Project 2

项目概述（见说明）：本项目将利用新开发的单分子工具，结合基于单核小体和阵列物理性质的染色质分离新方法，对中心常见的癌细胞系和组织进行表征，以探测癌症中的染色质和表观遗传变化。在模型系统中对染色质动力学的理解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癌症发生的关键步骤，而肿瘤抑制基因沉默的维持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，流行的假设是DNA甲基化通过结合甲基胞嘧啶DNA结合蛋白和随后的音调修饰因子的募集而使基因沉默，尽管这已经是十多年来的教条，但仍有待证明。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H2A.Z音调变体的结合或特性。我们和其他人的研究表明，H2A。Z使启动子处的核小体不稳定，因此有利于启动子的激活，因此通过阻止H2A。肿瘤抑制基因启动子的非预定DNA甲基化阻止了基因的激活。我们将通过研究H2A的全基因组变化来验证这一假设。并测定H2A的物理性质和翻译后修饰。由材料核心设施提供的癌细胞中含有z的核小体。我们的项目将应用原子力显微镜（AFM）和识别成像技术，我们最近在亚利桑那州立大学-哈奇合作中使用这些技术来表征含有CenHS his音调变体的单个天然染色质颗粒。通过跟踪DNA甲基化的变化，H2A。我们将使用全基因组和单分子方法在食管癌和结肠癌细胞和组织样本中选择翻译后修饰，验证我们的假设，探测表观遗传变化，并将这些变化与中心其他两个项目测量的物理特性相关联。

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.