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(见说明)：该项目将使用最新开发的单分子工具，以及基于单核小体和阵列的物理性质进行染色质分离的新方法，来确定该中心常见的癌细胞系和组织的特征，以探测癌症中的染色质和表观遗传学变化。在模型系统中对染色质动力学的理解方面的最新进展使我们提出了人类癌症中染色质变化的新机制。人们普遍认为，肿瘤抑制基因的沉默是肿瘤发生的关键步骤，而维持肿瘤抑制基因的沉默往往是癌症进展的基础。在负责维持这种沉默的表观遗传机制中，启动子DNA甲基化得到了强有力的实验支持。然而，流行的假说DNA甲基化通过结合甲基胞嘧啶DNA结合蛋白和随后的His音调修饰物的招募而使基因沉默，尽管这一事实已经被信奉了十多年。我们认为，基因沉默的发生是因为DNA甲基化和其他表观遗传修饰干扰了通用的His语调变体H2A.Z的掺入或性质。我们和其他人的工作表明，H2A.Z破坏了启动子上的核小体的稳定，从而有利于启动子的激活，从而通过阻止H2A.Z的掺入或失稳活性，肿瘤抑制基因启动子的非计划DNA甲基化防止了基因激活。我们将通过研究H_2A.Z在基因组范围内的变化来检验这一假设，并分析由材料核心设施提供的癌细胞中含有H_2A.Z的核小体的物理性质和翻译后修饰。我们的项目将应用原子力显微镜(AFM)和识别成像技术，在亚利桑那州立大学-哈奇正在进行的合作中，我们最近使用这些技术来表征包含CenHS His音调变体的单个天然染色质颗粒。通过使用全基因组和单分子方法跟踪食道和结肠癌细胞和组织样本中DNA甲基化、H2A.Z和选定的翻译后修饰的变化，我们将检验我们的假设，探测表观遗传学变化，并将这些变化与中心另外两个项目测量的物理特性相关联。