Project 2

项目2

• 批准号: 8744860
• 负责人: STUART LINDSAY
• 金额: $ 19.83万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8744860
• 关键词: Atomic Force Microscopy; Binding; Biological Assay; Biological Models; Cancer Biology; Cancer cell line; Chromatin; Collaborations; Colon; Colon Carcinoma; Core Facility; DNA Methylation; DNA-Binding Proteins; Diagnosis; Epigenetic Process; Epitopes; Fractionation; Gene Activation; Gene Silencing; Human; Image; Imaging technology; Instruction; Maintenance; Malignant Neoplasms; Malignant neoplasm of esophagus; Measures; Methods; Modification; Mutation; Normal Cell; Nucleosomes; Post-Translational Protein Processing; Prevention; Property; RNA Interference; Series; Testing; Tissue Sample; Tissues; Tumor Suppressor Genes; Variant; Work; aptamer; base; cancer cell; cancer initiation; genome-wide; novel; particle; physical property; physical science; prevent; promoter; single molecule; tool; tumor progression

PROJECT SUMMARY (See instructions): This project will characterize the cancer cell lines and tissues common to the Center using newly developed single-molecule tools, together with new methods for chromatin fractionation based on physical properties of mononucleosomes and arrays, to probe chromatin and epigenetic changes in cancer. Recent advances in the understanding of chromatin dynamics in model systems leads us to propose a novel mechanism for chromatin changes in human cancer. It is widely accepted that silencing of tumor suppressor genes is a key step in cancer initiation, and maintenance of tumor suppressor gene silencing often underlies cancer progression. Among the epigenetic mechanisms that are responsible for maintaining this silencing, promoter DNA methylation has strong experimental support. However, the popular hypothesis that DNA methylation silences genes by binding methylcytosine DNA binding proteins and consequent recruitment of his tone modifiers remains to be demonstrated, despite the fact that it has been the dogma for over a decade. We propose that gene silencing instead occurs because DNA methylation and other epigenetic modifications interfere with incorporation or properties ofthe universal his tone variant, H2A.Z. Our work and that of others suggests that H2A.Z destabilizes nucleosomes at promoters and thus favors promoter activation, so that by preventing H2A.Z incorporation or destabilization activity, unscheduled DNA methylation of tumor suppressor gene promoters prevents gene activation. We will test this hypothesis by investigating the genome-wide changes in H2A.Z and assay the physical properties and post-translational modifications of H2A.Z-containing nucleosomes from cancer cells provided by the Materials Core Facility. Our project will apply atomic force microscopy (AFM) and recognition imaging technologies that we have recently used to characterized single native chromatin particles containing the CenHS his tone variant in an ongoing ASU-Hutch collaboration. By following changes in DNA methylation, H2A.Z, and selected post-translational modifications in esophageal and colon cancer cells and tissue samples using both genome-wide and single-molecule methods, we will test our hypothesis, probe epigenetic changes, and correlate these changes with the physical properties measured by the two other projects in the Center.

项目摘要（请参阅说明）：该项目将使用新开发的单分子工具来表征该中心共同的癌细胞系和组织，以及基于单核体体和阵列的物理特性的新方法，用于探测癌症中的染色质和表观遗传变化。了解模型系统中染色质动力学的最新进展使我们提出了一种新型的人类癌症染色质变化的机制。人们普遍认为，抑制肿瘤基因的沉默是癌症启动的关键步骤，维持肿瘤抑制基因沉默通常是癌症进展的基础。在负责维持这种沉默的表观遗传机制中，启动子DNA甲基化具有强大的实验支持。然而，流行的假设是，通过结合甲基胞嘧啶DNA结合蛋白的DNA甲基化基因以及随之而来的他的音调修饰剂的募集仍然有待证明，尽管事实上它一直是教条已有十多年了。我们建议基因沉默而发生，因为DNA甲基化和其他表观遗传修饰会干扰其宇宙性张力变体H2A.Z的掺入或特性。我们的工作和其他工作表明，H2A.Z破坏了启动子的核小体稳定性，因此有利于启动子的激活，因此通过防止H2A.Z掺入或不稳定活性，肿瘤抑制基因启动子的未结束的DNA甲基化可以预防基因活化。我们将通过研究H2A.Z的全基因组变化并测定来自材料核心设施提供的癌细胞的H2A.Z含核小体的物理特性和翻译后修饰来检验这一假设。我们的项目将应用原子力显微镜（AFM）和识别成像技术，我们最近用来表征包含CENH的单个天然染色质颗粒，这些颗粒包含CENHS在正在进行的ASU-HUTCH协作中。通过遵循DNA甲基化，H2A.Z的变化以及使用全基因组和单分子方法的食管癌和结肠癌细胞和组织样品中选定的翻译后修饰，我们将测试假设，探针表观变化，并将这些变化与中心的其他两个项目测量的物理性质相关联。