Function of Mouse Heat Shock Factor 1 Alpha and Beta Isoforms

描述（由申请人提供）：热休克转录因子1 （HSF1）是热休克反应的主要调节因子，是一种高度保守的途径，可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是一个关键的长寿因子。本研究旨在阐明小鼠HSF1的同种异构体（指定为1和2）如何促进热休克反应。本提案将测试几个关键问题。这两种同工异构体的本构活性版本可以被构建吗？HSF11和2亚基之间是否形成功能性异源三聚体？HSF11或hsf2激活的基因有哪些？HSF1亚型主要是核蛋白吗？解决这些问题的具体目标是：目的1试图验证小鼠2异构体，像1异构体一样，是热休克蛋白（hsps）的激活剂的假设。目的2将验证HSF11和2亚型在NIH 3T3细胞中相互作用形成功能性异源三聚体的假设。最后的目标将检查HSF1亚型的亚细胞定位，以确定它们是否通常在细胞核中发现或在热休克后移动到那里。为了进行Aim 1，将在小鼠Hsf11和hsf2同工异构体基因中创建一系列位点定向突变，以创建不同的组成活性形式的蛋白质。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，使用定量实时RT-PCR分析。为了完成目的2，将使用转染的NIH 3T3细胞提取物的免疫沉淀和蛋白凝胶电泳来确定表位标记的Hsf11和2是否相互作用。不同的HSF1三聚体与几种热休克元件序列（HSEs）的结合将通过染色质免疫沉淀来评估。为了研究最终目的，在有或没有热休克的条件下，在瞬时转染的NIH 3T3细胞中，通过相对于核标记物的免疫荧光检测HSF1标记的组成型或天然同种异构体。这些研究将提供小鼠HSF1亚型的系统比较，检查它们在热休克蛋白基因转录激活中的各自作用，作为异质蛋白相互作用的潜力，以及亚细胞定位。对HSF1亚型功能的更完整表征的需求对于开发和测试针对HSF1的年龄相关疾病的新疗法至关重要。

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.