Wednesday, February 5, 2025
2/5/2025
Project Summary/Abstract Excess reactive oxygen species (ROS) arising from endogenous sources and environmental exposures, such as air pollution, chemicals and smoking, can cause oxidative stress, a detrimental condition linked to age-related diseases and cancer in humans. Oxidative stress damages the entire cell, but is markedly harmful for DNA, with the lesion 8-oxoguanine (8oxoG) being particularly prevalent. Cells adopt multiple mechanisms to maintain homeostasis, including mutagenesis prevention and transcriptome reprogramming. Guanine-rich sequences are enriched at telomeres and regulatory regions of the genome and are therefore, hot spots for 8oxoG formation. Telomeres, the nucleoprotein caps which protect linear chromosomes, are transcribed into long non-coding telomere repeat-containing RNAs (TERRA). 8oxoG formation and base excision repair processing at regulatory sequences, have been implicated in gene expression regulation. However, elucidating the mechanisms of transcriptional modulation by 8oxoG remains challenging, because oxidants used to induce oxidative stress damage multiple cell components and generate diverse DNA adducts. Our group validated a fluorogen activating peptide (FAP) system which produces singlet oxygen when bound to a photosensitizer dye and excited with far- red light, converting guanine into 8oxoG. We report 8oxoG production at telomeres with high spatial and temporal control by fusing the FAP with telomere protein TRF1. For this proposal, FAP has been adapted to target 8oxoG to specific promoter sequences (dead Cas9-FAP) or genome-wide (H2B-FAP). The goal of this proposal is to test the hypothesis that the processing of 8oxoG in G-rich genomic regions and promoters regulates transcriptional responses to oxidative genotoxic damage. During the mentored K99 phase, Aim 1 will delineate how 8oxoG damage at telomeres regulates the dynamics of TERRA transcription, stability and epigenetic modifications, and how these changes affect telomere homeostasis. During this phase, I will take advantage of resources at the University of Pittsburgh for professional development, to apply these skills through research, data presentation, and writing opportunities. During the independent R00 phase, I will apply technical skills obtained from additional training with key collaborators and models validated during the K99 phase to elucidate how 8oxoG regulates transcription of specific oxidative stress-responsive genes at the endogenous loci in cells (Aim 2, dCas-FAP). Also, during this phase I will extend these approaches genome-wide, employing RNA-seq, OG-seq and ChIP-seq to elucidate the coupling between 8oxoG processing and gene expression in response to singlet oxygen produced at chromatin (Aim 3, H2B-FAP). These experiments will provide me with the data required for an early independent publication and preliminary data for research proposals. Importantly, during the R00 phase I will develop independence from my mentor by focusing on the dynamic interplay between 8oxoG repair and gene expression regulation.
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