Circadian Clock Regulation in Skin

项目摘要/摘要 利用遗传小鼠模型，我们发现角质形成细胞内的昼夜节律时钟是 毛囊间表皮细胞增殖的日变化，这似乎与 昼夜节律的时钟调节新陈代谢。因此，我们发现参与氧化的基因的表达 磷酸化和活性氧(ROS)水平与S时相反，昼夜节律 ROS水平的变化取决于核心时钟调节器BMAL1。此外，我们发现老鼠的数量更多 夜间对UVB诱导的DNA表皮损伤敏感(当表皮数最高时 细胞经过S时相)比一天。基于这项工作，我们提出了这样一个中心假说： 生物钟协调表皮新陈代谢和细胞增殖的时间， ROS最大生成时间与S期的分离。虽然这一临时监管 排列将内源性ROS的DNA突变降至最低，它导致昼夜节律变化的敏感度 紫外线辐射导致的表皮DNA损伤。这意味着白天活动的人类可能特别 对UVR介导的DNA损伤敏感的白天，紫外线暴露的最大时间。我们计划 追求这些想法的三个具体目标：(1)理解生物钟与生物钟之间的关系， 毛囊间表皮中单个细胞分辨率的新陈代谢和细胞增殖。我们将绘制细胞地图 由BMAL1控制的周期阶段，并使用创新的成像方法定义时钟之间的关系 在表皮的基础细胞层中的单个细胞水平上的产出、新陈代谢和细胞周期进程。 我们还将检验这样一种假设，即不同的喂养计划可以改变新陈代谢和细胞的时间 表皮中的增殖。(2)鉴定卵泡间时间依赖性BMAL1靶基因 表皮。我们将使用染色质免疫沉淀测序和染色体构象。 捕捉(3C)试验检验BMAL1直接和间接调节关键代谢和 活体表皮内的细胞周期基因。(3)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的差异。我们将描述 中枢时钟调节器在UVB DNA损伤反应中的作用 紫外线诱导小鼠致癌模型验证皮肤时间依赖性变化假说 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 在快速增殖的上皮中时钟、代谢、细胞增殖与癌症的关系， 因为这表明昼夜节律可能是人类皮肤癌高发病率的原因之一， 因为这可能最终导致关于如何在增殖的上皮细胞中调节新陈代谢的想法 减少癌变和组织老化。这项工作具有创新性，因为它为 表皮祖细胞代谢和DNA合成时间分离的昼夜节律时钟。

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.