Investigating the Role of Transcription on DNA Damage at Early Replicating Fragile Sites

研究转录对早期复制脆弱位点 DNA 损伤的作用

• 批准号: 9257699
• 负责人: Commodore Perry St Germain
• 金额: $ 3.55万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9257699
• 关键词: Area; B-Lymphocytes; Cancerous; Cells; Characteristics; Chromosome Fragile Sites; Chromosome Fragility; Chromosome abnormality; Clustered Regularly Interspaced Short Palindromic Repeats; Conflict (Psychology); DNA; DNA Damage; DNA Sequence Rearrangement; DNA biosynthesis; DNA lesion; DNA replication fork; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Data; Deoxyuridine; Detection; Fluorescent in Situ Hybridization; Follicular Lymphoma; Gene Fusion; Genes; Genetic Transcription; Genome; Genomic Instability; Genomics; Goals; Immunoprecipitation; Karyotype; Label; Lead; Lesion; Location; Malignant Neoplasms; Maps; Mature B-Lymphocyte; Measures; Methods; Monitor; Mus; Mutation; Play; Recurrence; Role; S phase; STAT6 Transcription Factor; Sampling; Site; Source; Stress; System; Testing; Transcription Coactivator; Transcription Repressor; Transcriptional Regulation; Tumor Subtype; abstracting; cancer prevention; cancer therapy; experience; insight; large cell Diffuse non-Hodgkin' s lymphoma; novel; programs; repaired; research study; tool; tumor

Project Summary/Abstract Genome instability is a hallmark of cancer. This instability initiates with a DNA lesion that is unfaithfully repaired. Replication stress is a major source of these lesions and transcription greatly enhances replication stress. Early replicating fragile sites (ERFS) are specific locations in the genome that experience recurrent DNA damage in early S-phase in the presence of replication stress. When ERFS were mapped in mouse primary B-cells, it was found that they are found in in gene rich areas and largely overlap with recurrent sites of amplifications and deletions in diffuse large B-cell lymphoma. It is not known why these sites are prone to DNA damage at ERFS or in the cancer. It was shown at one site that a decrease in transcription decreased chromosome fragility. It has been previously shown that collisions between replication forks (RF) and RNA polymerase (RNAP) are mutagenic. Transcriptional dysregulation in early replicating regions of the genome may play a role in this chromosome fragility. The hypothesis is that an increase in transcriptional activity in early replicating regions will lead to elevated levels of DNA damage due to the spatial and temporal co-localization of RF and RNAP. These experiments will be performed in primary mouse B-cells, as their distinct transcriptional profile and normal karyotype will allow us to accurately document deviations from wild type. The first aim, to test the hypothesis, will be modulating transcription at early replicating regions by targeting a catalytically defective CRISPR-dCas9 tethered to either transcriptional activators or repressors. This will allow the modulation of transcription at specific loci. The DNA damage will be measured by observing chromosomal abnormalities at the same loci using fluorescence in situ hybridization (FISH). This approach will show how alterations in transcriptional activity influence the levels of DNA damage in early replicating regions. The second aim, to test the hypothesis, will be finding sites in the genome that have both RF and RNAP co-localized in early S-phase. To locate RF and RNAP spatially and temporally co- localizing, I will perform a sequential immunoprecipitation (IP) for RNAP and nascent DNA, labeled with 5-ethynyl-2'-deoxyuridine The DNA at these locations will be sequenced and the DNA damage will be quantified using FISH. The sequential IP experiment will provide us with genomic loci that have both RF and RNAP co-localized in early S-phase. Together the data will suggest that changes in transcriptional programming leads to conflicts between RF and RNAP generating recurrent DNA damage observed in cancer.

项目总结/摘要 基因组不稳定是癌症的标志。这种不稳定性始于DNA损伤， 不忠实的修复。复制应激是这些损伤和转录的主要来源 大大增强了复制压力。早期复制脆弱位点（ERFS）是指特定的位置 在基因组中，在S期早期， 复制应力当在小鼠原代B细胞中定位ERFS时，发现它们 在基因丰富的区域发现，并且在很大程度上与扩增的重复位点重叠， 弥漫性大B细胞淋巴瘤中的缺失。目前尚不清楚为什么这些位点容易出现DNA ERFS或癌症中的损伤。在一个位点显示， 降低染色体脆性。先前已经表明， 复制叉（RF）和RNA聚合酶（RNAP）是诱变性的。转录 基因组早期复制区域的失调可能在该染色体中起作用 脆弱这一假说认为，早期复制区转录活性的增加 由于RF的空间和时间共定位， 和RNAP。这些实验将在原代小鼠B细胞中进行，因为它们的不同之处在于， 转录谱和正常核型将使我们能够准确地记录偏离 野生型第一个目标，为了验证这一假设，将在早期调节转录。 通过靶向连接至以下任一者的催化缺陷型CRISPR-dCas 9来复制区域： 转录激活因子或抑制因子。这将允许在特定的转录水平上调节转录， 的位点DNA损伤将通过同时观察染色体异常来测量。 使用荧光原位杂交（FISH）检测基因座。这种方法将显示如何改变 转录活性影响早期复制区域的DNA损伤水平。的 第二个目标是检验这一假设，将在基因组中找到既有RF又有 RNAP共定位于早期S期。为了在空间和时间上共同定位RF和RNAP， 定位后，我将对RNAP和新生DNA进行连续免疫沉淀（IP）， 用5-乙炔基-2 '-脱氧尿苷标记。 将使用FISH定量DNA损伤。连续IP实验将为我们提供 RF和RNAP共同定位于早期S期的基因组位点。这些数据将 表明转录编程的变化导致RF和 RNAP产生癌症中观察到的复发性DNA损伤。