Regulatory Role of Transcription Factor Slug in Aging

描述(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隔间的再生能力下降可能是哺乳动物衰老的重要原因。虽然Ink4a/ARF基因座的表达下调被认为是一种内在联系的事件，但对调控p16INK4和p19ARF表达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，已知在各种组织中影响细胞存活和上皮-间充质转化，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，我们假设鼻涕虫通过抑制细胞衰老和维持成体干细胞和祖细胞的再生能力来影响衰老，主要是通过对p16INK4基因的直接负调控。这些进展促进了我们的长期目标--发现分化的干细胞和祖细胞衰老的新的分子途径--并导致了三个近期的研究目标，即确定鼻涕虫对生物衰老的确切贡献。目标1将利用基因敲除和基因敲除技术，确定p16INK4a在多大程度上有助于鼻涕虫缺乏的MEF加速衰老。目的2通过染色质免疫沉淀和凝胶迁移率改变分析，试图阐明SLUG负性调控p16INK4a表达的分子机制。目的3将确定鼻涕蛋白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，以及来自鼻涕虫和p16INK4a缺陷小鼠的造血细胞和骨骼肌干细胞。这一建议是创新的，因为它利用了一种转录因子Slug，它在INK4a/ARF基因座具有独特的调节活性。通过我们的项目，更好地理解该基因的体内调节，从而了解哺乳动物衰老的可能性似乎很高。

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.