正常细胞恶性转化过程中，基因组相关CpG位点从头甲基化的发生机制，是甲基化理论的瓶颈问题。申请人前期在胆管癌细胞中发现：Dicer通过募集HP1α形成Dicer-HP1α复合物，对RASSF1A启动子CpG位点从头甲基化具有显著调控作用。但是，Dicer-HP1α复合物并不能直接募集从头甲基转移酶，因此确定相关甲基化沉默因子（MSEs）并建立完整的调控通路，成为亟待澄清的关键问题。为此，我们利用人基因组shRNA文库筛选出20种候选MSEs。本项目在前述基础上，拟重点研究：1.鉴定HP1α下游MSEs种类；2.确定MSEs被募集的先后顺序，建立Dicer-HP1α-MSEs调控通路；3.构建敲除和转HP1α基因动物模型，验证Dicer-HP1α-MSEs调控RASSF1A启动子从头甲基化机制的正确性。项目主旨是探讨CpG位点从头甲基化机制，丰富甲基化理论，并探索胆管癌分子干预策略。

Exploring the mechanism of de novo methylation of tumor-related genomic CpG loci in the malignant transformation process of nomal cells has been a challenge for DNA methylation theory. We have shown that Dicer significantly affects the de novo methylation of CpG loci in RASSF1A promoter through recruiting HP1α to form a Dicer-HP1α complex. However, the Dicer-HP1α complex does not directly recruit endogenous de novo methyltransferase. So, identifying the related methylation silencing effectors (MSEs) to map Dicer-HP1α-MSEs pathway has been a key issue need to be resolved urgently. For this purpose, we have utilized human genome-wide shRNA library and found 20 candidate MSEs. Based on our pre-discoveries, the current project will focus on: 1) identifying the MSEs downstream of HP1α; 2) elucidating the sequential recruitment of MSEs to RASSF1A promoter and thus mapping the Dicer-HP1α-MSEs pathway; 3) validating the Dicer-HP1α-MSEs pathway in HP1α knockout and transgenic animal model. We aim to explore the mechanism of de novo methylation of tumor-related CpG loci, as well as the strategy of molecular therapy in cholangiocarcinoma.