Structure and Dynamics of Chromosomal Domains

本研究计划的目的是了解染色体结构域是如何由其组成蛋白组装而成的，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由DNA组成的特殊结构，这些DNA被包装成染色质并被特定的染色体蛋白质包裹。虽然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反应，它们的边界可以改变，它们可以移动到染色体上的新位置。这个项目目前正在研究一种模式染色体结构域的结构和动力学，沉默染色质，在出芽酵母，酿酒酵母。异色结构域在染色体的结构和传递以及细胞身份和增殖的调控中起着核心作用。在出芽酵母中，异染色质含有低乙酰化和去甲基化的核小体，并由三种蛋白质Sir2， Sir3和Sir4（沉默信息调节因子）复合物结合，称为SIR复合物。Sir2是一种保守的nad依赖蛋白去乙酰化酶，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。目前异染色质SIR复合体组装的模型表明，在募集到DNA元件后，组蛋白去乙酰化和SIR复合体结合的反复循环导致异染色质的扩散。虽然异染色质的成核已被很好地理解，但Sir蛋白在异染色质扩散和稳定性中的确切作用尚不清楚。我们主要关注Sir3和Sir4功能的两个方面：1。确定Sir3的扩散机制。尽管这三种SIR复合物蛋白的突变都能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Sir4结合Sir4的能力，并表明突变Sir3可以传播并形成沉默的染色质，具有接近野生型的效率。我们目前的模型提出Sir3的传播是由Sir3寡聚化驱动的，而不是组蛋白修饰的变化。我们已经确定了Sir3 n端可能介导Sir3寡聚化的小环，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. 确定细胞周期依赖性Sir3和Sir4磷酸化如何调节沉默染色质的稳定性。沉默的染色质在G1中比在有丝分裂中明显更稳定，但控制这种稳定性的机制尚不清楚。我们将研究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和生化分析来测试这些变化是否调节沉默染色质的稳定性。我们最初的重点将放在Sir3和Sir4的磷酸化上，Sir3和Sir4在细胞周期的不同阶段被磷酸化，而Asf2是沉默染色质的一个新的核心成分。的意义。对出芽酵母的研究开创了对异染色质的理解，这一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，但很少有人知道这些蛋白质是如何聚合的，如果它们作为聚合物绕过了特定组蛋白标记的需要，3)细胞在细胞周期中如何调节染色质结构？

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?