摘要
目的建立分析C57BL/6J小鼠肝脏组织mPer1基因启动子区甲基化状态的亚硫氢酸修饰法,检测mPer1表达峰、谷对应的甲基化状态。方法利用实时定量PCR方法检测mPer1基因表达时相特点,并利用亚硫氢酸修饰法对小鼠肝脏组织mPer1基因启动子区域CpG岛和E-box甲基化情况进行了研究。结果C57BL/6J小鼠肝脏组织mPer1基因表达有显著波动性,表达高峰位于ZT13,低谷位于ZT01。序列分析显示mPer1基因启动子区含有CpG岛,并且该CpG岛覆盖与mPer1波动性表达密切相关的顺式元件E-box1和E-box2。亚硫氢酸修饰测序结果显示,mPer1基因启动子区CpG岛和E-box均呈低甲基化或非甲基化状态。不同时间点mPer1基因CpG岛甲基化频率无显著变化(P=0.458)。结论肝脏中mPer1基因启动子区CpG岛和E-box均为开放状态,可以与时钟相关转录因子结合。甲基化不参与调节mPer1基因的节律性表达。
[Objective] To analyse the methylation of CpG dinucleotides in the promoter of mPerl gene in C57 BL/6J mouse liver. [Methods] Real-time PCR was employed to detect circadian expression of mPerl. Genomic DNA of mouse liver tissue were extracted and treated by bisulfite sodium and then sequenced. [Results] It indicated that mPerl oscillated around-the-clock with peak at ZT13 and through at ZT01. The 2 E-box in mPerl promoter region encompass 14 and 14 CpGs respectively, which were covered by a putative CpGs island. Both CpG island and E-boxes showed a low level of methylation, and even free from methylation. No time-dependent variations on DNA methylatian was observed in mPerl promoter region. [Conclusion] Methylation statuses of mPerl CpG island did not change over time in liver, suggesting that E-boxes are open to mBmall/mClock heterodimer, which is critical for mPerl daily oscillation. In addition, E-box methylation does not regulate the rhythmic expression of liver mPerl.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2009年第12期1813-1816,1820,共5页
China Journal of Modern Medicine
基金
国家自然科学基金(No:30400148
30430280)
科技部重点项目(No:2006CB500701)
北京市科技新星计划(No:H020821400190)
北京市自然基金(No:5063042)
