HEAT SHOCK FACTOR--STRESS AND THE CELL CYCLE

Thiele实验室之前的研究已经确定了羧基- 酵母热休克因子(HSF)的末端反式激活(CTA)区 作为转录激活所需的关键区域 酵母金属硫蛋白基因CUP1对高温和氧化的响应 压力。这个结构域对于热休克激活是必不可少的 HSP70基因家族。最近，我确定了这个域名的损失 也会导致细胞周期进程受阻，并停滞在G2期 一个接一个地转变到37摄氏度这是一个领域的第一次演示- HSF在细胞周期进程中的特殊作用，与 HSP70热休克蛋白必需家族的基础调控。 基于它作为一种强有力的基因调节因子的已知作用，我预测HSF 控制细胞周期进程所需基因的表达，并 在热应力条件下通过CTA进行分割。 进一步探讨HSF和潜在靶基因在细胞中的作用 分裂在压力下，我采取了遗传方法，并将 温度敏感性的多拷贝和外源抑制因子 缺乏HSF CTA的菌株表现出生长表型(HSF(1-583))。 我已经鉴定出到目前为止没有特征的基因YCR030C是一种 HSF(1-583)温度致死性的多拷贝抑制基因。我提议 鉴定该基因并确定其缓解G2的机制 在此背景下进行逮捕，最终目标是确定其 与HSF和细胞周期调控的关系。的表型效应 YCR030C的失活将通过基因破坏进行研究。 将进行YCR030C的基因表达和定位研究 确定基因产物的时间和空间协调性 与细胞周期进程有关。YCR030C在转录中的作用 将研究HSF的激活，以及这两种蛋白质的能力 物理上的相互作用将通过体外生化进行检测 实验。此外，我将进行基因筛查 HSF(1-583)TS表型的基因外抑制物，目的是 分离HSF可能与细胞周期进程相关的其他基因 在压力下。本提案中描述的工作将一个关键的 应激反应转录因子在细胞周期中的作用 进步。

Previous studies in the Thiele laboratory have identified the carboxyl- terminal transactivation (CTA) domain of yeast heat shock factor (HSF) as a critical region required for transcriptional activation of the yeast metallothionein gene CUP1 in response to heat and oxidative stress. This domain is dispensible for heat shock activation of the hsp70 gene family. Recently, I have determined that loss of this domain also results in a block in cell cycle progression, with arrest at G2 phase upon shift to 37 C. This is the first demonstration of a domain- specific role for HSF in cell cycle progression and is distinct from basal control of the essential family of hsp70 heat shock proteins. Based on its known role as a potent gene regulator, I predict that HSF controls the expression of genes required for cell cycle progression and division under conditions of thermal stress through the CTA. To further explore the roles HSF and potential target genes play in cell division during stress, I have taken a genetic approach and isolated both multicopy and extragenic suppressors of the temperature sensitive growth phenotype exhibited by strains lacking the HSF CTA (HSF(1-583)). I have identified the heretofore uncharacterized gene YCR030C as a multicopy suppressor of HSF(1-583) temperature lethality. I propose to characterize this gene and determine its mechanism for alleviating G2 arrest in this background with the ultimate goal of determining its relationship to HSF and cell cycle control. The phenotypic effects of inactivation of YCR030C will be investigated through gene disruption. Gene expression and localization studies of YCR030C will be conducted to determine the temporal and spatial coordination of the gene product with cell cycle progression. The role of YCR030C in transcriptional activation by HSF will be studied, and the ability of these two proteins to physically interact will be assayed by in vitro biochemical experiments. Furthermore, I will carry out a genetic screen for extragenic suppressors of the HSF(1-583) ts phenotype with the goal of isolating other genes which may link HSF with cell cycle progression during stress. The work described in this proposal links a critical stress-responsive transcription factor to a role in cell cycle progression.