组织再生中组蛋白甲基化酶的功能及调控机制研究

Because of its extraordinary regeneration ability, planarian has been a classic teaching material of biology. In recent years, the molecular markers of adult stem cells have been identified. The genes needed for regeneration have been preliminary analysed. we carried out the epigenetic research in planarian for the first time and we have achieved initial results. By the systematic RNAi screening of histone methyltransferase , we found that the PRC2 complex responsible for the inhibitory sites of H3K27me3 modification and the Set1/COMPASS complex responsible for the activation loci H3K4me3 modification are the key of two epigenetic component involved in regeneration. Knockdown of them inhibited the regeneration ability, suggesting that regeneration needs this "Yin and Yang" balance mediated by two histone modifications. However, the detailed cellular and molecular mechanism is still not clear. In this project, we plan to anlalyse the influence of PRC2 complex and Set1/COMPASS complex on the adult stem cell behavior during tissue regeneration. Combined with gene expression array and chromatin immunoprecipitation, we intend to discover and identify the downstream key target genes related to regeneration. In-depth study of the function and mechanism of "Yin and Yang" balance in adult stem cell maintainance and regeneration process, we try to establish a new regeneration related epigenetic regulation model. Relevant research contribute to the understanding of adult stem cell properties and the biological significance of chromatin structure adjustment during the regeneration process.

涡虫由于其强大的再生能力，一直是生物学的经典教学材料。本课题组前期首次开展涡虫再生的表观遗传学研究，取得了阶段性成果。通过针对组蛋白甲基化转移酶的系统性RNAi筛选，我们发现负责修饰活化性位点H3K4me3的Set1/COMPASS复合体和负责修饰抑制性位点H3K27me3的PRC2复合体是再生最为关键的两个组分，对其敲低都抑制了组织再生，提示再生需要这“一阴一阳”两种组蛋白修饰的平衡来调节，其详细的细胞和分子机制尚不明确。本项目计划细致分析这两种甲基化转移酶对组织再生过程中成体干细胞行为的影响，结合基因表达芯片和染色质免疫沉淀发现和鉴定其下游再生相关关键靶基因，深入研究这对“阴-阳”平衡机制在涡虫成体干细胞特性维持和再生过程中的功能及其分子机制，试图建立一个新的再生相关的表观遗传学调控模型。相关研究有助于理解干细胞特性维持和再生过程中染色质结构调节的生物学意义。

组织再生过程涉及的一系列基因需要在特定的时间和空间上表达。近年来，涡虫已成为强大的成体干细胞和组织再生研究模式生物。组蛋白甲基化已被证实在动态调控基因表达中具有重要的功能。然而，至今为止，组蛋白甲基化酶调控组织再生的精确分子机制尚不清楚。本项目在涡虫基因组中预测并鉴定了26个组蛋白赖氨酸甲基化酶。通过RNA干扰（RNAi）技术筛选涡虫体内26个组蛋白甲基化酶在组织再生中的功能，最终发现敲低Set1和Ezh2等6个组蛋白甲基化酶显著抑制组织再生。敲低Set1或者Ezh2均显著抑制组织再生过程中成体干细胞的稳态维持和增殖。据文献报道，Set1 负责组蛋白H3K4甲基化，具有激活基因表达的功能，而Ezh2修饰的组蛋白H3K27甲基化则具有抑制基因表达的功能。通过芯片分析显示，有84个基因既在敲低Set1后显著下调，又在敲低Ezh2后显著上调。进一步通过RNAi技术筛选这84个基因在组织再生中的功能，其中有7个基因在敲低后显著抑制了组织再生过程。染色质免疫共沉淀实验确认了这7个基因的启动子区同时含有H3K4me3和H3K27me3两种组蛋白甲基化修饰。总的来说，我们的结果首先从全基因组水平预测并鉴定了涡虫的组蛋白甲基化酶，进一步通过RNAi筛选了这些组蛋白甲基化酶在组织再生中的功能。其次，我们的结果还提示了组织损伤信号可以通过动态调控组蛋白甲基化的修饰来上调或者抑制组织再生的关键基因Six1/2-2、Soxp5、Lhx3-like、EgrG、Cpsf6、Den5B和RHA的表达。