Deciphering Networks Controlling DNA Amplification

破译控制 DNA 扩增的网络

• 批准号: 10330625
• 负责人: Johnathan R. Whetstine
• 金额: $ 46.75万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10330625
• 关键词: Address; Amplifiers; Automobile Driving; Behavior; Cells; Characteristics; Chemicals; Chromatin; Coupled; DNA; DNA Sequence Alteration; DNA amplification; Data; Diploidy; Disease; Enzymes; Epigenetic Process; Event; Gene Amplification; Generations; Genetic; Genome; Genomics; Goals; Heart Diseases; Heterogeneity; Histones; Human Genome; Individual; Knowledge; Laboratories; Lysine; Malignant Neoplasms; Maps; Methyltransferase; Microscopy; Molecular; Mutation; National Institute of General Medical Sciences; Nuclear; Oncogenes; Pathologic; Pathway interactions; Population; Process; RNA; Role; Site; base; cancer cell; epigenome; extrachromosomal DNA; insight; nervous system disorder; new therapeutic target; novel marker; prevent; therapeutic target; tool; transcriptome sequencing; treatment response

PROJECT SUMMARY DNA amplification is associated with pathological states such as neurological disorders, cardiac disease and cancer. At least 50% of the amplifications in cancer are transient extrachromosomal DNA (ecDNA). The transient behavior contributes to copy number plasticity, results in heterogeneous oncogene expression and alters therapeutic response. The unanswered question remains as to whether distinct mechanisms control ecDNA copy gains within cells and how they impact copy gain events associated with disease. My overall goal is to define the principles regulating selective DNA copy gains and the associated plasticity, so we may control these events. My laboratory was the first to discover a molecular basis for extrachromosomal transient site-specific DNA copy gains (TSSGs) in the human genome. Specifically, we identified the first enzyme capable of driving site-specific ecDNA amplification [the histone 3 lysine 9 and 36 tri-demethylase (H3K9/36me3) KDM4A] and demonstrated a fundamental role for epigenetic states in controlling the predilection of specific DNA regions to rereplicate and amplify. We discovered seven more chromatin enzymes- lysine methyltransferases (KMTs) and demethylases (KDMs)- that function in concert to control site-specific amplification in both non-cancer and cancer cells. These studies established a critical role for chromatin factors and their associated states in regulating DNA amplifications. With this NIGMS R35, my laboratory will expand our studies in order to elucidate: 1) the fundamental mechanisms controlling DNA amplification; 2) the molecular processes and characteristics promoting or preventing DNA amplification; and 3) the relationship between ecDNA generation and the associated RNA heterogeneity/DNA mutation burden. We will address these points by leveraging microscopy- based screens using genetic and chemical tools in order to identify key amplifiers, and in turn, generate epigenome profiles coupled to genome organization maps associated with these pathways so that molecular features controlling DNA amplification are resolved. These studies will also be coupled to state-of-the-art long read sequencing and single cell (DNA and RNA) sequencing strategies so that the associated heterogeneity within the cell population and individual cells can be correlated with the effect of the amplifier on TSSGs. These studies are being conducted in non-transformed cells that have a nearly diploid genome so that additional genomic anomalies and mutations do not impact these studies. Collectively, the data generated from these studies will increase our knowledge about the molecular features governing DNA copy gains and the associated heterogeneity, which will resolve novel biomarkers and therapeutic targets in order to control copy number- associated diseases in the years ahead.

项目概要 DNA 扩增与神经系统疾病、心脏病和心脏病等病理状态有关。 癌症。癌症中至少 50% 的扩增是短暂的染色体外 DNA (ecDNA)。瞬态的 行为有助于拷贝数可塑性，导致异质癌基因表达并改变 治疗反应。悬而未决的问题仍然是是否有不同的机制控制 ecDNA 拷贝 细胞内的增益以及它们如何影响与疾病相关的复制增益事件。我的总体目标是定义 调节选择性 DNA 拷贝增益和相关可塑性的原理，因此我们可以控制这些事件。 我的实验室是第一个发现染色体外瞬时位点特异性 DNA 拷贝的分子基础的实验室 人类基因组中的增益（TSSG）。具体来说，我们确定了第一种能够驱动位点特异性的酶 ecDNA 扩增 [组蛋白 3 赖氨酸 9 和 36 三去甲基酶 (H3K9/36me3) KDM4A] 并证明 表观遗传状态在控制特定 DNA 区域的重新复制和偏好方面的基本作用 放大。我们发现了另外七种染色质酶 - 赖氨酸甲基转移酶 (KMT) 和去甲基酶 （KDM）——协同控制非癌细胞和癌细胞中的位点特异性扩增。这些 研究确定了染色质因子及其相关状态在调节 DNA 中的关键作用 放大。借助 NIGMS R35，我的实验室将扩大我们的研究，以阐明：1) 控制DNA扩增的基本机制； 2）分子过程和特征 促进或阻止DNA扩增； 3）ecDNA生成与 相关的RNA异质性/DNA突变负担。我们将通过利用显微镜来解决这些问题 - 使用遗传和化学工具进行基于屏幕的筛选，以识别关键放大器，进而生成 表观基因组图谱与与这些途径相关的基因组组织图相结合，以便分子 控制 DNA 扩增的特征已得到解决。这些研究还将与最先进的长期研究相结合 读取测序和单细胞（DNA 和 RNA）测序策略，以便相关的异质性 细胞群和单个细胞内的细胞可以与放大器对 TSSG 的影响相关。这些 研究正在具有近二倍体基因组的非转化细胞中进行，以便额外 基因组异常和突变不会影响这些研究。总的来说，从这些生成的数据 研究将增加我们对控制 DNA 拷贝增益的分子特征以及相关的了解 异质性，这将解决新的生物标志物和治疗靶点，以控制拷贝数- 未来几年的相关疾病。