Circadian behavior is light-reprogrammed by plastic DNA methylation (sequencing)

The timing of daily “circadian” behavior can be highly variable among different individuals, and twin studies suggest that about half of this variability is environmentally controlled. Similar plasticity can be seen in mice exposed to an altered lighting environment – for example, 22-hour days instead of 24-hour ones – which stably alters the genetically determined period of circadian behavior for months. The mechanisms mediating these environmental influences are unknown. Here, we show that transient exposure of mice to such lighting stably alters global transcription in the suprachiasmatic nucleus of the hypothalamus (the SCN, the “master clock” tissue determining circadian behavior in mammals). We have also showed that, these changes in transcription are due to change in DNA methylation in the SCN. Indeed, genome-wide methylation profiling revealed global alterations in promoter DNA methylation in the SCN. Importantly, infusion of a methyltransferase inhibitor to the SCN during 22-hour days suppressed period changes. We also found that these behavioral and DNA methylation changes are reversible upon entrainment to 24-hours days. We conclude that the SCN utilizes DNA methylation as a mechanism to drive circadian clock plasticity. Methods: First, 16 mice 5 to 6 weeks old were exposed to DD for 7 days in order to measure their FRP. Then, mice were separated into 2 groups of 8 and entrained to ST and NT cycle. After 4 weeks we recorded wheel-running behavior in constant darkness. After 5 days in DD, we used Clocklab software to predict time of activity onset for each mouse. Brains were isolated at CT4 and sliced with a microtome using a fresh HBSS medium (thickness 250 uM) and the SCN was cut out with the aid of a binocular microscope and stored immediately at –80°C. Total RNA was extracted using the SurePrep™ Nuclear or Cytoplasmic RNA Purification Kit (Fisher Scientific) according to the manufacturer''s protocol. A total of 500 ng of RNA was used for subsequent steps. These steps included DNAase treatment, polyA selection, depletion of ribosomal RNA, overall quality check, and library preparation, and were performed by Fasteris (CH). Libraries were prepared according to standard Illumina protocols. sequence quality, read mapping and data analysis: all steps were done using the GeneProf platform. Briefly, the sequencing reads of each library were first quality checked for read length and nucleotide composition, and then submitted for mapping to the mouse genome refrence sequence (Ensemble 58 mouse Genes, NCBIM37 assembly) using the alignment tool TopHat. Results: Entrainment to non-24 hours light/dark cycle induces a global changes in the SCN transcriptome. Overall design: SCN mRNA profile of mice entrained to normal and short T-cycle using RNAseq technology.

不同个体的日常"昼夜节律（circadian）"行为时序存在显著个体差异，双生子研究表明，此类差异中约一半受环境调控。在暴露于改变的光照环境（例如以22小时日长替代24小时日长）的小鼠中也可观察到类似的可塑性——该处理可稳定改变遗传决定的昼夜节律行为周期长达数月。介导上述环境影响的分子机制目前仍不明确。本研究证实，将小鼠短暂暴露于此类光照环境，可稳定改变下丘脑视交叉上核（suprachiasmatic nucleus of the hypothalamus, SCN，哺乳动物调控昼夜节律行为的"主时钟"组织）的全局转录水平。我们还发现，这些转录变化源于SCN内的DNA甲基化（DNA methylation）改变。全基因组甲基化分析确实揭示了SCN内启动子区域DNA甲基化的全局改变。值得注意的是，在22小时光周期下向SCN输注甲基转移酶抑制剂（methyltransferase inhibitor）可抑制周期变化。我们还观察到，这些行为与DNA甲基化改变在同步至24小时光周期后可完全逆转。综上，我们认为SCN通过利用DNA甲基化作为机制，驱动昼夜节律时钟的可塑性。

方法：首先，将16只5至6周龄的小鼠置于全暗（DD）环境中饲养7天，以测定其自由运行周期（FRP）。随后将小鼠分为两组，每组8只，分别同步至短光周期（ST cycle）与正常光周期（NT cycle）。4周后，在持续黑暗条件下记录小鼠的转轮行为。在持续黑暗环境中饲养5天后，我们使用Clocklab软件预测每只小鼠的活动启动时间。于节律时间4（CT4）时刻分离小鼠脑组织，使用新鲜汉克平衡盐溶液（HBSS）通过切片机制备厚度为250 μm的脑片，借助立体显微镜分离出SCN并立即置于-80℃保存。采用SurePrep™细胞核/细胞质RNA纯化试剂盒（Fisher Scientific），按照制造商的说明书提取总RNA。取总计500 ng RNA用于后续实验步骤，包括脱氧核糖核酸酶处理、polyA富集、核糖体RNA去除、整体质量检测以及文库制备，上述步骤均由瑞士Fasteris公司完成。文库构建按照标准Illumina流程进行。

测序质量控制、读段比对与数据分析：所有步骤均通过GeneProf平台完成。简言之，首先对每个文库的测序读段进行读长与核苷酸组成的质量检测，随后使用比对工具TopHat将读段比对至小鼠参考基因组序列（Ensemble 58小鼠基因集，NCBIM37组装版本）。

结果：暴露于非24小时光/暗周期可诱导SCN转录组的全局改变。

整体实验设计：采用RNA测序（RNA-seq）技术分析同步至正常光周期与短光周期的小鼠的SCN mRNA表达谱。