RACK7选择性结合活跃增强子的分子机制及生物学意义的研究

As important epigenetics regulators, enhancers are recently reported to be actively transcribed to produce enhancer RNA (eRNA), which is positively related to enhancer activity. In our published work we have illustrated that RACK7/KDM5C localize on active enhancers and repress their activity by down-regulating eRNA level. We propose to investigate the mechanism of RACK7/KDM5C’s binding selectivity towards active enhancers. Moreover, by investigating the biological significance of this regulation process in a well-established breast cancer cell line, MCF-7, this study might shed light to new therapies of breast cancer. Our previous study show that RACK7 have strong binding affinity towards INTS1, which is an indispensable factor during eRNA biogenesis. Transcription inhibitors such as DRB strongly repressed the enrichment level of RACK7 on enhancers. On the other hand, Estrogen (E2) treatment greatly promoted RACK7 enrichment level on E2 induced enhancers. Therefore, we bring out the following hypothesis: There is a reverse feedback between RACK7/KDM5C and active transcription on enhancers. As RACK7/KDM5C repressed eRNA transcription, which on the other hand, promote RACK7 binding.

增强子是一类重要的基因转录调控元件，近年多项研究发现增强子本身也可以发生转录产生enhancer RNA (eRNA)，而eRNA的转录水平与增强子的活性正相关。在已发表的工作中我们已证明RACK7/KDM5C复合物可以定位到活跃增强子区域并通过下调eRNA转录水平抑制其过度活化。本项目拟在MCF-7乳腺癌细胞中深入研究RACK7/KDM5C选择性结合活跃增强子的分子机制并探讨该调控过程对于乳腺癌治疗的意义以寻找新的治疗靶点。前期结果表明，RACK7与eRNA转录的关键调控因子INTS1/12蛋白有较强相互作用；转录抑制剂DRB的处理能显著降低RACK7在增强子上的结合水平；而雌二醇（E2）处理能显著提高依赖E2激活的增强子上RACK7的结合水平。我们就此提出如下假设：增强子上的活跃转录过程能促进RACK7/KDM5C复合物的结合，负反馈抑制eRNA的过度转录，从而抑制增强子的过度活化。

增强子是一类重要的基因转录调控元件，增强子本身也可以发生转录产生enhancer RNA (eRNA)，而eRNA的转录水平与增强子的活性正相关。我们前期的工作发现增强子的活性受到RACK7/KDM5C复合物负调控，增强子上H3K4m3/H3K4me1修饰的比值标记了增强子的活性。但是RACK7如何结合到增强子区域负反馈抑制增强子活性的机制则还未知，同时也不知道哪个甲基转移酶负责了增强子上的H3K4me3修饰，本项目主要针对RACK7如何结合到增强子以及哪个甲基转移酶负责增强子上H3K4me3修饰这两个问题展开研究。.我们在本项目中完成了MCF-7乳腺癌细胞在激素剥夺后，EtoH和E2刺激下RACK7在染色质上的图谱，发现RACK7在增强子上的结合跟eRNA的转录呈正相关动态变化，提示eRNA可能参与招募RACK7到增强子区域；随后我们用Tether实验证实特定的eRNA确实参与招募RACK7到特定的增强子区域，这些证据表明特定增强子转录产生的eRNA可能确实参与招募RACK7到特定的增强子，从而负反馈抑制eRNA的转录并进而抑制增强子的活性。.我们同时筛选了增强子上组蛋白H3K4me3甲基转移酶，相关研究还在继续开展中。