Genesis and Consequences of Aberrant DNA Methylation

描述（由申请人提供）：由DNA甲基化和染色质结构改变引起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致癌。过去几年的新研究指出，局部染色质环境是决定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物之间的相互依赖关系，以及它们在促进或预防癌症表观遗传沉默中的作用。我们发现了组蛋白H4修饰（H4K16 Ac; H4K20me3）和催化这些标记的复合物在调控RNA聚合酶II在CpG岛启动子上的暂停动力学中的新作用。具体来说，我们发现SUV420H2组蛋白甲基转移酶沉积H4K20甲基化，通过抑制hmof介导的H4K16乙酰化，阻断Pol II启动子逃逸，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一个由H4K20me3标记的基因亚群，这些基因在原发性非小细胞肺癌中非常容易发生异常DNA甲基化。这些发现支持了一个模型，即肿瘤发生中的一个关键事件是DNA甲基化施加的更稳定和可遗传的抑制取代了基于染色质的抑制机制。本提案的目的是验证SUV420H2和h4k20me3的局部靶向性强制Pol II暂停使某些基因在癌症中增加表观遗传沉默风险的假设。结合定向基因研究和全局分析，我们将评估SUV420H2和h4k20me3介导的抑制是否以及通过何种机制促进易发甲基化的CpG岛启动子的异常DNA甲基化。我们将直接测试改变的Pol II暂停动力学和暂停Pol II的不稳定对CpG岛启动子异常DNA甲基化的影响。将采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驱动异位Pol II暂停和在独特染色体位点的异常DNA甲基化。最后，结合敲低实验、染色质免疫沉淀和新型GRO-seq技术，我们将探索suv420h2介导的H4K20me3与癌细胞中易于异常甲基化的CpG岛启动子DNA甲基化传播之间的关系。目前表观遗传疗法在肿瘤治疗中的应用引起了人们极大的兴趣。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，也将为癌症表观基因组重编程的改进策略的发展提供框架。

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.