Control of mating-type switching by Sir2 and condensin

Sir2 和 condensin 控制交配型转换

• 批准号: 9894360
• 负责人: Jeffrey Scott Smith
• 金额: $ 8.5万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-13 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9894360
• 关键词: Architecture; Binding; Binding Sites; Biological Assay; Cell Cycle; Cell Cycle Progression; Cells; Chromatin; Chromatin Interaction Analysis by Paired-End Tag Sequencing; Chromosome Structures; Chromosomes; Cis-Acting Sequence; DNA; DNA Double Strand Break; Double Strand Break Repair; Enhancers; Eukaryotic Cell; Gene Silencing; Genes; Genetic Recombination; Genetic Transcription; Genome; Heterochromatin; Interphase Cell; Kinetics; Mass Spectrum Analysis; Mating Types; Measures; Mediating; Mitotic Chromosome; Molecular; Molecular Conformation; Mothers; Nuclear; Pattern; Process; Property; Proteins; RNA; Role; Saccharomycetales; Sir2-like Deacetylases; Site; Structure; System; Testing; Time; Transcriptional Regulation; Untranslated RNA; activating transcription factor; chromosome movement; condensin; crosslink; daughter cell; ds-DNA; endonuclease; homologous recombination; preference; promoter; rapid technique; recruit; repaired; transcription factor

Project Summary. Since the first descriptions of mating-type switching in budding yeast approximately 40 years ago, characterization of this process has led to numerous key advances in understanding the mechanisms of gene silencing (heterochromatin), cell-fate determination (mating-type), and homologous recombination. For example, the highly conserved NAD+-dependent protein deacetylase, Sir2, and other “silent information regulator (SIR) proteins, were initially identified due to their roles in silencing the heterochromatic HMLα and HMRa loci, which are maintained as silenced copies of the active MATα and MATa loci, respectively. Mating-type switching occurs when a programmed dsDNA break is generated at the MAT locus by an endonuclease called HO. The break is then repaired through homologous recombination using either HMLα or HMRa as a template, with a strong preference for switching to the opposite mating type. Such “donor preference” in switching is directed by a cis-acting sequence adjacent to HMLα called the recombination enhancer (RE). Numerous chromatin factors have been implicated in mating-type switching, but mechanisms involving chromosome structural dynamics have remained elusive. In this proposal we investigate a newly discovered process of coordinating the “sensing” of an HO-induced dsDNA break at the MAT locus with requisite chromosome III structural changes. Within the RE we identified a strong binding site for Sir2, condensin, and cohibin (Lrs4/Csm1) at the promoter of a long non-coding RNA (lncRNA) gene called RDT1. This site maintains chromosome III in a switching- competent structural conformation. Sir2 normally represses RDT1 transcription in non-switching cells, but is redistributed to the HO-induced break site at MAT, which activates RDT1 transcription and triggers mating-type switching. We propose 3 specific aims that will determine not only how RDT1 lncRNA induces switching, but also investigate the roles of condensin and cohibin in the programmed structural reorganization of chromosome III. This provides a unique and well-defined system for elucidating condensin function outside of mitotic chromosome compaction, something currently missing in the field.

项目摘要。 自从大约40年前首次描述萌芽酵母的交配类型转换以来， 对这一过程的表征导致了许多关键的进展，了解了 基因沉默(异染色质)、细胞命运决定(交配型)和同源重组。 例如，高度保守的依赖于NAD+的蛋白去乙酰基酶Sir2等 信息调节蛋白(SIR)最初被发现是因为它们在沉默中的作用 异染色质HMLα和HmrA基因座，它们作为活性MATα和HmrA的沉默拷贝保存 MATA基因座。交配型转换发生时，程序化dsDNA断裂在 MAT基因座由一种名为HO的内切酶决定。然后通过同源基因修复断裂 使用hmlα或hmrA作为模板进行重组，并强烈倾向于切换到 相反的交配型。这种转换中的“供体偏好”是由相邻的顺式作用序列引导的 将HMLα称为重组增强子(RE)。许多染色质因子被认为与 交配型转换，但涉及染色体结构动力学的机制仍然存在 难以捉摸。在这项建议中，我们研究了一个新发现的过程，即协调一种 HO诱导的dsDNA在MAT基因座断裂，并伴随着必要的第三染色体结构变化。在 我们在A基因启动子上发现了Sir2、凝聚素和Cohibin(Lrs4/Csm1)的一个强结合位点 长的非编码RNA(LncRNA)基因称为RDT1。这个位置保持着第三号染色体的开关状态- 称职的结构构造。Sir2通常抑制非转换细胞中的RDT1转录，但 被重新分配到HO诱导的MAT断裂位点，激活RDT1转录并触发 交配型开关。我们提出了三个具体目标，这三个目标不仅将决定RDT1 lncRNA如何 诱导转换，但也研究凝聚素和胶凝素在程序化结构中的作用 第三号染色体的重组。这提供了一个独特和明确的系统来阐明 凝聚素在有丝分裂染色体紧凑之外发挥作用，这是目前该领域所缺少的。