Assembly and function of heterochromatin

异染色质的组装和功能

• 批准号: RGPIN-2019-07287
• 负责人: Rudner, Adam
• 金额: $ 3.06万
• 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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744329
• 关键词: Assembly; function; heterochromatin

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.

该研究计划的目的是了解异染色质（一种特殊的抑制性染色体结构域）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发挥重要作用，并用于调节发育基因和途径的表达。在芽殖酵母（酿酒酵母）中，异染色质含有核小体，这些核小体与三种蛋白质（Sir2、Sir3 和 Sir4（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表明，在招募到 DNA 元件后，Sir2 依赖性组蛋白脱乙酰化和 SIR 复合物招募的迭代循环会导致异染色质的扩散。我们已经鉴定出 Asf2，它是 Sir4 的旁系同源物，也组装成核心异染色质结构域。 Sir3、Sir4 和 Asf2 在异染色质的扩散和稳定性中的确切作用尚不清楚。 该资助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功能。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，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，Asf2 在转录基因沉默中发挥次要功能，我们将测试它是否调节 DNA 复制晚期起点的激发以及异染色质基因座之间发生的特殊重组事件。我们还将使用全基因组方法来探索 Asf2 依赖性异染色质的其他新功能。 2.确定Sir3的传播机制。我们已经证明，Sir3 可以独立于 Sir4/Sir2 和特定组蛋白修饰而沿着染色质扩散，这表明 Sir3 寡聚化驱动异染色质扩散。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，我们将使用该环中的突变体来测试该模型。 3. 亚端粒异染色质是否会产生表型多样性？亚端粒异染色质存在于所有真核生物中，但其确切功能尚不清楚。亚端粒异染色质表现出位置效应杂色，其中在群体内的给定细胞中只有端粒的子集受到转录抑制。我们将测试以下模型：沉默亚端粒不同组合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，我们将测试亚端粒异染色质是否存在共同调节，以及特定端粒的沉默是否与特定表型相关。