Circadian Clock Regulation in Skin

描述（由申请人提供）：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毛囊间表皮细胞增殖中的时间依赖性变化所必需的，这似乎与昼夜节律钟调节的代谢相协调。因此，我们发现，参与氧化磷酸化和活性氧（ROS）水平的基因的表达是反相的S期，和ROS水平的昼夜变化取决于核心时钟调节BMAL 1。此外，我们发现小鼠在夜间（当最高数量的表皮细胞通过S期时）对UVB诱导的DNA表皮损伤比白天更敏感。基于这项工作，我们提出了这个中心假设：昼夜节律钟协调表皮中代谢和细胞增殖的时间，在最大ROS产生和S期的时间之间施加分离。虽然这种时间调节安排最大限度地减少了内源性ROS的DNA突变，但它会导致表皮对紫外线介导的DNA损伤敏感性的昼夜变化。这意味着白天的人可能对紫外线介导的DNA损伤特别敏感，因为白天是紫外线暴露最多的时候。我们计划在三个具体的目标来追求这些想法：（1）在单个细胞分辨率的毛囊间表皮中了解生物钟，代谢和细胞增殖之间的关系。我们将绘制由BMAL 1控制的细胞周期阶段，并使用创新的成像方法来定义表皮基底细胞层内单细胞水平的时钟输出，代谢和细胞周期进程之间的关系。我们还将测试的假设，不同的喂养时间表可以改变代谢和表皮细胞增殖的时间。(2)确定毛囊间表皮中的时间依赖性BMAL 1靶基因。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）分析来测试BMAL 1直接和间接调节活表皮内关键代谢和细胞周期基因的假设。(3)确定核心生物钟基因是否是紫外线诱导的皮肤癌发生中时间依赖性差异所必需的。我们将描述中央时钟调节器在人类角质形成细胞中UVB DNA损伤反应中的作用，并利用紫外线诱导的小鼠致癌模型来测试皮肤致癌作用的昼夜节律机制依赖于时间的变化这一假设。这项工作意义重大，因为它揭示了快速增殖上皮细胞中的生物钟，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，并且因为它可能最终导致关于如何在增殖上皮细胞中调节代谢以减少致癌作用和组织衰老的想法。这项工作是创新的，因为它表明了生物钟在表皮祖细胞中代谢和DNA合成的时间分离中的新作用。

DESCRIPTION (provided by applicant): 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.