Structure and Dynamics of Chromosomal Domains

染色体结构域的结构和动力学

• 批准号: RGPIN-2014-06096
• 负责人: Rudner, Adam
• 金额: $ 2.99万
• 依托单位: University of Ottawa
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=588807
• 关键词: Structure; Dynamics; Chromosomal; Domains

The objective of this research program is to understand how specialized chromosomal domains are assembled from their constituent proteins, and to determine how the dynamics of these regions change in response to changing physiological conditions. Specific regions of chromosomes are assembled into specialized structures composed of DNA packaged into chromatin and coated by specific chromosomal proteins. Although these structures are often viewed as static elements they are responsive to changing cellular conditions, their boundaries can change and they can move to new locations on chromosomes. This program is currently investigating the structure and dynamics of one model chromosomal domain, silent chromatin, in the budding yeast, Saccharomyces cerevisiae. Heterochromatic domains play a central role in the structure and transmission of chromosomes, and in the regulation of cell identity and proliferation.  In budding yeast heterochromatin contains nucleosomes that are hypoacetylated and demethylated and are bound by a complex of three proteins, Sir2, Sir3 and Sir4 (Silent information regulator), named the SIR complex.  Sir2 is a conserved NAD-dependent protein deacetylase and creates the hypoacetylated domains of nucleosomes within heterochromatin.  Sir3 and Sir4 are histone-binding proteins that are believed to spread on chromatin and form a repressive structure.  Current models for SIR complex assembly of heterochromatin propose that after recruitment to a DNA element, iterative rounds of histone deacetylation and SIR complex binding lead to spreading of heterochromatin.  Although nucleation of heterochromatin is well understood, the precise role of the Sir proteins in the spreading and stability of heterochromatin is poorly understood. We are focusing on two aspects of Sir3 and Sir4 function: 1. To determine the mechanism of Sir3 spreading.  Although mutations in all three SIR complex proteins abolish heterochromatin formation, it has been difficult to determine if all three proteins are required for both nucleation and spreading. We have created and characterized mutants of Sir3 that disrupt its ability to bind Sir4, and have shown that mutant Sir3 can spread and form silent chromatin with near wild type efficiency.  Our current model proposes that Sir3 spreading is driven by Sir3 oligomerization, and not changes in histone modifications. We have identified a small loop in the Sir3 N-terminus that may mediate Sir3 oligomerization, and our characterization of mutations in this region will allow us to rigorously test our model. 2. To determine how cell cycle dependent phosphorylation of Sir3 and Sir4 regulate the stability of silent chromatin.  Silent chromatin is significantly more stable in G1 than in mitosis, but the mechanism that controls this stability is unknown.  We will examine cell cycle changes in the composition and modification of the SIR complex and test if these changes modulate the stability of silent chromatin using simple transcriptional readouts, as well as FRAP and biochemical assays.  Our initial focus will be on phosphorylation of Sir3 and Sir4, which are phosphorylated at different stages of the cell cycle, and Asf2, a novel core component of silent chromatin. Significance.  Work on budding yeast has pioneered the understanding of heterochromatin, and this proposal addresses a number of fundamental questions in chromatin biology: 1) How important are histone modifications for the spreading of chromatin domains? 2) Heterochromatin is thought to be formed from polymers of heterochromatin proteins, but little is known about how these proteins polymerize and if as polymers they bypass the need for specific histone marks, 3) How do cells regulate chromatin structures during the cell cycle?

这项研究计划的目的是了解特殊的染色体区域是如何从它们的组成蛋白组装起来的，并确定这些区域的动态如何随着生理条件的变化而变化。染色体的特定区域被组装成特殊的结构，由DNA包装成染色质，并被特定的染色体蛋白覆盖。尽管这些结构通常被视为静态元件，它们对不断变化的细胞条件做出反应，但它们的边界可以改变，它们可以移动到染色体上的新位置。 该计划目前正在研究酿酒酵母中一种模式染色体结构域--沉默染色质的结构和动力学。异染色域在染色体的结构和传递，以及对细胞身份和增殖的调控中发挥着核心作用。 在发芽酵母中，异染色质含有去乙酰化和去甲基化的核小体，并被三种蛋白质Sir2、Sir3和Sir4(沉默信息调节因子)组成的复合体结合，称为SIR复合体。Sir2是一种保守的依赖于NAD的蛋白质脱乙酰酶，在异染色质中产生核小体的低乙酰化区域。Sir3和Sir4是组蛋白结合蛋白，被认为分布在染色质上，形成抑制结构。目前的SIR异染色质复合体组装模型认为，在募集到DNA元件后，组蛋白脱乙酰化和SIR复合体结合导致异染色质的扩散。尽管人们对异染色质的成核有很好的理解SIR蛋白在异染色质扩散和稳定中的确切作用还知之甚少。我们主要关注Sir3和Sir4功能的两个方面： 1.确定Sir3扩散的机制：虽然所有三个SIR复合体蛋白的突变都会取消异染色质的形成，但很难确定这三个蛋白质是否都是成核和扩散所必需的。我们已经创建并鉴定了Sir3的突变体，这些突变体破坏了它与Sir4的结合能力，并表明突变的Sir3可以以接近野生型的效率扩散并形成沉默的染色质。我们目前的模型认为，Sir3的扩散是由Sir3齐聚驱动的，而不是组蛋白修饰的变化。我们已经在Sir3N-末端发现了一个可能介导Sir3齐聚的小环，我们对该区域突变的表征将使我们能够严格测试我们的模型。 2.确定依赖于细胞周期的Sir3和Sir4的磷酸化如何调节沉默染色质的稳定性。沉默染色质在G1期明显比有丝分裂中更稳定，但控制这种稳定性的机制尚不清楚。我们将检测细胞周期中SIR复合体的组成和修饰的变化，并使用简单的转录读数以及FRAP和生化分析来测试这些变化是否调节了沉默染色质的稳定性。我们最初的重点将是Sir3和Sir4的磷酸化，它们在细胞周期的不同阶段被磷酸化，以及沉默染色质的新的核心成分ASF2。 意义。有关发芽酵母的工作开创了对异染色质的理解，这个建议解决了染色质生物学中的一些基本问题：1)组蛋白修饰对于染色质区域的传播有多重要？2)异染色质被认为是由异染色质蛋白的聚合物形成的，但人们对这些蛋白质如何聚合知之甚少，如果作为聚合物它们绕过了对特定组蛋白标记的需要，3)细胞如何在细胞周期中调节染色质结构？