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: 2704567
• 负责人: BREHON C LAURENT
• 金额: $ 22.93万
• 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/2704567
• 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.

几种多蛋白复合物与ATP依赖的 核小体的转录重塑，包括 进化保守的S.酿酒酵母Snf/Swi复合物。其他基本 这些过程也需要染色质的时间重组， 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。 小说十六- 能够重塑染色质结构的蛋白质复合物（RSC） 是从酵母中提纯出来的 尽管有一个共同的ATP依赖的核小体 重构活动，RSC和Snf/Swi的功能是不同的：Rsc 多肽是必不可少的，两个RSC中的热不稳定突变 基因STH 1（SNF 2同源物）和SFH 1（SNF 5同源物）将细胞阻滞在 细胞周期的G2/M转换。 sth 1和sfh 1条件的研究 因此，突变体提供了一个很好的机会来研究 将染色质重塑与通过细胞凋亡的进展联系起来的机制。 细胞分裂周期 为了确定RSC功能的目标，筛选 sth 1-ts突变的基因外抑制因子，包括多拷贝 抑制基因和抑制基因突变，以及第二位点突变， 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sfh 1-ts等位基因，在遗传筛选中鉴定， 基于相应SNF 5-TS突变的靶向诱变， 将在功能上和生物化学上进行表征。 条件 等位基因可导致SFH 1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- 特异性磷酸化、转录激活和组装成 RSC。 前提是丰度，磷酸化状态，或 Sth 1 p或Sfh 1 p的活性在细胞周期中波动，将被检测 目标三。 将测定sth 1和sfh 1突变体的能力， 执行动态重组所涉及的具体职能 细胞周期中染色质的变化，包括细胞周期中 完成DNA复制或染色体浓缩， 隔离。 在第四个目标中， 将使用一种新的探针在体内研究核小体的重组， 微型染色体的染色质结构。 长期目标是 项目是定义RCS的生理功能，包括 将染色质重塑与细胞周期联系起来的转导途径 进展 阐明这种联系将直接影响我们的 了解控制分化和发育的机制 在多细胞生物中。