Deciphering Networks Controlling DNA Amplification

项目总结 DNA扩增与病理状态有关，如神经疾病、心脏病和 癌症。在癌症中，至少有50%的扩增是瞬时染色体外DNA(EcDNA)。《过客》 行为导致拷贝数可塑性，导致癌基因异质性表达和改变 治疗反应。关于不同的机制是否控制ecDNA复制仍然是一个悬而未决的问题 细胞内的获得以及它们如何影响与疾病相关的复制获得事件。我的总体目标是定义 调节选择性DNA复制获得和相关的可塑性的原理，所以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间定点dna复制的分子基础的实验室。 人类基因组中的收益(TSSG)。具体地说，我们确定了第一种能够驱动特定部位的酶 EcDNA扩增[组蛋白3赖氨酸9和36三甲基酶(H3K9/36me3)KDM4A]，证实了 表观遗传状态在控制特定DNA区域倾向于重新复制和 放大。我们又发现了七种染色质酶--赖氨酸甲基转移酶(KMTs)和去甲基酶 (KDM)-在非癌症细胞和癌细胞中协同控制位点特异性扩增的功能。这些 研究证实了染色质因子及其相关状态在调节DNA中的关键作用 放大。有了NIGMS R35，我的实验室将扩展我们的研究，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；以及3)ecDNA的产生与 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- 以遗传和化学工具为基础的筛选，以识别关键放大器，进而产生 表观基因组图谱与与这些途径相关的基因组组织图相连，因此分子 解决了控制DNA扩增的特征。这些研究还将与最先进的Long相结合 阅读测序和单细胞(DNA和RNA)测序策略，以便相关的异质性 在细胞群体和单个细胞内，可以与放大器对TSSG的影响相关联。这些 正在对具有接近二倍体基因组的未转化细胞进行研究，因此额外的 基因组异常和突变不会影响这些研究。总体而言，从这些数据中生成的数据 研究将增加我们对控制DNA复制获得的分子特征和相关的 异质性，将解决新的生物标记物和治疗靶点，以控制拷贝数- 未来几年可能会出现相关疾病。

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.