Genesis and Consequences of Aberrant DNA Methylation

描述（由申请人提供）：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有助于人类致癌。过去几年的新兴工作指出，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。在上一个资助期间，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中的表观遗传沉默中的作用。我们发现了组蛋白H4修饰（H4 K16 Ac; H4 K20 me 3）和催化这些标记的复合物在调节RNA聚合酶II在CpG岛启动子处暂停动力学中的新作用。具体而言，我们发现，沉积的H4 K20甲基化的SUV 420 H2组蛋白甲基转移酶施加了一个块启动子逃逸Pol II通过抑制hMOF介导的乙酰化H4 K16，作为一个开关，以强制Pol II启动子近端暂停。此外，我们已经在正常细胞中鉴定了一个由H4 K20 me 3标记的基因子集，这些基因在原发性非小细胞肺癌中高度倾向于异常DNA甲基化。这些发现支持了肿瘤发生中的一个关键事件是由DNA甲基化施加的更稳定和可遗传的抑制取代基于染色质的抑制机制的模型。该提案的目标是测试以下假设：SUV 420 H2和H4 K20 me 3的局部靶向-强制Pol II暂停使某些基因在癌症中的表观遗传沉默风险增加。使用定向基因研究和全球分析的组合，我们将评估是否，以及通过什么机制，SUV 420 H2和H4 K20 me 3介导的抑制促进异常的DNA甲基化在甲基化倾向的CpG岛启动子。我们将直接测试改变的Pol II暂停动力学和暂停的Pol II对CpG岛启动子处的异常DNA甲基化的不稳定性的影响。将采用转基因方法来确定SUV 420 H2和H4 K20 me 3介导的阻遏的异常靶向是否足以驱动独特染色体基因座处的异位Pol II暂停和异常DNA甲基化。最后，结合敲除实验、染色质免疫沉淀和新型GRO-seq技术，我们将探索趋异转录、SUV 420 H2介导的H4 K20 me 3与癌细胞中易于发生异常甲基化的CpG岛启动子处DNA甲基化扩散之间的关系。目前，表观遗传疗法在乳腺癌治疗中的应用引起了极大的兴趣。我们的研究结果将为推动癌症表观遗传沉默的基本机制提供重要的见解，也将为开发重新编程癌症表观基因组的改进策略提供框架。 公共卫生相关性：我们的基因组包装成染色体的方式的改变通过引起通常阻止这种异常生长的基因沉默而促成肿瘤形成。与使这些基因永久丧失功能的突变不同，这些表观遗传改变是可逆的，这是目前在某些恶性肿瘤治疗中利用的一种特性。然而，关于驱动这些基因沉默事件的分子机制存在不确定性。我们的研究结果将为推动癌症基因沉默的基本机制提供重要的见解，也将为开发旨在“重编程”癌症基因组的新型治疗策略提供框架。

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. PUBLIC HEALTH RELEVANCE: Alterations in the way our genome is packaged into chromosomes contributes to tumor formation by causing the silencing of genes that normally prevent such aberrant growth. Unlike mutations, which render such genes permanently non-functional, these epigenetic alterations are reversible, a property that is currently being exploitd in the treatment of certain malignancies. However, uncertainties exist regarding the molecular mechanisms that drive these gene silencing events. The results of our studies will provide important insight into the basic mechanisms driving gene silencing in cancer, and will also provide a framework for the development of novel treatment strategies aimed at "reprogramming" the cancer genome.