottom 96-well plates. We will then quantify the morphological variability present in each mutant background. Additionally, we will utilize this system to monitor the growth of individual micro-colonies and determine the distribution of growth rates in each genotype. Finally, we will use fluorescent reporter proteins, driven by a variety of promoters, to determine the effect that each SWI/SNF mutant has on gene expression noise. The data generated in these experiments will allow us to determine whether stochastic gene expression is a requisite for phenotypic heterogeneity or whether variability in phenotype can arise by other means. PUBLIC HEALTH RELEVANCE: It is widely believed that biological phenotypes are determined solely by genetics, the environment, and gene- environment interactions. However, isogenic populations of cells will invariably display a range of phenotypes, even in highly homogenous environments. We will test the hypothesis that stochastic fluctuations in gene expression contribute to phenotypic variability.