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

基本信息

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
项目类别：
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&apos s lymphoma novel repaired tool tumor

项目摘要

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.

项目概要/摘要基因组不稳定是癌症的一个标志。这种不稳定性始于 DNA 损伤，即不忠实地修复。复制压力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) 是特定位置在存在以下因素的情况下，在早期 S 期经历反复 DNA 损伤的基因组复制压力。当 ERFS 在小鼠原代 B 细胞中进行定位时，发现它们发现于基因丰富的区域，并且与扩增的重复位点很大程度上重叠弥漫性大 B 细胞淋巴瘤的缺失。目前尚不清楚为什么这些位点容易出现 DNA ERFS 或癌症损伤。在一个位点显示转录减少染色体脆性降低。之前已经表明，之间的碰撞复制叉 (RF) 和 RNA 聚合酶 (RNAP) 具有诱变性。转录的基因组早期复制区域的失调可能在该染色体中发挥作用脆弱性。该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。这些实验将在原代小鼠 B 细胞中进行，因为它们具有独特的转录谱和正常核型将使我们能够准确记录与野生型。第一个目标是检验这一假设，即在早期调节转录。通过靶向连接有催化缺陷的 CRISPR-dCas9 来复制区域转录激活子或阻遏子。这将允许在特定的转录调节基因座。 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）的基因座。这种方法将展示如何改变转录活性影响早期复制区域的 DNA 损伤水平。这第二个目标是检验这一假设，寻找基因组中同时具有 RF 和 RF 的位点。 RNAP 共定位于早期 S 期。定位 RF 和 RNAP 的空间和时间协同定位后，我将对 RNAP 和新生 DNA 进行连续免疫沉淀 (IP)，用 5-乙炔基-2'-脱氧尿苷标记这些位置的 DNA 将被测序，并且 DNA 损伤将使用 FISH 进行定量。顺序 IP 实验将为我们提供 RF 和 RNAP 共定位于早期 S 期的基因组位点。数据一起将表明转录编程的变化导致 RF 和在癌症中观察到 RNAP 会产生反复的 DNA 损伤。