DNA甲基化对刺参夏眠调控关键基因的转录抑制机理

Exploration of the molecular mechanisms of long-term gene transcriptional suppression is a current challenge for aestivation research and other forms of hypometabolism. DNA methylation is a major epigenetic mechanism that is a key regulatory candidate for such transcriptional suppression. The present application proposes a novel analysis of the role of DNA methylation as the molecular mechanism of gene silencing during aestivation in the sea cucumber. Based on our previous profile of gene expression during aestivation, several crucial hypometabolism-associated genes were identified as significantly down-regulated during deep aestivation and then significantly up-regulated again during arousal. The full-length cDNA and promoter regions of these genes will be cloned and identified using RACE and genome-walking methods, and CpG islands (putative methylation sites) within them will be predicted. Technologies for DNA methylation analysis will then be applied to determine the methylation status of these crucial genes and at least 5 aestivation-associated candidate genes that are regulated by DNA methylation will be identified for further study. To validate the relationship between DNA methylation and transcriptional gene depression during aestivation, the DNA methylation status and mRNA expression patterns of these candidate genes will be investigated in 5-ADC treated intestine cells collected from different stages of the aestivation-arousal cycle. On the basis of these results, the functional role of methylated CpG binding proteins (MBDs) as mediators of methylated DNA will then be explored using ChIP technology. The proposed study will reveal the importance of DNA methylation as a regulatory mechanism for reversible gene transcriptional depression during aestivation.

代谢减退过程中长期的基因转录抑制机制将是未来几年夏眠调控机理研究热点之一，DNA甲基化作为表观遗传修饰的主要方式之一在基因转录抑制的调控中起到了关键的作用。项目拟从夏眠刺参代谢减退期间的基因整体表达抑制机制入手，以DNA甲基化的表观调控为突破点，基于前期构建的夏眠刺参基因表达谱筛选出的代谢减退调控的关键基因，克隆鉴定关键基因的全长和启动子区域，预测其CpG岛；借助DNA甲基化的研究手段，测定夏眠不同时期关键基因的甲基化状态，发掘5个以上受DNA甲基化调控的夏眠相关的候选基因；以不同浓度的去甲基化药物处理非夏眠和深度夏眠期刺参肠道组织细胞，测定候选基因甲基化状态和mRNA表达水平的变化，验证DNA甲基化和夏眠代谢减退期间基因转录抑制的相关性；应用ChIP技术阐明甲基结合蛋白（MBDs）在抑制基因转录活性途径中的关键作用；揭示DNA甲基化在刺参夏眠代谢减退过程中的基因转录调控机理。

代谢减退过程中长期的基因转录抑制机制将是未来几年夏眠调控机理研究热点之一，DNA甲基化作为表观遗传修饰的主要方式之一在基因转录抑制的调控中起到了关键的作用。本项目以夏眠刺参代谢减退期间DNA甲基化的表观调控作用研究为突破点，发掘了DNA甲基化系统关键调节酶（DNA甲基转移酶家族成员，甲基结合蛋白家族成员和DNA去甲基酶家族成员）在夏眠不同阶段的表达模式；利用RACE 技术和Genome walking技术分别获得了部分夏眠相关关键基因的cDNA 全长，启动子区和内含子区，分析鉴定了夏眠相关关键基因的完整基因结构。应用 qRT-PCR技术和BSP（Bisulfite sequencing PCR）技术分别测定了夏眠关键基因在刺参夏眠不同阶段不同组织中 mRNA 的表达水平和甲基化特征，发掘了4个夏眠相关关键基因在刺参夏眠期间的甲基化模式，分析了关键基因甲基化特征变化与夏眠代谢减退期间转录表达变化之间的相关性。项目执行后期在刺参基因组释放的基础上，利用高分辨率的全基因组重亚硫酸盐测序技术对刺参夏眠不同时期不同组织的DNA甲基化特征进行了全基因组的扫描分析，研究发现RPPT基因家族成员在夏眠期间表现出显著的甲基化差异，DMR pathway富集分析表明mRNA监视路径被显著富集，进一步从基因组层面上初步阐明了DNA 甲基化在夏眠代谢减退期间基因转录表达中的整体调控策略；本研究将为刺参代谢减退过程中长期的基因表达抑制机制研究提供新思路，为阐明环境胁迫，基因表达和有机体应激反应三者之间复杂关系提供一个新的契机，为水产动物特殊生理机能发生机制的研究开拓一个新的突破点。..