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.
The proposed research is relevant to public health because it will provide a method for sequencing the genome for the exact sequence locations of DNA lesions induced by oxidative stress. Oxidative stress is a causative agent in age-related disorders including cancer, diabetes, and Alzheimer?s disease, as well as playing a role in injury and inflammation-related disorders such as traumatic brain injury (TBI). The project is therefore relevant to NIH?s mission to understand the interplay of oxidative stress and gene expression and their roles in disease.
