Gene Regulation from Long-Distance Chromosomal Interactions in Yeast

 描述(申请人提供)：严格控制基因表达对细胞增殖和分化是必不可少的。近年来，越来越多的证据表明，基因组的三维组织对于基因活性的调控具有重要意义。染色体构象捕获(3C)技术及其衍生物(4C、Hi-C等)揭示了在各种真核物种中广泛的染色体内和染色体间的相互作用。其中一些相互作用，如在β-珠蛋白基因位点控制区的增强子-启动子环，在基因调控中发挥着重要作用。另一种类型的长距离相互作用，即同源配对，也可以影响体细胞二倍体细胞的基因表达。在芽殖酵母的营养生长过程中，观察到了染色体的远距离相互作用和同源配对。然而，这些相互作用的功能意义尚不清楚。这项建议的目标是识别和机械地剖析调控萌芽酵母基因表达的长距离相互作用。我们将分析单个活细胞中的基因表达，以探讨其对平均水平、细胞间变异性(Noise)和 基因表达。在目标1中，我们重点研究同源等位基因之间的相互作用对基因的调控作用。我们的初步数据提供了强有力的证据，证明报告基因可以与其等位基因拷贝接触并影响其活性。我们计划充分描述这一现象，并阐明其潜在机制。此外，我们还将开创一种操纵远距离染色体相互作用的方法，并探索相应的基因调控效应。在目标2中，我们将研究非同源的远距离染色体相互作用对基因的调控。我们设计了一个新的酵母文库，其中包含双报告，以筛选导致基因表达变化的远距离相互作用。在部分文库菌株中发现了报告表达的改变(平均水平和噪声)，这可能是由染色体环状/聚集引起的。我们建议对这些菌株的基因调控进行进一步的表征，并对调控机制进行深入分析。这些研究的完成将为了解长途电话的调节功能提供关键的见解 并建立芽殖酵母作为研究远距离基因调控的重要模型。这些工作也可能为在多细胞真核生物中设计类似的实验产生原则证明概念。

 DESCRIPTION (provided by applicant): Rigorously controlled gene expression is essential for cellular proliferation and differentiation. In recent years, more and more evidence show that the 3D organization of the genome is important for regulation of gene activity. Chromosome Conformation Capture (3C) technique and its derivatives (4C, Hi-C, etc.) have revealed extensive intra- and inter-chromosomal interactions in various eukaryotic species. Some of these interactions, such as the enhancer-promoter looping at the beta-globin Locus Control Region, play an essential role in gene regulation. Another type of long-distance interaction, homologous pairing, can also affect gene expression in somatic diploid cells. Both long-distance chromosomal interactions and homologous pairing were observed in budding yeast during vegetative growth. However, the functional significance of these interactions are not understood. The goal of this proposal is to identify and mechanistically dissect long-distance interactions tha regulate gene expression in budding yeast. We will assay gene expression in single live cells to probe the regulatory effect on the average level, cell-to-cell variability (noise), and dynamics of gene expression. In Aim 1, we focus on gene regulatory effect by interactions between homologous alleles. Our preliminary data have provided strong evidence that a reporter gene can make contacts with its allelic copy and influence its activity. We plan to fully characterize this phenomenon and elucidate the underlying mechanism. In addition, we will pioneer a method to manipulate long-distance chromosomal interactions and probe the corresponding effect on gene regulation. In Aim 2, we will study gene regulation by non-homologous long-distance chromosomal interactions. We designed a novel yeast library containing dual reporters to screen for long-distance interactions that lead to change in gene expression. Altered reporter expression (both the average level and noise) was found in a fraction of the library strains, potentially caused by chromosomal looping / clustering. We propose further characterization of the gene regulation in these strains and an in-depth analysis of the regulatory mechanism. Completion of these studies will provide key insights into the regulatory function of long-distance interactions and establish budding yeast as an important model for studying long-distance gene regulation. These works may also generate proof-of-principle concepts for designing similar experiments in multi-cellular eukaryote.