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: 9456514
• 负责人: Commodore Perry St Germain
• 金额: $ 3.65万
• 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/9456514
• 关键词: Area; B-Lymphocytes; CRISPR/Cas technology; Cancerous; Cells; Chromosome Fragile Sites; Chromosome Fragility; Chromosome abnormality; Clustered Regularly Interspaced Short Palindromic Repeats; Conflict (Psychology); DNA; DNA Damage; DNA Replication 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/Corepressor; Transcriptional Regulation; Tumor Subtype; cancer cell; cancer prevention; cancer therapy; experience; experimental study; insight; large cell Diffuse non-Hodgkin' s lymphoma; novel; repaired; 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.

项目摘要/摘要 基因组不稳定性是癌症的标志。这种不稳定性以DNA病变的启动 不忠实的维修。复制应力是这些病变和转录的主要来源 大大增加了复制应力。早期复制脆弱的位点（ERF）是特定位置 在存在的基因组中，在存在的早期S阶段中经历了复发性DNA损伤 复制应力。当将ERF映射到小鼠初级B细胞中时，发现它们 在富含基因的地区发现，并在很大程度上与复发的放大位点重叠 弥漫性大B细胞淋巴瘤中的缺失。尚不清楚为什么这些位点容易出现DNA ERF或癌症的损害。在一个地点显示了转录的减少 染色体脆弱性降低。以前已经证明 复制叉（RF）和RNA聚合酶（RNAP）是诱变的。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 脆弱性。假设是早期复制区域转录活性的增加 由于RF的空间和时间共定位，将导致DNA损伤水平升高 和rnap。这些实验将在主要小鼠B细胞中进行，因为它们的独特 转录轮廓和正常的核型将使我们能够准确记录与 野生型。检验假设的第一个目的将在早期调节转录 通过靶向催化有缺陷的CRISPR-DCAS9来复制区域 转录激活因子或阻遏物。这将允许在特定的 基因座。 DNA损伤将通过观察相同的染色体异常来衡量 使用荧光原位杂交（FISH）的基因座。这种方法将显示如何改变 转录活性会影响早期复制区域中DNA损伤的水平。这 检验假设的第二个目的是在基因组中找到具有RF和RF的位点 RNAP在早期S期共定位。要在空间和时间上找到RF和RNAP 本地化，我将对RNAP和新生DNA进行连续免疫沉淀（IP）， 用5-乙基-2'-脱氧尿苷标记的DNA将在这些位置进行测序，并将 DNA损伤将使用鱼类量化。顺序IP实验将为我们提供 具有RF和RNAP在早期S期共定位的基因组基因局。数据将在一起 提示转录编程的变化会导致RF和 RNAP在癌症中观察到的复发性DNA损伤。