Assembly and function of heterochromatin

这项研究的目的是了解异染色质是如何从其组成蛋白中组装出来的，并确定这些区域的动态如何随着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，并被用来调节发育基因和途径的表达。 在萌芽酵母中，异染色质含有核小体，由三种蛋白质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。目前的异染色质组装模型认为，在募集到一个DNA元件后，依赖于Sir2的组蛋白去乙酰化和SIR复合体募集的迭代循环导致了异染色质的扩散。我们已经鉴定出ASF2，Sir4的一个平行序列，也组装成核心异色结构域。Sir3、Sir4和AsF2在异染色质扩散和稳定性中的确切作用还知之甚少。 这笔赠款提出了以下短期目标： 1.确定Sir4和ASF2在异染色质动力学中的作用。我们已经证明，Sir4的丰度调节异染色质的从头组装，并且在特定的细胞周期阶段，Sir4蛋白水平急剧下降。我们将研究细胞用于调节Sir4丰度的机制，如果干扰这一途径改变了异染色质新结构域的建立。相反，ASF2在转录基因沉默中起着次要的作用，我们将测试它是否相反地调节DNA复制的晚期起始点的激发和发生在异染色质基因座之间的特殊重组事件。我们还将使用全基因组方法来探索ASF2依赖的异染色质的其他新功能。 2.确定了Sir3扩散的机制。我们已经证明，Sir3可以独立于Sir4/Sir2和特定的组蛋白修饰而沿着染色质扩散，这表明Sir3寡聚推动了异染色质的扩散。我们已经在Sir3N-末端发现了一个可能介导Sir3齐聚的小环，我们将使用这个环中的突变体来测试这一模型。 3.亚端粒异染色质是否产生表型多样性？亚端粒异染色质存在于所有真核生物中，但对其确切功能知之甚少。亚端粒异染色质表现出位置效应的多样性，其中只有一部分端粒在群体中的给定细胞中转录抑制。我们将测试这个模型，即沉默不同组合的亚端粒的表达是细胞用来产生表型多样性的机制。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同调节，以及特定端粒的沉默是否与特定的表型相关。

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.