Structure and Dynamics of Chromosomal Domains

这项研究计划的目的是了解专门的染色体结构域是如何从其组成蛋白质组装，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由包装在染色质中并被特定染色体蛋白质包被的DNA组成的专门结构。虽然这些结构通常被视为静态元素，但它们对变化的细胞条件做出反应，它们的边界可以改变，并且可以移动到染色体上的新位置。该项目目前正在研究芽殖酵母（Saccharomycescerevisiae）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起核心作用。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，并被三种蛋白质Sir 2、Sir 3和Sir 4（沉默信息调节因子）的复合物结合，称为SIR复合物。 Sir 2是一种保守的NAD依赖性蛋白质脱乙酰酶，在异染色质内产生核小体的低乙酰化结构域。 Sir 3和Sir 4是组蛋白结合蛋白，被认为分布在染色质上并形成抑制结构。 异染色质的SIR复合物组装的当前模型提出，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导致异染色质的扩散。 虽然异染色质的成核作用已被很好地理解，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。我们主要关注Sir 3和Sir 4的两个方面的功能：1.确定Sir 3的传播机制。 虽然在所有三个SIR复合物蛋白的突变废除异染色质的形成，它一直难以确定，如果所有三种蛋白质都需要成核和扩展。我们已经创建并表征了破坏其结合Sir 4的能力的Sir 3的突变体，并且已经表明突变体Sir 3可以以接近野生型的效率扩散并形成沉默染色质。 我们目前的模型提出，Sir 3扩散是由Sir 3寡聚化驱动的，而不是组蛋白修饰的变化。我们已经确定了一个小环在Sir 3的N-末端，可能介导Sir 3寡聚化，我们的突变在这个区域的表征将使我们能够严格测试我们的模型。研究细胞周期依赖性Sir 3和Sir 4磷酸化如何调节沉默染色质的稳定性。 沉默染色质在G1期比在有丝分裂期更稳定，但控制这种稳定性的机制尚不清楚。 我们将研究细胞周期的变化的组成和修改的SIR复合物和测试，如果这些变化调节沉默染色质的稳定性，使用简单的转录读出，以及FRAP和生化测定。 我们最初的重点将是Sir 3和Sir 4的磷酸化，它们在细胞周期的不同阶段被磷酸化，以及Asf 2，沉默染色质的一种新的核心成分。 对芽殖酵母的研究开创了对异染色质的理解，这一提议解决了染色质生物学中的一些基本问题：1）组蛋白修饰对染色质结构域的扩展有多重要？2)异染色质被认为是由异染色质蛋白质的聚合物形成的，但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？

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?