Extreme genomic instability at large transcribed genes: mechanisms and consequences for the cancer genome

项目总结/摘要 染色体重排是诱变的一种基本形式，对人类的遗传学有着深远的影响。 健康结构突变的过程在体细胞中是活跃的，在那里它们最终 导致癌症。来自癌症基因组图谱（TCGA）和其他项目的大量基因组数据揭示了 染色体内重排特别常见，某些基因座高度倾向于 他们的出现。因此，不稳定基因座的性质和机制对癌症至关重要 病因学我们正在使用获得性基因组拷贝数变异（CNV）模型来探索这些问题，这是一种 术语包括间隙性缺失和重复，并代表与复制相同的机制。 数中性倒位和易位。在这些模型中，外源性复制应激以低- 剂量的阿非迪霉素或羟基脲是培养的体细胞中新CNV的有效诱导剂。CNV是 其特征在于典型的致病性重排的微同源连接， 复制错误。诱导CNV形成的热点与常见的脆性位点是相同的位点， 大基因的活跃转录，导致其极端的细胞类型特异性不稳定性。该项目探索 假设在这些外源性体细胞CNV模型中观察到相同的不稳定机制， 作为内源性复制应激结果，诱导导致癌症中的复发性基因组改变。这个想法 在三个目标中进行了测试，这些目标研究了导致极端轨迹不稳定性的机制 转录基因和这些机制对癌症基因组的影响。目标1和2地址 关于转录如何与复制应激相互作用以赋予基因座不稳定性的非排他性假设。要求1 他认为，大基因会产生一种动态冲突，在这种冲突中，转录进入S期会消除后期启动 复制起始并产生对复制抑制高度敏感的大复制子。新方法将 通过确定复制、转录和起点存在的细胞周期时间来检验这一假设， 击发目的2认为，转录导致持续的R环，导致分叉停滞，从而导致前体 CNV的形成。监测和操纵在表达β-内酰胺酶的细胞系中的R环形成 特定的大基因将检验这一假设。目标3涉及这些机制对 癌症基因组首先通过TCGA癌症数据集的生物信息学探索来关联缺失热点 肿瘤和癌症类型特异性转录。强制性癌基因激活的组织培养模型和 结肠癌的小鼠模型将使新生CNV形成与内源性复制应激相关 是癌症固有的

PROJECT SUMMARY/ABSTRACT Chromosomal rearrangements are a fundamental form of mutagenesis with a profound impact on human health. Poorly understood processes of structural mutagenesis are active in somatic cells where they ultimately lead to cancer. A flood of genomic data from The Cancer Genome Atlas (TCGA) and other projects is revealing that intrachromosomal rearrangements are especially common and that certain genomic loci are highly prone to their occurrence. The nature and mechanisms of unstable loci are thus of central importance to cancer etiology. We are exploring these questions using models of acquired genomic copy number variants (CNVs), a term encompassing interstitial deletions and duplications and representing the same mechanisms as copy- number-neutral inversions and translocations. In these models, exogenous replication stress in the form of low- dose aphidicolin or hydroxyurea is a potent inducer of new CNVs in cultured somatic cells. CNVs are characterized by microhomologous junctions typical of pathogenic rearrangements that likely arise as replication errors. Hotspots of induced CNV formation are the same loci as common fragile sites, and it is the active transcription of large genes that leads to their extreme cell-type-specific instability. This project explores the hypothesis that the same instability mechanism(s) observed in these models of exogenous somatic CNV induction lead to recurrent genomic alterations in cancer as a result of endogenous replication stress. This idea is tested in three aims that examine the mechanisms leading to the extreme locus instability at large transcribed genes and the consequences of these mechanisms for the cancer genome. Aims 1 and 2 address non-exclusive hypotheses for how transcription interacts with replication stress to confer locus instability. Aim 1 argues that large genes create a dynamic conflict in which transcription into S-phase removes late-firing replication origins and creates large replicons highly sensitive to replication inhibition. Novel approaches will test this hypothesis by determining the cell-cycle timing of replication, transcription, and origin presence and firing. Aim 2 argues that transcription leads to persistent R-loops that cause fork stalling and thus precursor lesions for CNV formation. Monitoring and manipulating R-loop formation in cell lines that variably express specific large genes will test this hypothesis. Aim 3 addresses the consequences of these mechanisms on the cancer genome first through bioinformatic explorations of TCGA cancer data sets to correlate deletion hotspots with tumor- and cancer-type-specific transcription. Tissue-culture models of forced oncogene activation and mouse models of colon cancer will relate de novo CNV formation with the endogenous replication stress inherent to cancer.