Circadian Clock Regulation in Skin

描述（由申请人提供）：使用小鼠遗传模型，我们发现角化细胞内的生物钟是滤泡间表皮细胞增殖的时间依赖性变异所必需的，这似乎与生物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）水平的基因的表达与s期是反相的，并且ROS水平的昼夜变化取决于核心时钟调节因子BMAL1。此外，我们发现小鼠在夜间（表皮细胞数量最多的时候进入s期）比白天对uvb诱导的DNA表皮损伤更敏感。基于这项工作，我们提出了这个中心假设：生物钟协调表皮代谢和细胞增殖的时间，将最大ROS生成时间和s期分开。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uvr介导的DNA损伤的敏感性发生昼夜变化。这意味着，白天的人可能对紫外线辐射介导的DNA损伤特别敏感，白天是紫外线照射最大的时间。我们计划在三个特定目标中追求这些想法：(1)在单个细胞分辨率下了解卵泡间表皮的生物钟，代谢和细胞增殖之间的关系。我们将绘制由BMAL1控制的细胞周期阶段，并使用创新的成像方法在表皮基底细胞层的单细胞水平上定义时钟输出、代谢和细胞周期进程之间的关系。我们还将测试不同的摄食时间表可以改变表皮代谢和细胞增殖的时间的假设。(2)在卵泡间表皮中鉴定每日时间依赖性BMAL1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）分析来验证BMAL1直接或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需要在紫外线辐射诱发的皮肤癌变的时间依赖性差异中发挥作用。我们将描述中央时钟调节因子在人类角化细胞中UVB DNA损伤反应中的作用，并利用紫外线诱导的小鼠癌变模型来验证皮肤癌变的时间依赖性取决于生物钟机制的假设。这项工作意义重大，因为它揭示了快速增殖上皮中生物钟、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调节增殖上皮中的代谢以减少癌变和组织衰老的想法。这项工作具有创新性，因为它提出了生物钟在表皮祖细胞的代谢和DNA合成中施加时间分离的新作用。

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.