OXIDATIVE STRESS AND BASE MODIFICATIONS IN REGULATORY DNA

调控 DNA 中的氧化应激和碱基修饰

基本信息

批准号：
10153820
负责人：
Cynthia J Burrows
金额：
$ 30.5万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-01 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10153820
关键词：
Address Alzheimer&apos s Disease Antibodies Base Excision Repairs Binding Sites Breast Cancer cell line Cancerous Cell Culture Techniques Cell Line Cells Chemicals Chemistry Chromatin Colon Coupled CpG Islands DNA DNA Modification Process DNA Polymerase II DNA lesion DNA sequencing Data Diabetes Mellitus Disease Enzymes Epigenetic Process Exposure to Flavins G-Quartets Gene Expression Genes Genome Genomic DNA Glioblastoma Guanine Helicobacter pylori Heme Histone Acetylation Histone Deacetylase Inhibitor Histones Hydrogen Peroxide Hypersensitivity Infection Inflammation Inflammatory Response Injury Introns KDM1A gene Laboratories Lead Lesion Ligation Link Location Lysine Malignant Neoplasms Metabolic Diseases Metabolism Methods Mission Mitochondrial DNA Modification Monitor Mutagenesis Nucleic Acid Regulatory Sequences Nucleotides OGG1 gene Outcome Oxidative Stress Oxides Pathway interactions Plasmids Play Process Promoter Regions Public Health RNA Radiation Reactive Oxygen Species Research Resolution Role Site Small Interfering RNA Structure TNF gene Toxic Environmental Substances Transcript Traumatic Brain Injury United States National Institutes of Health Untranslated Regions Work age related base bisulfite sequencing chromatin remodeling experimental study gene induction genome sequencing genomic data knock-down mammalian genome member methylation pattern nervous system disorder next generation sequencing novel overexpression oxidation promoter stressor transcription factor transcriptome sequencing whole genome

项目摘要

ABSTRACT Reactive oxygen species (ROS) with the potential to modify genomic and mitochondrial DNA are formed from exposure to radiation, environmental toxins, and endogenous chemistries resulting from inflammation, allergies, injury and metabolism. Oxidative stress leads to myriad effects on the cell, notably changes in gene expression and mutagenesis, and ultimately contributes to numerous age- related diseases including cancer, neurological disorders, diabetes and others. Recent work in this laboratory has shown that the common guanine oxidation product 8-oxo-7,8-dihydroguanine (OG) is not only a pre-mutagenic lesion, but also a base modification that impacts gene expression when present in potential G-quadruplex-forming sequences of regulatory regions of the genome. This epigenetic-like DNA modification is proposed to be written by chromatin remodeling in addition to exposure to ROS, while it is read and erased by the base excision repair (BER) pathway, including OGG1 and APE1, critical members of BER. The specific aims of this project will first augment recent results toward whole- genome sequencing of DNA for OG at single-nucleotide resolution via “OG-Seq” and related methods for other modifications read by BER. The approach uses a novel chemical ligation strategy that is shown to be superior to OG antibodies in providing unbiased pull-down of OG-containing fragments for PCR amplification and next-generation sequencing. The sequencing methods will be applied in Aim 2 to address the hypothesis that base modifications resulting from oxidative stress are non-uniformly distributed in the genome, that G-quadruplex sequences are hotspots for oxidation, and that they subsequently impact gene induction. Both cancerous and noncancerous cell lines will be studied using various types of oxidative stress that mimic, for example, the inflammatory response or heme overload. The impact of chromatin remodeler LSD1 and chromatin opening via HDAC inhibitors will be examined in Aim 3. The overall outcome will be a deeper understanding of the relationship between oxidative stress and disease by defining where and when these epigenetic-like modifications occur in DNA.

抽象的具有修饰基因组和线粒体DNA的潜力的活性氧（ROS）被形成从暴露于辐射，环境毒素和内源性化学因素炎症，过敏，伤害和代谢。氧化应激会对细胞产生无数的影响，显着的基因表达和诱变的变化，并最终导致了许多年龄相关疾病，包括癌症，神经系统疾病，糖尿病等。最近的工作实验室表明，普通的鸟嘌呤氧化产物8-氧-7,8-二氢甘氨酸（OG）不是仅是杀虫剂前病变，但也是一种基础修饰，会影响基因表达基因组调节区域的潜在G四链体形成序列。这种表观遗传状提议除了暴露于ROS之外，还建议通过染色质重塑编写DNA修饰。虽然基本的惊喜维修（BER）途径（包括OGG1和APE1）读取和擦除，但 Ber的重要成员。该项目的具体目的将首先增强最新成果通过“ OG-SEQ”和相关方法的单核苷酸分辨率在单核苷酸分辨率下进行DNA的基因组测序 Ber读取的其他修改。该方法使用一种新型的化学连接策略，该策略被证明在为PCR提供公正的含OG的片段下拉的下拉，要优于OG抗体放大和下一代测序。测序方法将应用于目标2 解决以下假设，即氧化应激产生的基础修饰是不均匀的分布在基因组中，g四链体序列是用于氧化的热点，它们是随后影响基因诱导。取消和非癌细胞系都将使用模仿炎症反应或血红素过载的各种类型的氧化应激。将检查通过HDAC抑制剂开放染色质重塑剂LSD1和染色质的影响目标3。总体结果将是对氧化应激之间关系的更深入的了解通过定义这些表观遗传样的修饰发生在DNA中的何时何地。