Bmal1 controls circadian cell proliferation and susceptibility to UVB-induced DNA damage in the epidermis [telogen]

While several physiological skin parameters vary in a circadian manner, the identity of genes participating in chronobiology of skin remains unknown, leading us to define the circadian transcriptome of mouse skin at two different stages of the hair cycle, telogen and anagen. The circadian transcriptomes of telogen and anagen skin are largely distinct, with the former dominated by genes involved in cell proliferation and metabolism. The expression of many metabolic genes is antiphasic to cell cycle related genes, the former peaking during the day and the latter peaking at the night. Consistently, accumulation of reactive oxygen species, a byproduct of oxidative phosphorylation, and S-phase are antiphasic to each other in telogen skin. Furthermore, the circadian variation in S-phase is controlled by BMAL1 intrinsic to keratinocytes as keratinocyte-specific deletion of Bmal1 obliterates time of day dependent synchronicity of cell division in the epidermis leading to a constitutively elevated cell proliferation. Consistent with higher cellular susceptibility to UV-induced DNA damage during S phase, we found that mice are most sensitive to UVB-induced DNA damage in the epidermis at night. As maximum numbers of keratinocytes go through S phase in the late afternoon in the human epidermis, we speculate that in humans the circadian clock imposes regulation of epidermal cell proliferation such that skin is at a particularly vulnerable stage during times of maximum UV exposure, thus contributing to the high incidence of human skin cancers. Whole skin was collected at 4 hour intervals for 48 hours. Where ZT number indicates the number of hours elapsed from when lights are switched on. ZT0 = lights on (6am). ZT12=lights off (6pm). Total RNA was purified from the skin of each mouse and equal amount of RNA from the 3 replicates for each time point were pooled. Telogen samples were collected from skin of P46 male mice.尽管多项皮肤生理参数呈现昼夜节律性变化，但参与皮肤时间生物学调控的基因种类仍未明确，为此我们对处于毛囊周期两个不同阶段——休止期（telogen）与生长期（anagen）——的小鼠皮肤的昼夜转录组（circadian transcriptome）开展了分析与定义。 休止期与生长期皮肤的昼夜转录组整体差异显著，其中休止期皮肤的基因表达以细胞增殖与代谢相关基因为主导。 诸多代谢基因的表达时相与细胞周期相关基因呈反相：前者在日间达到表达峰值，后者则于夜间达到峰值。 与此一致的是，氧化磷酸化副产物活性氧簇（reactive oxygen species, ROS）的积累与S期在休止期皮肤中同样呈反相关系。 进一步研究表明，角质形成细胞（keratinocytes）内源性的BMAL1基因可调控S期的昼夜节律变化：角质形成细胞特异性敲除Bmal1会消除表皮细胞分裂的昼夜同步性，导致细胞增殖水平持续升高。 鉴于S期细胞对紫外线（UV）诱导的DNA损伤敏感性更高，我们发现小鼠表皮在夜间对UVB诱导的DNA损伤最为敏感。 人体表皮的角质形成细胞通常在午后晚些时候进入S期，据此我们推测，人体的昼夜节律钟可调控表皮细胞增殖，使得皮肤在紫外线暴露峰值时段处于易受损伤的脆弱状态，这或许是人类皮肤癌症高发的重要诱因之一。 本研究每隔4小时采集全皮肤样本，持续48小时。其中ZT值表示灯光开启后的时长：ZT0对应灯光开启时刻（早晨6点），ZT12对应灯光关闭时刻（傍晚6点）。 从每只小鼠的皮肤中提取总RNA（total RNA），并将每个时间点3次生物学重复的等量RNA进行混合。 休止期皮肤样本采集自46日龄（P46）的雄性小鼠。