Regulatory Role of Transcription Factor Slug in Aging

描述（由申请人提供）：最近的证据表明，细胞衰老机制和某些自我更新干细胞区室再生能力的下降可能对哺乳动物衰老起重要作用。尽管INK4A/ ARF基因座的下调被认为是一个内在关联的事件，但对p16INK4和p19ARF表达调节的具体机制知之甚少。PI实验室的初步工作表明，Slug是一种锌指转录因子，已知会影响多种组织中的细胞存活和上皮-间质转化，直接抑制p16INK4的转录，以至于在体外和动物模型中，Slug缺乏会导致细胞衰老和过早衰老。因此，我们假设Slug通过抑制细胞衰老和维持成体干细胞和祖细胞的再生潜力来影响衰老，主要是通过直接负调控p16INK4基因。这些进展促进了我们的长期目标——发现分化细胞、干细胞和祖细胞衰老背后的新分子途径——并导致了三个直接的研究目标，以确定鼻涕虫对机体衰老的精确贡献。Aim 1将通过敲低和敲除技术确定p16INK4a在鼻涕虫缺陷mef中加速衰老的程度。目的2将试图阐明Slug负调控p16INK4a表达的分子机制，通过依赖染色质免疫沉淀和电泳迁移转移试验。目的3将定义鼻涕虫在组织衰老中的功能作用以及成体干细胞和祖细胞的再生潜力。这些实验将使用一系列转基因动物模型以及来自Slug和p16INK4a缺陷小鼠的造血和骨骼肌干细胞。这一建议是创新的，因为它利用了一种转录因子，Slug，在INK4a/ ARF位点具有独特的调控活性。从我们的项目中，我们很有可能更好地了解这个基因座的体内调节，从而了解哺乳动物的衰老。

DESCRIPTION (provided by applicant): Recent evidence suggests that the cellular senescence machinery and the decline of the regenerative capacity of certain self-renewing stem cell compartments may contribute importantly to mammalian aging. Although derepression of INK4A/ ARF locus is thought to be an intrinsically linked event, very little is known about the specific mechanisms responsible for modulation of p16INK4 and p19ARF expression. Preliminary work from the PI's laboratory suggests that Slug, a zinc-finger transcription factor known to affect cell survival and the epithelial-mesenchymal transition in a variety of tissues, directly inhibits p16INK4 transcription, to the extent that Slug deficiency leads to cellular senescence and premature aging in vitro and in animal models. We therefore hypothesize that Slug influences aging by restraining cellular senescence and sustaining the regenerative potential of adult stem and progenitor cells, primarily through direct negative regulation of the p16INK4 gene. These developments promote our long-term objective -- to discover novel molecular pathways underlying the senescence of both differentiated and stem and progenitor cells -- and have led to three immediate research aims to determine the precise contribution of Slug to organismal aging. Aim 1 will establish the extent to which p16INK4a contributes to accelerated senescence in Slug-deficient MEFs using both knockdown and knockout technologies. Aim 2 will attempt to elucidate the molecular mechanisms by which Slug negatively regulates expression of p16INK4a, by relying on chromatin immunoprecipitation and electrophoretic mobility shift assays. Aim 3 will define the functional role of Slug in tissue senescence and the regenerative potential of adult stem and progenitor cells. These experiments will use a series of genetically modified animal models as well as hematopoietic and skeletal muscle stem cells from Slug and p16INK4a- deficient mice. This proposal is innovative because it exploits a transcription factor, Slug, with unique regulatory activity at the INK4a/ ARF locus. The likelihood that a better understanding of the in vivo regulation of this locus, and hence of mammalian aging, will emerge from our project appears high.