Assembly and function of heterochromatin

这项研究计划的目的是了解异染色质，一个专门的抑制性染色体结构域，如何从其组成蛋白组装，并确定这些区域的动态如何响应细胞周期位置和生理条件的变化而变化。异染色质在染色体结构和分离中起重要作用，并用于调节发育基因和途径的表达。 在芽殖酵母（Saccharomyces cerevisiae）中，异染色质含有核小体，核小体被三种蛋白质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。目前的异染色质组装模型提出，在招募到DNA元件后，Sir 2依赖性组蛋白脱乙酰化和SIR复合物招募的迭代轮次导致异染色质的扩散。我们已经确定了Asf 2，一个parabolic的Sir 4，也组装成核心异染色质结构域。Sir 3、Sir 4和Asf 2在异染色质扩散和稳定中的确切作用知之甚少。 这项赠款提出了以下短期目标： 1.确定Sir 4和Asf 2在异染色质动力学中的功能。我们已经表明，丰富的Sir 4调节异染色质从头组装，并在特定的细胞周期阶段Sir 4蛋白水平显着下降。我们将研究用于调节Sir 4丰度的细胞机制，以及干扰该途径是否会改变异染色质新结构域的建立。相比之下，Asf 2在转录基因沉默中起着次要的作用，我们将测试它是否调节DNA复制的晚期起点和异染色质基因座之间发生的专门重组事件。我们还将使用全基因组方法来探索Asf 2依赖性异染色质的其他新功能。 2.确定Sir 3扩散的机制。我们已经证明，Sir 3可以独立于Sir 4/Sir 2和特定的组蛋白修饰而沿着染色质扩散，这表明Sir 3寡聚化驱动异染色质扩散。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，我们将测试这个模型使用突变体在这个循环。 3.亚端粒异染色质产生表型多样性吗？亚端粒异染色质存在于所有的真核生物中，但其确切的功能知之甚少。亚端粒异染色质显示位置效应杂色，其中在群体内的给定细胞中只有一个端粒子集被转录抑制。我们将测试沉默亚端粒的不同组合的表达是细胞用于产生表型多样性的机制的模型。通过选择特定端粒的沉默，我们将测试是否存在亚端粒异染色质的共调节，以及特定端粒的沉默是否与特定表型相关。

The objective of this research program is to understand how heterochromatin, a specialized repressive chromosomal domain, assembles from its constituent proteins and to determine how the dynamics of these regions change in response to changing cell cycle position and physiological conditions. Heterochromatin plays important roles in chromosome structure and segregation and is used to regulate the expression of developmental genes and pathways. In the budding yeast, Saccharomyces cerevisiae, heterochromatin contains nucleosomes that are bound by a complex of three proteins, Sir2, Sir3 and Sir4 (Silent information regulator), named the SIR complex. Current models for heterochromatin assembly propose that after recruitment to a DNA element, iterative rounds of Sir2-dependent histone deacetylation and SIR complex recruitment lead to the spreading of heterochromatin. We have identified Asf2, a paralogue of Sir4, that also assembles into core heterochromatic domains. The precise roles of Sir3, Sir4 and Asf2 in the spreading and stability of heterochromatin are poorly understood. This grant proposes the following short-term objectives: 1. Determine the function of Sir4 and Asf2 in heterochromatin dynamics. We have shown the abundance of Sir4 regulates de novo assembly of heterochromatin and that during specific cell cycle stages Sir4 protein levels decline dramatically. We will investigate the mechanism cells used to regulate Sir4 abundance, and if perturbing this pathway alters the the establishment of new domains of heterochromatin. Asf2, in contrast, plays a minor function in transcriptional gene silencing and we will test if instead it regulates the firing of late origins of DNA replication and a specialized recombination event that occurs between heterochromatic loci. We will also use whole genome approaches to explore other novel functions of Asf2-dependent heterochromatin. 2. Determine the mechanism of Sir3 spreading. We have shown that Sir3 can spread along chromatin independently of Sir4/Sir2 and specific histone modifications, suggesting that Sir3 oligomerization drives heterochromatin spreading. We have identified a small loop in the Sir3 N-terminus that may mediate Sir3 oligomerization, and we will test this model using mutants in this loop. 3. Does sub-telomeric heterochromatin generate phenotypic diversity? Sub-telomeric heterochromatin is found in all eukaryotes, but its precise function is poorly understood. Sub-telomeric heterochromatin displays position effect variegation, in which only a subset of telomeres are transcriptionally repressed in a given cell within a population. We will test the model that silencing the expression of different combinations of sub-telomeres is a mechanism cells use to generate phenotypic diversity. By selecting for silencing of specific telomeres, we will test if there is co-regulation of sub-telomeric heterochromatin, and if silencing of specific telomeres is associated with specific phenotypes.