Regulators of Cancer-Specific DNA Hypermethylation.

描述（由申请人提供）：在正常细胞中，启动子相关CpG岛中的DNA甲基化很少见，但对X失活、基因组印记和重复元件的抑制很重要。在肿瘤发生过程中，数百至数千个基因在启动子相关的CpG岛中获得甲基化，影响许多途径，包括肿瘤抑制途径。DNA甲基化的这种巨大转变的原因仍然是个谜。有趣的是，一些基因对癌症中的异常从头甲基化是难治的，而另一些基因则经常被靶向。解读CpG岛对异常甲基化敏感或抵抗之间的差异将有助于更好地理解调节癌症特异性DNA超甲基化的细胞因子。基于初步数据，我们解释这种差异的中心假设是，与发育转录程序相关的局部序列特征（重复元件，DNA结合蛋白的识别位点）和基线染色质状态（组蛋白修饰）之间的相互作用调节了癌症中的CpG岛甲基化。为了验证这一假设，我们提出了以下具体目标：（1）确定基线遗传和表观遗传特征，这些特征将癌症中具有从头甲基化倾向的基因与受保护免于从头甲基化的基因分离;以及（2）使用DNA甲基化诱导的细胞模型来验证候选遗传和表观遗传特征的个体和协同作用。在具体目标1中，我们提出（a）测量髓性白血病和结直肠癌中启动子相关CpG岛对DNA超甲基化的倾向）和（B）鉴定区分甲基化倾向与甲基化抗性CpG岛的基因组（转录因子结合位点、反转录转座子、短直接重复序列）和表观基因组（正常细胞中的组蛋白修饰）因子。将在数学模型中输入重要特征，以揭示调节癌症甲基化的个体和合作活性，并将在其他样品和肿瘤类型中验证该模型。与DNA甲基化差异易感性相关的最显著特征和已知因素（转录因子Sp1、LINE/SINE反转录转座子和绝缘子蛋白CTCF、USF 1/2和VEZF 1）将在具体目标2中进行测试。为了进行这项测试，我们将使用我们实验室开发的一系列细胞模型，其中工程转基因可以插入特定的基因组位点，然后由于四环素诱导的阻遏物的存在而移入和移出阻遏环境。我们希望我们研究的成功发展将为异常DNA甲基化如何靶向特定基因而不影响其他基因带来新的见解，并将导致鉴定基于表观遗传学的治疗的多个靶点。 公共卫生相关性：在大部分人类癌症中观察到DNA甲基化模式的改变，并且靶向这种表观遗传标记的疗法已被证明在治疗几种人类肿瘤中有效。癌细胞如何获得改变的DNA甲基化谱尚未完全了解，我们已经发现证据表明特定基因具有甲基化的固有倾向。在这项研究中，我们将对与DNA甲基化易感性和抗性相关的基因组特征进行全面评估，我们希望得到一个解释癌症中观察到的异常高甲基化事件的模型。

DESCRIPTION (provided by applicant): In normal cells, DNA methylation is rare in promoter-associated CpG islands but important to X-inactivation, genomic imprinting and repression of repetitive elements. During tumorigenesis hundreds to thousands of genes gain methylation in promoter-associated CpG islands, affecting many pathways including tumor- suppressor pathways. The causes of this massive switch in DNA methylation remain mysterious. Interestingly, some genes are refractory to abnormal de-novo methylation in cancer while others are frequently targeted. Deciphering the differences between CpG islands sensitive or resistant to aberrant methylation will lead to a better understanding of the cellular factors that modulate cancer-specific DNA hypermethylation. Based on preliminary data, our central hypothesis to explain this difference is that an interplay between local sequence features (repeat elements, recognition sites for DNA binding proteins) and baseline chromatin states (histone modifications) related to developmental transcription programs modulates CpG island methylation in cancer. To test this hypothesis, we propose the following specific aims: (1) Identify baseline genetic and epigenetic features which segregate genes with propensity to become de-novo methylated in cancer from genes protected from de-novo methylation; and (2) use cellular models of DNA methylation induction to validate the individual and cooperative action of candidate genetic and epigenetic features. In specific aim 1, we propose (a) to measure the propensity of promoter-associated CpG islands to DNA hypermethylation in myeloid leukemia and colorectal carcinomas) and (b) to identify the genomic (transcription factor binding sites, retrotransposons, short direct repeats) and epigenomic (histone modifications in normal cells) factors that distinguish methylation-prone versus methylation-resistant CpG islands. Significant features will be entered in a mathematical model to reveal individual and cooperative activity in modulating methylation in cancer, and the model will be validated in other samples and tumor types. The most significant features and known factors associated with differential predisposition to DNA methylation (the transcription factor Sp1, LINE/SINE retrotransposons and the insulator proteins CTCF, USF1/2 and VEZF1) will be tested in specific aim 2. For this testing we will use a series of cellular models developed in our laboratory where engineered transgenes can be inserted in specific genomic loci, and later on moved in and out of repressive contexts due to the presence of tetracycline-induced repressors. We expect that the successful development of our research will bring novel insights in how abnormal DNA methylation is targeted to specific genes while sparing others, and will also result in the identification of multiple targets for epigenetic-based therapies. PUBLIC HEALTH RELEVANCE: Alteration of the pattern of DNA methylation is observed in a large fraction of human cancers, and therapies targeting this epigenetic mark have proved effective in the treatment of several human neoplasias. How cancer cells acquire an altered DNA methylation profile is not fully understood, and we have found evidence that specific genes have an inherent predisposition to become methylated. In this study, we will perform a comprehensive evaluation of the genomic features associated with predisposition and resistance to DNA methylation, and we expect to derive a model that explains aberrant hypermethylation events observed in cancer.