Circadian Clock Regulation in Skin

项目总结/摘要 使用遗传小鼠模型，我们发现角质形成细胞内的昼夜节律钟是维持细胞周期的时间所必需的。 毛囊间表皮细胞增殖的日依赖性变化，这似乎与 生物钟调节的新陈代谢。因此，我们发现，参与氧化的基因的表达， 磷酸化和活性氧（ROS）水平是反相的S期，昼夜节律 ROS水平的变化取决于核心时钟调节器BMAL 1。此外，我们发现小鼠比其他动物 敏感的UVB诱导的DNA表皮损伤在夜间（当最高数量的表皮 细胞通过S期）。基于这项工作，我们提出了这个中心假设： 生物钟协调新陈代谢和表皮细胞增殖的时间， 最大ROS产生时间和S期之间的分离。虽然这种时间调节 这种排列使内源性ROS的DNA突变最小化，它导致敏感性的昼夜变化， 紫外线介导的表皮DNA损伤这意味着白天活动的人类可能特别 对紫外线介导的DNA损伤敏感，在白天，最大紫外线暴露的时间。我们计划 在三个具体目标中追求这些想法：（1）了解生物钟之间的关系， 代谢，和细胞增殖在一个单一的细胞分辨率在滤泡间表皮。我们将绘制细胞 周期阶段由BMAL 1控制，并使用创新的成像方法来定义时钟之间的关系 输出、代谢和表皮基底细胞层内单细胞水平的细胞周期进程。 我们还将测试不同的喂养时间表可以改变代谢和细胞生长时间的假设。 表皮增生。(2)为了鉴定滤泡间质中的时间依赖性BMAL 1靶基因， 表皮我们将使用染色质免疫沉淀测序（ChIP-seq）和染色体构象 捕获（3C）试验，以检验BMAL 1直接和间接调节关键代谢的假设， 活表皮内的细胞周期基因。(3)为了确定是否需要核心生物钟基因 在紫外线诱导的皮肤癌发生中的时间依赖性差异。我们将描述的作用， 在人类角质形成细胞中UVB DNA损伤反应的中心时钟调节器，并利用 紫外线诱导的小鼠致癌模型，以检验皮肤中的时间依赖性变化 癌症的发生依赖于生物钟机制。这项工作意义重大，因为它揭示了 在快速增殖的上皮细胞中，生物钟、代谢、细胞增殖和癌症之间的关系， 因为它表明昼夜节律调节可能导致人类皮肤癌的高发病率， 因为它可能最终导致关于如何在增殖的上皮细胞中调节代谢的想法， 以减少致癌作用和组织老化。这项工作是创新的，因为它提出了一个新的作用， 昼夜节律钟在表皮祖细胞代谢和DNA合成的时间分离中的作用。

PROJECT SUMMARY/ABSTRACT Using genetic mouse models, we found that the circadian clock within keratinocytes is required for the time-of- day dependent variation in interfollicular epidermal cell proliferation, which seems to be coordinated with circadian clock-regulated metabolism. Thus, we find that the expression of genes involved in oxidative phosphorylation and reactive oxygen species (ROS) levels are antiphasic to S-phase, and that the circadian variation in ROS levels depends on core clock regulator BMAL1. Furthermore, we found that mice are more sensitive to UVB-induced DNA epidermal damage during the night (when the highest numbers of epidermal cells go through S-phase) than the day. Based on this work we propose this central hypothesis: The circadian clock coordinates the timing of metabolism and cell proliferation in the epidermis, imposing separation between the times of maximum ROS generation and S-phase. While this temporal regulatory arrangement minimizes DNA mutations from endogenous ROS, it causes circadian variation in the sensitivity to UVR-mediated DNA damage in the epidermis. The implication is that diurnal humans may be especially sensitive to UVR-mediated DNA damage during the day, the time of maximum UV exposure. We plan to pursue these ideas in three Specific Aims: (1) To understand the relationships between the circadian clock, metabolism, and cell proliferation at a single cell resolution in the interfollicular epidermis. We will map the cell cycle stage controlled by BMAL1 and use innovative imaging methods to define the relationship between clock output, metabolism, and cell cycle progression at a single cell level within the basal cell layer of the epidermis. We will also test the hypothesis that different feeding schedules can change timing of metabolism and cell proliferation in the epidermis. (2) To identify time-of-day dependent BMAL1 target genes in the interfollicular epidermis. We will use chromatin immunoprecipitation sequencing (ChIP-seq) and chromosome conformation capture (3C) assays to test the hypothesis that BMAL1 directly and indirectly regulates key metabolism and cell cycle genes within the living epidermis. (3) To determine whether core circadian clock genes are required for time-of-day dependent differences in UVR-induced skin carcinogenesis. We will characterize the role of the central clock regulators in the UVB DNA damage response in human keratinocytes and take advantage of a UV-induced mouse carcinogenesis model to test the hypothesis that time-of-day-dependent variation in skin carcinogenesis depends on circadian clock mechanisms. The work is significant because it reveals the relationship between the clock, metabolism, cell proliferation and cancer in a rapidly proliferating epithelium, because it suggests that circadian regulation may contribute to the high incidence of skin cancer in humans, and because it may ultimately lead to ideas about how metabolism can be modulated in proliferating epithelia to decrease carcinogenesis and tissue aging. The work is innovative because it suggests a new role for the circadian clock in imposing temporal separation of metabolism and DNA synthesis in epidermal progenitors.