The Role of Drosophila SIR2 in gene silencing, DNA meta*

果蝇 SIR2 在基因沉默、DNA 元*中的作用

• 批准号: 6401254
• 负责人: Peter J. Harte
• 金额: $ 30.1万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-15 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6401254
• 关键词: Drosophilidae; alleles; amidohydrolases; arthropod genetics; chromatin; chromatography; enzyme activity; gene dosage; gene expression; gene induction /repression; gene interaction; gene mutation; histones; homeobox genes; immunoprecipitation; longevity; nicotinamide adenine dinucleotide; nucleic acid metabolism; transcription factor

The long term goal of the proposed research is to understand the role of the Drosophila SIR2 protein in gene silencing and investigate its possible role in lifespan-extension by caloric restriction (CR) in higher eukaryotes. In the budding yeast Saccharomyces cerevisiae, SIR2 has been demonstrated to be a determinant of lifespan, a single extra copy of the Sir2 gene extending lifespan by up to 50 percent. CR promotes a similar extension of yeast lifespan that is dependent on SIR2. It is of great interest to discover whether the SIR2 orthologs in higher eukaryotes are also involved in determining lifespan and lifespan extension by CR. We have identified the Drosophila SIR2 ortholog (dSIR2) by database searches and have identified mutations affecting it. We plan to use these mutations, as well as alterations in SIR2 gene dosage and directed overexpression of dSIR2 to investigate its role in gene silencing and longevity. We will characterize the biochemical activities of dSIR2, in particular determining whether it has an NAD+-dependent histone deacetylase activity that is conserved in yeast and mouse SIR2 proteins. We also plan to carry out biochemical purification of complexes containing dSIR2 by conventional chromatography and identify their polypeptide components by peptide sequencing. In preliminary studies we have made intriguing connections between dSIR2 and Polycomb Group proteins, which are involved in stable long-term silencing of the homeotic genes in Drosophila and are highly conserved in higher eukaryotes. We plan to explore this connection further through genetic and biochemical studies to determine its functional significance. We expect that these studies will provide new insights into the role of dSIR2 in gene silencing and reveal whether and how it plays a role in determining lifespan and lifespan extension by CR in higher eukaryotes.

本研究的长期目标是了解果蝇 SIR2 蛋白在基因沉默中的作用，并研究其在高等真核生物中通过热量限制 (CR) 延长寿命的可能作用。 在芽殖酵母酿酒酵母中，SIR2 已被证明是寿命的决定因素，Sir2 基因的一个额外副本可将寿命延长高达 50%。 CR 也能类似地延长依赖于 SIR2 的酵母寿命。 发现高等真核生物中的 SIR2 直系同源物是否也参与决定寿命和通过 CR 延长寿命是非常有趣的。 我们通过数据库搜索鉴定了果蝇 SIR2 直系同源物 (dSIR2)，并确定了影响它的突变。 我们计划利用这些突变以及 SIR2 基因剂量的改变和 dSIR2 的定向过度表达来研究其在基因沉默和长寿中的作用。我们将表征 dSIR2 的生化活性，特别是确定它是否具有在酵母和小鼠 SIR2 蛋白中保守的 NAD+ 依赖性组蛋白脱乙酰酶活性。 我们还计划通过常规色谱法对含有dSIR2的复合物进行生化纯化，并通过肽测序鉴定其多肽成分。 在初步研究中，我们在 dSIR2 和 Polycomb Group 蛋白之间建立了有趣的联系，这些蛋白参与果蝇中同源基因的稳定长期沉默，并且在高等真核生物中高度保守。 我们计划通过遗传和生化研究进一步探索这种联系，以确定其功能意义。 我们期望这些研究将为 dSIR2 在基因沉默中的作用提供新的见解，并揭示它在高等真核生物中是否以及如何在决定寿命和通过 CR 延长寿命方面发挥作用。