CELL CYCLE PROGRESSION AND CHROMATIN REMODELING IN YEAST

酵母细胞周期进程和染色质重塑

• 批准号: 6386783
• 负责人: BREHON C LAURENT
• 金额: $ 25.03万
• 依托单位: SUNY DOWNSTATE MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386783
• 关键词: adenosine triphosphate; affinity chromatography; alleles; cell cycle; cell proliferation; chromatin; chromosome movement; fluorescence microscopy; gene expression; gene interaction; genetic transcription; high performance liquid chromatography; immunoprecipitation; ion exchange chromatography; molecular cloning; northern blottings; nucleic acid structure; nucleosomes; phosphorylation; protein purification; protein structure function; temperature sensitive mutant; transcription factor; western blottings; yeast two hybrid system; yeasts

Several multiprotein complexes are implicated in the ATP-dependent remodeling of nucleosomes for transcription, including the evolutionarily conserved S. cerevisiae Snf/Swi complex. Other essential processes also require the temporal restructuring of chromatin as cells progress through the cell division cycle or undergo differentiation, any of which might require Snf/Swi or related factors. A novel sixteen- protein complex capable of remodeling the structure of chromatin (RSC) has been purified from yeast. Despite a shared ATP-dependent nucleosome remodeling activity, the functions of RSC and Snf/Swi are distinct: Rsc polypeptides are indispensable, and thermolabile mutations in two RSC genes, STH1 (SNF2 homolog) and SFH1 (SNF5 homolog), arrest cells at the G2/M transition of the cell cycle. A study of sth1 and sfh1 conditional mutants therefore presents an excellent opportunity to investigate the mechanisms that link chromatin remodeling to progression through the cell division cycle. To identify targets of RSC function, screens for extragenic suppressors of an sth1-ts mutation, including multicopy suppressors and suppressor mutations, and for second-site mutations that are lethal in combination with this ts allele will be completed. In the second aim, new sfh1-ts alleles, identified in a genetic screen and from a targeted mutagenesis based on the corresponding snf5-ts mutations, will be characterized functionally and biochemically. Conditional alleles can lead to partial or complete loss of SFH1 function by impairing distinct activities, including cell cycle progression, G1- specific phosphorylation, transcriptional activation, and assembly into RSC. The premise that the abundance, phosphorylation state, or activities of Sth1p or Sfh1p fluctuate in the cell cycle, will be tested in aim three. sth1 and sfh1 mutants will be assayed for the ability to carry out specific functions implicated in the dynamic reorganization of chromatin during the cell cycle, including those required for the completion of DNA replication or chromosome condensation and segregation. In the fourth objective, mechanism(s) by which RSC restructures nucleosomes will be studied in vivo using a novel probe of the chromatin structure of mini-chromosomes. The long-term goal of this project is to define the physiological function of RCS, including the transduction pathway(s) that links chromatin remodeling to cell cycle progression. Elucidation of this connection will impact directly on our understanding of mechanisms that control differentiation and development in multicellular organisms.

几种多蛋白复合体与ATP依赖有关 核小体重塑以进行转录，包括 进化上保守的酿酒酵母SnF/Swi复合体。其他基本要素 这一过程还需要染色质作为细胞的暂时重组。 通过细胞分裂周期的进展或经历分化，任何 其中可能需要SNF/SWI或相关因素。一部十六岁的小说-- 能够重塑染色质结构的蛋白质复合体 是从酵母中提纯出来的。尽管有一个共同的依赖于ATP的核小体 重塑活动，RSC和SNF/Swi的功能截然不同：RSC 多肽是不可缺少的，两个RSC中存在不耐热突变 STH1(SNF2同源基因)和SFH1(SNF5同源基因)使细胞停滞在 细胞周期的G2/M期转变。关于sth1和sfh1条件句的研究 因此，突变体提供了一个极好的机会来研究 将染色质重塑与进展联系起来的机制 细胞分裂周期。为了识别RSC功能的目标，筛选 Sth1-ts突变的基因外抑制物，包括多拷贝 抑制子和抑制子突变，以及第二位点突变 都是致命的，与这个ts等位基因结合将完成。在 第二个目标，新的sfh1-ts等位基因，通过遗传筛查和来自 基于相应的SNF5-TS突变的靶向突变， 将在功能和生化方面进行表征。有条件的 等位基因可通过以下方式导致SFH1功能的部分或完全丧失 损害不同的活动，包括细胞周期进程，G1- 特异的磷酸化、转录激活和组装成 RSC.前提是丰度、磷酸化状态或 Sth1p或Sfh1p的活性在细胞周期中波动，将被测试 在三号目标。将检测sth1和sfh1突变体的能力 执行动态重组中涉及的特定职能 细胞周期中染色质的含量，包括 DNA复制或染色体凝聚的完成和 种族隔离。在第四个目标中，机制(S)通过RSC 重组核小体将使用一种新的探针在体内进行研究 小染色体的染色质结构。这样做的长期目标是 项目是定义RCS的生理功能，包括 染色质重塑与细胞周期相关的信号转导途径(S) 进步。对这一联系的澄清将直接影响我们的 对控制分化和发展的机制的理解 在多细胞生物体中。