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: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=663897
• 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?

该研究计划的目的是了解特殊的染色体结构域如何从其组成蛋白组装而成，并确定这些区域的动态如何响应不断变化的生理条件而变化。染色体的特定区域被组装成特殊的结构，这些结构由包装到染色质中的 DNA 组成，并被特定的染色体蛋白包裹。 尽管这些结构通常被视为静态元素，但它们对不断变化的细胞条件做出响应，但它们的边界可以改变，并且可以移动到染色体上的新位置。 **该计划目前正在研究芽殖酵母（酿酒酵母）中一种模型染色体结构域（沉默染色质）的结构和动力学。异染色质结构域在染色体的结构和传递以及细胞身份和增殖的调节中发挥着核心作用。 **在芽殖酵母中，异染色质含有低乙酰化和去甲基化的核小体，并与 Sir2、Sir3 和 Sir4（沉默信息调节因子）这三种蛋白质的复合物结合，称为 SIR 复合物。  Sir2 是一种保守的 NAD 依赖性蛋白脱乙酰酶，可在异染色质内产生核小体的低乙酰化结构域。  Sir3 和 Sir4 是组蛋白结合蛋白，被认为在染色质上扩散并形成抑制结构。  目前的异染色质 SIR 复合物组装模型表明，在招募到 DNA 元件后，组蛋白脱乙酰化和 SIR 复合物结合的迭代轮次会导致异染色质的扩散。  尽管异染色质的成核作用已被充分了解，但 Sir 蛋白在异染色质的扩散和稳定性中的确切作用却知之甚少。我们重点关注 Sir3 和 Sir4 功能的两个方面：**1。确定Sir3传播的机制。  尽管所有三种 SIR 复合体蛋白的突变都会消除异染色质的形成，但很难确定这三种蛋白是否都是成核和扩散所必需的。我们已经创建并表征了 Sir3 的突变体，这些突变体破坏了其结合 Sir4 的能力，并表明突变体 Sir3 可以以接近野生型的效率扩散和形成沉默染色质。  我们当前的模型提出 Sir3 扩散是由 Sir3 寡聚化驱动的，而不是组蛋白修饰的变化。我们已经在 Sir3 N 末端发现了一个可能介导 Sir3 寡聚化的小环，并且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 Sir3 和 Sir4 的细胞周期依赖性磷酸化如何调节沉默染色质的稳定性。  沉默染色质在 G1 期的稳定性明显高于有丝分裂期，但控制这种稳定性的机制尚不清楚。  我们将检查 SIR 复合物的组成和修饰的细胞周期变化，并使用简单的转录读数以及 FRAP 和生化测定来测试这些变化是否调节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 Asf2（沉默染色质的新型核心成分）。**意义。  对芽殖酵母的研究开创了对异染色质的理解，该提案解决了染色质生物学中的许多基本问题：1）组蛋白修饰对于染色质结构域的扩散有多重要？ 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以及它们是否作为聚合物绕过了对特定组蛋白标记的需要知之甚少，3) 细胞在细胞周期中如何调节染色质结构？