Regulators of Cancer-Specific DNA Hypermethylation.

描述(申请人提供)：在正常细胞中，DNA甲基化在启动子相关的CpG岛上很少见，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，成百上千个基因在启动子相关的CpG岛上发生甲基化，影响许多途径，包括肿瘤抑制途径。DNA甲基化的这种巨大变化的原因仍然是个谜。有趣的是，一些基因对癌症中异常的去新生甲基化是无效的，而另一些基因则经常成为靶点。破译对异常甲基化敏感或抵抗的CpG岛之间的差异，将有助于更好地理解调节癌症特异性DNA超甲基化的细胞因素。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)识别基线遗传和表观遗传特征，这些特征将癌症中有去新甲基化倾向的基因与受去新甲基化保护的基因分开；(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。在特定的目标1中，我们建议(A)在髓系白血病和结直肠癌中测量启动子相关的CpG岛对DNA高甲基化的倾向，以及(B)确定区分甲基化倾向和甲基化抵抗的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)和表观基因组(正常细胞中的组蛋白修饰)因子。重要的特征将被输入到一个数学模型中，以揭示在调节癌症甲基化方面的个人和合作活动，该模型将在其他样本和肿瘤类型中得到验证。与DNA甲基化易感性相关的最重要的特征和已知因素(转录因子Sp1、行/正弦反转录转座子和绝缘蛋白CTCF、USF1/2和VEZF1)将在特定的目标2中进行测试。在此测试中，我们将使用我们实验室开发的一系列细胞模型，在这些模型中，工程转基因可以插入特定的基因组位置，然后由于四环素诱导阻遏物的存在而移入和移出抑制环境。我们希望我们的研究的成功发展将带来关于异常DNA甲基化如何针对特定基因而不针对其他基因的新见解，并将导致基于表观遗传的治疗的多个靶点的确定。 公共卫生相关性：在很大一部分人类癌症中观察到DNA甲基化模式的改变，针对这种表观遗传标记的治疗已被证明对几种人类肿瘤的治疗有效。癌细胞如何获得改变的DNA甲基化还不完全清楚，我们已经发现证据表明，特定的基因具有固有的甲基化倾向。在这项研究中，我们将对与DNA甲基化易感性和抵抗力相关的基因组特征进行全面评估，并期望得出一个模型来解释在癌症中观察到的异常高甲基化事件。

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.