Lifespan and healthspan regulation by hypoxic response transcription factor HIF-1

描述(申请人提供)：我们最近发现了缺氧反应转录因子HIF-1，它是秀丽隐杆线虫寿命和健康寿命的新修饰物。在发表在《科学》杂志上的一项研究中，我们表明，通过在常氧条件下抑制蛋白酶体的降解来稳定HIF-1，可以通过一种与饮食限制和胰岛素样信号减少不同的遗传机制来延长寿命，这一发现后来被两个小组独立复制。我们还表明，野生动物在低氧条件下的生长足以稳定和激活HIF-1，也可以延长寿命。在我们的研究发表后，Kapahi实验室报告说，在他们的实验条件下，HIF-1的缺失可以延长寿命。自那以后，我们已经确定HIF-1的稳定以温度无关的方式延长寿命，而HIF-1的丢失以温度依赖的方式延长寿命。与常氧下通过激活HIF-1来延长寿命不同，通过缺失HIF-1或缺氧来延长寿命需要FOXO家族转录因子DAF-16。在这里，我们建议进一步定义HIF-1调节线虫寿命和健康寿命的机制，作为一个促进和反对长寿的因素。在特定的目标1中，我们将(1)进一步定义缺失HIF-1可以延长寿命的实验条件，并确定在这些条件下是否也延长了健康寿命，(2)使用染色质免疫沉淀检验DAF-16由丢失HIF-1或缺氧激活DAF-16的假设，以确定DAF-16是否定位于已知或新的靶基因，以及(3)通过使用质谱仪定量DAF-16已知的和新的翻译后修饰来响应这些干预，确定DAF-16如何重新定位到细胞核以响应HIF-1的缺失或缺氧。在特定的目标2中，我们将(1)使用响应HIF-1稳定的差异表达的RNAi敲除mRNAs来确定当HIF-1在常氧条件下稳定时与寿命和健康延长相关的HIF-1下游靶点，以及(2)确定这些基因是否在调节其他已知的长寿途径中发挥作用。在具体目标3中，我们将通过产生表达(1)针对HIF-1降解的VHL-1 E3连接酶或(2)在已知细胞类型特异性启动子下的不可降解形式的HIF-1的转基因动物，并量化由此产生的对寿命和健康寿命的影响，来确定哪些细胞类型参与调节HIF-1在常氧条件下稳定的寿命和健康寿命的延长。HIF-1和缺氧反应从线虫到人类都高度保守，并与人类疾病有关，最明显的是各种癌症。因此，更好地理解HIF-1作为线虫寿命和健康期限修饰物的双重作用将导致新的假说，这些假说可以在哺乳动物身上直接测试，并且很可能与人类健康和年龄相关疾病直接相关。

DESCRIPTION (provided by applicant): We recently identified the hypoxic response transcription factor, HIF-1, as a novel modifier of longevity and healthspan in Caenorhabditis elegans. In a study published in the journal Science, we showed that stabilization of HIF-1 by inhibiting its proteasomal degradation under normoxic conditions increases life span through a mechanism that is genetically distinct from dietary restriction and reduced insulin-like signaling, a finding that has since been independently replicated by two groups. We also showed that growth of wild type animals under hypoxia, which is sufficient to stabilize and activate HIF-1, also increases life span. Following publication of our study, the Kapahi lab reported that loss of HIF-1 increases life span under their experimental conditions. We have since determined that stabilization of HIF-1 increases life span in a temperature- independent manner, while loss of HIF-1 increases life span in a temperature-dependent manner. Unlike life span extension from activation of HIF-1 in normoxia, life span extension from deletion of HIF-1 or hypoxia requires the FOXO-family transcription factor DAF-16. Here we propose to further define the mechanisms by which HIF-1 modulates longevity and healthspan in C. elegans, both as a pro- and anti-longevity factor. In Specific Aim 1, we will (1) further define the experimental conditions under which deletion of HIF-1 can increase life span and determine whether healthspan is also extended under these conditions, (2) test the hypothesis that DAF-16 is activated by loss of HIF-1 or by hypoxia using chromatin immunoprecipitation to determine whether DAF-16 is localized to known or novel target genes, and (3) determine how DAF-16 becomes relocalized to the nucleus in response to loss of HIF-1 or hypoxia by using mass spectrometry to quantify known and novel post-translational modifications of DAF-16 in response to these interventions. In specific Aim 2, we will (1) use RNAi knock-down mRNAs that are differentially expressed in response to stabilization of HIF-1 to identify the downstream targets of HIF-1 that are involved in life span and healthspan extension when HIF-1 is stabilized under normoxic conditions and (2) determine whether these genes play a role in modulating longevity in response to other known longevity pathways. In specific Aim 3, we will determine which cell types are involved in modulating life span and healthspan extension from stabilization of HIF-1 under normoxic conditions by generating transgenic animals expressing either (1) the VHL-1 E3 ligase which targets HIF-1 for degradation or (2) a non-degradable form of HIF-1 under promoters with known cell- type specificities and quantifying the resulting effects on longevity and measures of healthspan. HIF-1 and the hypoxic response are highly conserved from nematodes to humans and have been implicated in human diseases, most notably a variety of cancers. Thus, a better understanding of the dual role that HIF-1 can play as a modifier of life span and healthspan in nematodes will lead to new hypotheses that can be directly tested in mammals, and is likely to be directly relevant to human health and age-related disease.