Circadian Clock Regulation in Skin

描述(申请人提供)：利用遗传的小鼠模型，我们发现角质形成细胞内的生物钟是毛囊间表皮细胞增殖随时间变化所必需的，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物种(ROS)水平的基因表达与S相反，并且ROS水平的昼夜变化依赖于核心时钟调节因子BMAL1。此外，我们还发现，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，我们提出了这一中心假说：生物钟协调表皮新陈代谢和细胞增殖的时间，将ROS产生最多的时间与S时相分开。虽然这种时间调控安排最大限度地减少了内源性ROS造成的DNA突变，但它导致了表皮对UVR介导的DNA损伤的敏感度的昼夜变化。这意味着，白天的人类可能对紫外线辐射最大的白天的紫外线介导的DNA损伤特别敏感。我们计划在三个具体目标上追求这些想法：(1)了解毛囊间表皮单个细胞分辨率下的生物钟、新陈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图，并使用创新的成像方法在表皮基底细胞层的单个细胞水平上定义时钟输出、新陈代谢和细胞周期进程之间的关系。我们还将测试这样一个假设，即不同的喂养计划可以改变表皮中新陈代谢和细胞增殖的时间。(2)确定毛囊间表皮中BMAL1靶基因的时间依赖性。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直接和间接调节活的表皮内的关键新陈代谢和细胞周期基因的假设。(3)确定核心生物钟基因在UVR诱导的皮肤癌变过程中是否存在时间依赖性差异。我们将研究中枢时钟调节器在人体角质形成细胞中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检验皮肤癌变中依赖于时间的变化取决于生物钟机制的假说。这项工作意义重大，因为它揭示了快速增殖的上皮细胞的时钟、新陈代谢、细胞增殖和癌症之间的关系，因为它表明昼夜节律可能导致人类皮肤癌的高发病率，还因为它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以减少癌症发生和组织老化的想法。这项工作具有创新性，因为它表明生物钟在施加新陈代谢和表皮祖细胞DNA合成的时间分离方面发挥了新的作用。

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.