DOT1L及其介导的H3K79甲基化在乳腺癌转移中的作用及机理研究

Histone proteins are subject to reversible post-translational modifications, which play an important role in the establishment and maintenance of gene expression. DOT1L and its homologs appear to be the only enzyme responsible for H3K79 methylation which marks active transcriptions. Although, over the years, progresses have been made in characterzing the enzymatic activity of DOT1L and its associated H3K79 methylation in various organisms and cellular processes, the mechanisms underlying its enzymatic activity and its diverse biological functions are still not completely understood. Our previous work showed that knockdown of DOT1L leads to increased expressions of epithelial markers and decreased expression of mesenchymal markers in MCF-7 cells; we demonstrated that DOT1L promotes migration of MDA-MB-231 cells based on transwell assays, indicating that DOT1L is implicated in the control of epithelial-mesenchymal transition (EMT) process of breast cancer. We propose here the investigation on the transcriptional factors that recruit DOT1L to the genome, the cofactors that are required for DOT1L to methylate H3K79, and the cellular targets of DOT1L. Furthermore, since study of the distinct epigenetic states of cells before and after cell reprogramming is an important part of epigenetics, we are also going to elucidate change of master transcriptional factors of cells in epithelial and mesenchymal states, and the change of distribution of DOT1L and its associated H3K79 methylation during EMT. Our work will shed new lights on the molecular mechanisms of tumor metastasis, and may provide potential targets for metastasis intervention.

DOT1L是近年来发现的细胞内唯一的H3K79甲基化酶，尽管已经有报导指出DO1L所介导的H3K79的甲基化与基因的转录活化相关，其中的机制以及细胞生物学功能研究还远未深入。我们前期的工作发现DOT1L对乳腺癌的EMT具有促进作用。我们的研究将通过寻找细胞内与DOT1L相互作用的转录因子和其它协同因子，以及DOT1L在细胞内的靶基因，探讨DOT1L及其介导的H3K79的甲基化与乳腺癌转移之间的关系及其中的分子机制。另外，探究细胞重编程的过程中表观遗传修饰的变化是表观遗传学研究最重要的一部分内容。在乳腺癌上皮细胞转化为间质细胞过程中，负责维持细胞特性的主要转录因子的转变，以及DOT1L及H3K79甲基化修饰在全基因组水平上的分布变化和其中分子机制也将是我们研究的关键问题。我们的研究将为认识肿瘤转移的分子机制提供理论基础，为寻找遏制肿瘤转移的药物提供可能的靶点。

DOT1L是近年来发现的细胞内唯一的H3K79甲基化酶，尽管已经有报导指出DOT1L所介导的H3K79的甲基化与基因的转录活化相关，其中的机制以及它的细胞生物学功能研究还远未深入。我们的研究工作发现DOT1L对乳腺癌的EMT具有促进作用。我们的研究表明DOT1L在转移性乳腺癌细胞中表达较高。我们在乳腺癌细胞系内敲低DOT1L之后上皮细胞marker表达升高，间质细胞marker表达下降；transwell实验证实敲低DOT1L确实抑制乳腺癌细胞的EMT。我们筛选了稳定表达DOT1L shRNA的MDA-MB-231细胞，注射入小鼠的鼠尾静脉，检测乳腺癌细胞的肺转移，发现敲低DOT1L之后，肺部转移明显减少。分子机制方面，我们利用RNA-seq找到DOT1L的靶基因ZEB2，解释了DOT1L促进乳腺癌转移的原因。另一方面，我们研究发现DOT1L对血管生成具有促进作用。我们证实在人脐静脉内皮细胞（HUVECs）中敲低DOT1L的表达会导致细胞存活降低，细胞迁移、形成管状结构、以及形成血管样新梢的能力降低；移植到小鼠体内的HUVECs在matrigel中形成有功能的血管网络的能力也降低。我们通过分析HUVECs中H3K79me2的ChIP-seq数据找到了DOT1L在基因组内的靶基因，其中包括对于血管生成至关重要的VEGFR2。我们的结果显示DOT1L与ETS-1协同发挥作用激活VEGFR2的表达，活化其下游的ERK1/2和AKT信号通路，促进血管生成。我们的研究将为认识肿瘤转移的分子机制提供理论基础，为寻找遏制肿瘤转移的药物提供可能的靶点。