Function of Mouse Heat Shock Factor 1 Alpha and Beta Isoforms

描述(申请人提供)：热休克转录因子1(HSF1)是热休克反应的主要调节因子，热休克反应是一种高度保守的途径，保护细胞免受各种应激源的影响。HSF1是sirtuin 1的主要靶点，sirtuin 1是一种关键的长寿因素。这项提议试图阐明小鼠HSF1的亚型(命名为1和2)如何对热休克反应做出贡献。几个关键问题将在这项提案中得到检验。是否可以构建两种异构体的结构活性版本？HSF11和HSF2亚基之间是否形成了功能性杂三聚体？HSF11或HSF2激活的基因图谱是什么？HSF1亚型主要是核蛋白吗？解决这些问题的具体目标是：目标1试图测试这样的假设，即小鼠2的异构体和1的异构体一样，是热休克蛋白(HSPs)的激活剂。目的2验证HSF11和HSF2亚型在NIH3T3细胞中相互作用形成功能性异三聚体的假设。最终目的将研究HSF1亚型的亚细胞定位，以确定它们是通常存在于细胞核中，还是在热休克后移动到那里。为了实现目标1，将在小鼠Hsf11和2异构体基因中创建一系列定点突变，以创建不同的构成活性蛋白质形式。这些改变形式的小鼠HSF11和2将在瞬时转染的NIH3T3细胞中进行测试，以确定它们是否可以在没有热休克的情况下激活目标HSP基因，使用实时定量RT-PCR分析。为了实现目标2，将采用免疫沉淀和蛋白凝胶电泳法检测Hsf11和Hsf2的表位标记版本是否相互作用。不同的HSF1三聚体与几个HSP的热休克元件序列(HSE)的结合将通过染色质免疫沉淀进行评估。为了探索最终目标，在有或没有热休克的条件下，将通过相对于核标记的免疫荧光在瞬时转染的NIH3T3细胞中检测到标记的构成或天然的HSF1亚型。这些研究将对小鼠HSF1亚型进行系统的比较，考察它们各自在HSP基因转录激活中的作用，作为异构体蛋白相互作用的可能性，以及亚细胞定位。需要对HSF1亚型功能进行更完整的表征，这对于开发和测试针对HSF1的与年龄相关的疾病的新治疗方法至关重要。 与公共健康相关：热休克因子1是长寿因子sirtuin 1的主要靶点，它为身体细胞提供保护，使其免受高温和过氧化氢等应激因素的影响。这项提议将确定小鼠热休克因子1的异构体在激活应激反应基因中的作用。了解这些基本机制将为针对热休克途径的年龄相关疾病的新治疗提供关键框架。

DESCRIPTION (provided by applicant): Heat shock transcription factor 1 (HSF1) is the principal regulator of the heat shock response, a highly conserved pathway that protects cells from a variety of stressors. HSF1 is a major target of sirtuin 1, a critical longevity factor. This proposal seeks to clarify how isoforms of mouse HSF1, designated 1 and 2, contribute to the heat shock response. Several key questions will be tested in this proposal. Can constitutively active versions of both isoforms be constructed? Are functional heterotrimers between HSF11 and 2 subunits formed? What is the profile of genes activated by HSF11 or 2? Are HSF1 isoforms predominantly nuclear proteins? The specific aims that will address these issues are: Aim 1 seeks to test the hypothesis that the mouse 2 isoform, like the 1 isoform, is an activator of heat shock proteins (hsps). Aim 2 will test the hypothesis that HSF11 and 2 isoforms have the potential to interact with each other in NIH 3T3 cells to form functional heterotrimers. The final Aim will examine the subcellular localization of HSF1 isoforms, to determine whether they are usually found in the nucleus or move there after heat shock. To carry out Aim 1, a series of site-directed mutations will be created in the mouse Hsf11 and 2 isoform genes to create distinct constitutively active forms of the protein. These altered forms of mouse HSF11 and 2 will be tested in transiently transfected NIH3T3 cells to determine whether they can activate target hsp genes in the absence of heat shock, using quantitative real-time RT-PCR assays. To accomplish Aim 2, immunoprecipitation and protein gel electrophoresis of transfected NIH 3T3 cell extracts will be used to determine whether epitope tagged versions of Hsf11 and 2 interact. The binding of different HSF1 trimers to heat shock element sequences (HSEs) of several hsps will then be assessed by chromatin immunoprecipitation. To investigate the final Aim, tagged constitutive or native isoforms of HSF1 will be detected in transiently transfected NIH 3T3 cells by immunofluorescence relative to a nuclear marker, under conditions with or without heat shock. These studies will provide a systematic comparison of mouse HSF1 isoforms, examining their respective roles in transcriptional activation of hsp genes, potential for interacting as heteromeric proteins, and subcellular localization. The need for more complete characterization of HSF1 isoform function is essential to developing and testing new treatments for age-related diseases that target HSF1. PUBLIC HEALTH RELEVANCE: Heat shock factor 1, a major target of the longevity factor, sirtuin 1, provides body cells with protection from stressors such as heat and peroxide. This proposal will identify the contribution of isoforms of mouse heat shock factor 1, a regulatory protein, in activating stress response genes. Understanding these basic mechanisms will provide a critical framework for new treatments for age- associated diseases targeted to the heat shock pathway.