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

描述（由申请人提供）：我们最近发现了低氧反应转录因子HIF-1，作为秀丽隐杆线虫寿命和健康寿命的一种新的修饰因子。发表在《科学》杂志上的一项研究表明，在常压条件下，通过抑制HIF-1的蛋白酶体降解来稳定HIF-1，可以通过一种与饮食限制和减少胰岛素样信号传导不同的遗传机制延长寿命，这一发现已被两组独立重复。我们还发现，野生型动物在缺氧条件下生长，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，失去HIF-1可以延长寿命。我们已经确定HIF-1的稳定以温度无关的方式增加寿命，而HIF-1的损失以温度依赖的方式增加寿命。与正常缺氧条件下激活HIF-1延长寿命不同，HIF-1缺失或缺氧条件下延长寿命需要foxo家族转录因子DAF-16。在这里，我们提出进一步定义HIF-1调节秀丽隐杆线虫的寿命和健康期的机制，无论是作为一个促长寿因子还是抗长寿因子。在Specific Aim 1中，我们将(1)进一步确定HIF-1缺失可以延长寿命的实验条件，并确定在这些条件下健康寿命是否也会延长；(2)使用染色质免疫沉淀来验证DAF-16是由HIF-1缺失或缺氧激活的假设，以确定DAF-16是否定位于已知或新的靶基因。(3)确定DAF-16如何在HIF-1缺失或缺氧的情况下重新定位到细胞核，方法是使用质谱法量化DAF-16在这些干预措施下已知的和新的翻译后修饰。在特定的Aim 2中，我们将(1)使用RNAi敲除响应HIF-1稳定而差异表达的mrna，以确定当HIF-1在常氧条件下稳定时参与寿命和健康寿命延长的HIF-1下游靶标；(2)确定这些基因是否在响应其他已知的长寿途径中调节寿命。在特定的Aim 3中，我们将通过产生转基因动物来表达(1)靶向HIF-1降解的VHL-1 E3连接酶或(2)在具有已知细胞类型特异性的启动子下不可降解的HIF-1形式，并量化由此产生的对寿命和健康寿命的影响，从而确定哪些细胞类型参与调节HIF-1在常压条件下稳定的寿命和健康寿命延长。HIF-1和低氧反应从线虫到人类是高度保守的，并且与人类疾病有关，最明显的是各种癌症。因此，更好地了解HIF-1作为线虫寿命和健康寿命调节剂的双重作用，将导致可以在哺乳动物中直接测试的新假设，并且可能与人类健康和与年龄相关的疾病直接相关。

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.