Chromatin condensation as a DNA protective response to UV radiation in human melanocytes and aging C. elegans

紫外线诱导的DNA损伤是与年龄相关的皮肤病和光老化的主要原因。虽然身体 可以随着时间的推移形成对紫外线应激的防御能力(即黑色素浓度和地层增厚 角质层)，在理解细胞是否能立即有效地 紫外线的保护性反应。我们最近发现了一种即时而强大的紫外线诱导的DNA保护 一种机制，涉及钙内流引发的全球染色质压缩。染色质 在人HeLa细胞中表现出紧致和DNA保护反应，紧致是 在NIH2/4小鼠胚胎成纤维细胞和线虫中也有表达。我们的长期合作 目的是了解这种机制的分子基础，并研究是否可以操纵它来 增强我们对紫外线伤害的天然保护。我们将探讨这种机制是否会随着年龄的增长而衰退 就像其他抗压机制一样。该应用程序的目标是开发线虫作为一种 探索这种应激抵抗机制的年龄依赖性的模型系统并利用人的表皮 黑素细胞与线虫结合，首次窥探这种紫外线的分子途径-- 诱导DNA保护性染色质致密。中心假设是染色质压缩是 由光感受器通过GαQ/11偶联作用的保守分子机制触发 激活钙内流的光传导途径。我们假设这是一个进化上保守的 在衰老的生物体中效率较低的反应。选择线虫的理由是它是一口井- 用精湛的遗传和发育工具建立了研究衰老的模型体系。人类原始性 选择表皮黑素细胞是因为它们是人类皮肤细胞，在这种细胞中，一条特定的途径涉及 光感受器和G-αQ/11被证明控制紫外线诱导的钙内流。这方面的具体目标 研究内容包括：1)测试紫外线诱导的染色质压缩是否能保护DNA免受进一步损伤 表皮黑素细胞(HEM)和幼年和老年线虫。我们会用紫外线照射大麻和线虫。 并通过西南印迹法检测光产物的去除率(对于哼哼和线虫)和 活体(对于线虫)。2)确定紫外线诱导的染色质压缩是否涉及GαQ/11偶联 人表皮黑素细胞和线虫的光信号转导途径和钙离子内流。这一目标将 这是通过线虫和人类原代表皮黑素细胞中的RNAi/siRNA敲除实现的。这个 拟议的研究具有创新性，因为它探索了一种以前未描述的DNA防御机制 它的目标是建立线虫作为将这一现象与 衰老。增加我们的创新能力的是线虫实验室和 哺乳动物染色质实验室。结果将是重要的，因为控制DNA的新的潜在目标- 保护途径将被发现，这可能导致更好的药物策略，以防止紫外线。

UV-induced DNA damage is a major cause of age-related skin diseases and photoaging. Although the body can develop defenses over time to UV stress (i.e., melanin concentration and thickening of the stratum corneum), there is a fundamental gap in understanding whether cells can mount an effective immediate protective response to UV. We recently discovered an immediate and robust UV-induced DNA protection mechanism that involves a global chromatin compaction triggered by calcium influx. The chromatin compaction and DNA protection responses were demonstrated in human HeLa cells, and the compaction was also demonstrated in NIH2/4 mouse embryonic fibroblasts and in the roundworm C. elegans. Our long-term goal is to understand the molecular basis of this mechanism, and to investigate if it can be manipulated to increase our natural protection from UV damage. We will explore whether this mechanism declines with age like other stress resistance mechanisms. The objectives of this application are to develop the C. elegans as a model system to probe the age-dependence of this stress resistance mechanism and to use human epidermal melanocytes in combination with C. elegans to gain the first glimpse into the molecular pathway of this UV- induced DNA protective chromatin compaction. The central hypothesis is that chromatin compaction is triggered by a conserved molecular machinery of a photoreceptor acting through the Gαq/11-coupled phototransduction pathway to activate a calcium influx. We hypothesize that this is an evolutionarily conserved response that is less efficient in aged organisms. The rationale for choosing C. elegans is that it is a well- established model system for studying aging with superb genetic and developmental tools. Human primary epidermal melanocytes were chosen because they are human skin cells in which a specific pathway involving photoreceptors and Gαq/11 was shown to control a UV-induced calcium influx. The specific aims for this research are: 1) Test whether UV-induced chromatin compaction protects DNA from further damage in human epidermal melanocytes (HEMs) and in young and old C. elegans. We will UV irradiate HEMs and C. elegans and detect the rate of removal of photoproducts by Southwestern blotting (for HEMs and C. elegans) and in vivo (for C. elegans). 2) Determine whether UVR-induced chromatin compaction involves the Gαq/11-coupled phototransduction pathway and calcium influx in human epidermal melanocytes and C. elegans. This aim will be achieved by RNAi/siRNA knock downs in C. elegans and human primary epidermal melanocytes. The proposed research is innovative because it explores a previously undescribed DNA defense mechanism from UV radiation and it sets as a goal establishing C. elegans as a model system for relating this phenomenon to aging. Adding to our innovative capacity is the ongoing cross-pollination between a C. elegans lab and a mammalian chromatin lab. The results will be significant because new potential targets that control the DNA- protective pathway will be discovered which can lead to better pharmaceutical strategies to protect from UV.