Epigenetic Targeting in Non-Hodgkin's Lymphoma

描述（由申请人提供）：启动子CpG岛的超甲基化在癌症中起着重要作用。与组蛋白乙酰化/甲基化的改变合作，这种表观遗传事件建立了一种抑制性染色质结构，导致关键癌症相关基因的沉默。基因组内DNA甲基化的发生不是随机的，而是产生基因和肿瘤类型特异性的甲基化模式。DNA甲基化模式是如何建立的仍然知之甚少。由于在体内发现了多种与DNA甲基转移酶（DNMT）相关的转录因子或调节因子，我们假设：1）致癌转录因子可以招募DNMT靶向基因启动子并在肿瘤细胞中定义独特的表观遗传标记; 2）剖析这种复杂的表观遗传层次将为诊断、预后和治疗干预确定新的分子靶点。为了验证我们的假设，我们开发了一种高通量技术，用于全基因组分析与特定蛋白质（如组蛋白、转录因子或任何DNA结合蛋白）相关的DNA甲基化。名为ChIP-Chop-DMH的新方法将结合联合收割机全基因组定位分析（也称为ChIP-on-Chip）和差异甲基化杂交（DMH）分析，这两种新兴技术用于表观遗传学研究。所提出的方法具有明显的优势，目前的协议：第一，这种方法直接检查在体内的相互作用的特定蛋白质与甲基化的DNA在整个基因组中;第二，这种方法可能会发现新的生物学特性的转录因子;第三，这种方法可以应用于发现新的表观遗传生物标志物相关的肿瘤发生。在初步研究中，我们已经验证了这种方法的实用性与甲基化组蛋白H3在赖氨酸9和赖氨酸4在人类癌细胞。在R21阶段，我们将继续对该方法进行微小的改进，并追求三个目标：1）改进和优化ChIP-Chop-DMH方法，用于分析DNA甲基化与组蛋白修饰的全基因组关联; 2）利用所提出的方法研究DNA甲基化与染色质重塑因子的关联：3）显示使用阵列检查原发性非霍奇金淋巴瘤（NHL）的概念验证。在此开发阶段，我们将重点关注拟定方法的灵敏度、重现性和准确度。在R33阶段，我们的目标是利用这项技术来测试生物学假设。我们将充分实施该方法并追求这些目标：1）发现与已知致癌转录因子c-Myc和BCL 6相关的表观遗传靶基因：2）鉴定的表观遗传靶点并研究相关致癌转录因子的调控作用。这种系统的方法将为未来的机制研究以及癌症诊断提供一个强大的工具。

DESCRIPTION (provided by applicant): Hypermethylation of promoter CpG islands plays a prominent role in cancer. In partnership with alterations in histone acetylation/methylation, this epigenetic event establishes a repressive chromatin structure that leads to silencing of key cancer-related genes. The occurrence of DNA methylation within the genome is not random, but rather patterns of methylation are generated that are gene and tumor type specific. How DNA methylation patterns are established is still poorly understood. Since various transcriptional factors or regulators are found in association with DNA methyltransferases (DNMTs) in vivo, we hypothesize that: 1) Oncogenic transcription factors can recruit DNMTs to target gene promoters and define a unique epigenetic signature in tumor cells; 2) Dissecting such complex epigenetic hierarchy will identify novel molecular targets for diagnosis, prognosis and therapeutic intervention. To test our hypothesis, we developed a high throughput technique for genome wide analysis of DNA methylation associated with specific proteins such as histones, transcription factors or any DNA binding proteins. The new approach named ChlP-Chop-DMH will combine both genome wide location analysis (also known as ChlP-on-Chip) and Differential Methylation Hybridization (DMH) analysis, two emerging technologies used in epigenetic research. The proposed method has distinct advantages over current protocols: first, this method directly examines the in vivo interaction of specific proteins with methylated DNA throughout the genome; second, this method may uncover novel biological properties of transcription factors; third, this method can be applied to discover novel epigenetic biomarkers relevant to tumorigenesis. In preliminary studies, we have verified the utility of this method with methylated histone H3 at lysine 9 and lysine 4 in human cancer cells. In the R21 phase, we will continue minor refinement of the method and pursue three aims: 1) Improve and optimize the ChlP-Chop-DMH method for analyzing genome wide association of DNA methylation with histone modification; 2) Utilize the proposed method to investigate the association of DNA methylation with chromatin remodeling factors: 3) Show proof-of-concept using the array to examine primary non-Hodgkin's lymphomas (NHLs). In this development phase we will focus on the sensitivity, reproducibility and accuracy of the proposed method. In the R33 phase, our goal is to utilize the technology to test biological hypotheses. We will fully implement the method and pursue these aims: 1) Discover epigenetic target genes associated with known oncogenic transcription factors c-Myc and BCL6: 2) Validate the identified epigenetic targets and investigate the regulatory role of the associated oncogenic transcription factors. This systematic approach will provide a powerful tool for future mechanistic studies as well as cancer diagnosis.