Genesis and Consequences of Aberrant DNA Methylation

描述(申请人提供)：DNA甲基化和染色质结构改变导致的异常基因沉默在人类癌症中肿瘤抑制基因和其他基因的失活中起着重要作用。我们的长期目标是研究这些基因沉默事件背后的分子机制，并研究某些基因的表观遗传沉默如何促进人类癌症的发生。过去几年的新工作表明，当地的染色质环境是决定CpG岛在发育和转化过程中表观遗传命运的关键因素。在上一次资助期间，我们研究了DNA甲基转移酶和组蛋白修饰复合体之间的相互依赖关系，以及它们在促进或防止癌症表观遗传沉默方面的作用。我们已经发现了组蛋白H4修饰(H4K16ac；H4K20me3)和催化这些标记的复合体在调节RNA聚合酶II在CpG岛启动子上的暂停动力学中的一个新作用。具体地说，我们发现SUV420H2组蛋白甲基转移酶沉积了H4K20甲基化，通过抑制hMOF介导的H4K16乙酰化，阻止了POL II对启动子的逃逸，作为强制POL II启动子-近端暂停的开关。此外，我们已经在正常细胞中发现了由H4K20me3标记的一组基因，这些基因在原发非小细胞肺癌中高度容易发生异常的DNA甲基化。这些发现支持这样的模型，即肿瘤发生中的一个关键事件是基于染色质的抑制机制被DNA甲基化施加的更稳定和可遗传的抑制机制所取代。这项提议的目的是测试一种假设，即局部靶向SUV420H2和H4K20me3强制暂停POL II会使某些基因在癌症中增加表观遗传沉默的风险。利用定向基因研究和全球分析相结合的方法，我们将评估SUV420H2和H4K20me3介导的抑制是否以及通过什么机制促进易于甲基化的CpG岛启动子的异常DNA甲基化。我们将直接测试改变的POL II停顿动态和停顿的POL II的不稳定对CpG岛启动子区异常DNA甲基化的影响。将使用转基因方法来确定SUV420H2和H4K20me3介导的抑制的异常靶向是否足以在一个独特的染色体上驱动异位Pol II暂停和异常DNA甲基化。最后，结合击倒实验、染色质免疫沉淀和新的Gro-seq技术，我们将探索分化转录、SUV420H2介导的H4K20me3和癌细胞中易于异常甲基化的CpG岛启动子上DNA甲基化的扩散之间的关系。目前，表观遗传疗法在癌症治疗中的应用引起了人们的极大兴趣。我们的研究结果将为癌症表观遗传沉默的基本机制提供重要的见解，并将为开发改进的癌症表观基因组重新编程策略提供一个框架。

DESCRIPTION (provided by applicant): Aberrant gene silencing resulting from alterations in DNA methylation and chromatin structure plays an important role in the inactivation of tumor suppressor and other genes in human cancers. Our long term goals are to investigate the molecular mechanisms underlying these gene silencing events, and to study how the epigenetic silencing of certain genes contributes to human carcinogenesis. Emerging work over the last several years points to local chromatin environment as a critical factor in determining the epigenetic fate of CpG islands during development and transformation. During the last funding period we have studied the interdependent relationship between DNA methyltransferases and histone modifying complexes, and their role in promoting or preventing epigenetic silencing in cancer. We have uncovered a novel role for histone H4 modifications (H4K16 Ac; H4K20me3) and the complexes that catalyze these marks, in regulation of RNA polymerase II pausing dynamics at CpG island promoters. Specifically, we find that the deposition of H4K20 methylation by the SUV420H2 histone methyltransferase imposes a block to promoter escape by Pol II through inhibition of the hMOF-mediated acetylation of H4K16, acting as a switch to enforce Pol II promoter-proximal pausing. Further, we have identified a subset of genes marked by H4K20me3 in normal cells that are highly prone to aberrant DNA methylation in primary non-small cell lung cancers. These findings support the model that a critical event in tumorigenesis is the replacement of chromatin-based repression mechanisms by the more stable and heritable repression imposed by DNA methylation. The goal of this proposal is to test the hypothesis that local targeting of SUV420H2 and H4K20me3-enforced Pol II pausing places certain genes at increased risk of epigenetic silencing in cancer. Using a combination of directed gene studies and global analyses, we will assess whether, and by what mechanism, SUV420H2 and H4K20me3-mediated repression promotes aberrant DNA methylation at methylation-prone CpG island promoters. We will directly test the impact of altered Pol II pausing dynamics and destabilization of paused Pol II on aberrant DNA methylation at CpG island promoters. A transgene approach will be employed to determine whether the aberrant targeting of SUV420H2 and H4K20me3-mediated repression is sufficient to drive ectopic Pol II pausing and aberrant DNA methylation at a unique chromosomal locus. Finally, in a combination of knock down experiments, chromatin immunoprecipitation and novel GRO-seq technology, we will explore the relationship between divergent transcription, SUV420H2-mediated H4K20me3 and the spread of DNA methylation at CpG island promoters prone to aberrant methylation in cancer cells. There is currently a great deal of interest in the application of epigenetic therapy in cancr treatment. The results of our studies will provide important insight into the basic mechanisms driving epigenetic silencing in cancer, and will also provide a framework for the development of improved strategies for reprogramming the cancer epigenome.