Reactive oxygen species (ROS), generated in mitochondria and cytosol, globally induce single-strand breaks (SSBs) and oxidized bases in the genome. Previously unrecognized, localized oxidative damage in promoter/enhancer regions, are induced by nuclear ROS produced during oxidative demethylation of methyl Lys/Arg in histones H3/H4 by lysine specific demethylase (LSD1in KMD family), and Jumanji (JMJ) family enzymes, and of methyl CpG sites in promoter sequences by TET dioxygenases during transcriptional activation. Ligands including estrogen (E2), retinoic acid (RA) and TNF? activate hundreds of target genes by binding to cognate receptors, which then form multi-protein complexes to unfold chromatin via complex histone modifications and CpG demethylation as a prerequisite for transcription initiation. We observed transient formation of single-strand breaks (SSBs), localized to the cis elements, presumably caused by ROS products of demethylases followed by repair which is initiated by SSB sensor PARP1, XRCC1 and end processing enzymes, e.g., APE1, PNKP. By monitoring site-specific repair using quantitative PCR, global genome repair by alkaline Comet assay and recruitment of repair proteins at the enhancer site via ChIP analysis, we observed that genome damage and repair rates are fast (completed in 10 min) for E2 activation of the BCL2 gene, moderate (~1 h) for TNF? activation of IL1? promoter and slow for RA activation of the RAR?2 gene (~4 h). Co-IP analysis showed that RA activation increased interaction between LSD1 and APE1and ChIP assay confirmed increased recruitment of PARP1 and other SSBR proteins at the enhancer site. This universal phenomenon of regulatory region specific damage induction and repair during gene activation has implications in damage signaling and toxicity which we will characterize by pursuing the following aims.
Aim 1. To test the hypothesis that enhancer/promoter-specific oxidized bases and SSBs in BCL2, IL1? and RAR?2 genes and their repair are universally induced during gene activation. We will determine relative contribution of histone vs. CpG demethylation to the SSB production, elucidate the mechanism of recruitment of repair proteins and characterize the repair sub-pathways and end processing enzymes recruited at the SSB site.
Aim 2. To test the hypothesis that repair proteins are recruited at promoter/enhancer sites by demethylases to form repair complexes. We will characterize complexes of demethylases with repair proteins, possibly including those of nucleotide excision repair.
Aim 3. To unravel the role of PARP1 in repair of promoter/enhancer-specific SSBs induced during gene activation. We will test if PARP inhibition or depletion abrogates repair and transcriptional activation in all systems, and if SSB repair inhibition induces apoptosis after transcription activation only in replicating cells. Our state-of the-art approaches and sophisticated reagents will establish a new paradigm about gene activation-dependent genome damage and repair with therapeutic potential.

Public Health Relevance

Deficiency in single-strand break (SSB) repair in the human genome has been etiologically linked to many diseases. This project by elucidating the link between gene activation and genome damage leading to various diseases may also illuminate the role of PARP in SSB repair that could help identify new therapeutic targets for cancer. Thus comprehensive understanding of SSB formation induced during gene activation and its repair has profound health relevance.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Cancer Etiology Study Section (CE)
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Willis, Kristine Amalee
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Methodist Hospital Research Institute
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Sengupta, Shiladitya; Yang, Chunying; Hegde, Muralidhar L et al. (2018) Acetylation of oxidized base repair-initiating NEIL1 DNA glycosylase required for chromatin-bound repair complex formation in the human genome increases cellular resistance to oxidative stress. DNA Repair (Amst) 66-67:1-10
Rangaswamy, Suganya; Pandey, Arvind; Mitra, Sankar et al. (2017) Pre-Replicative Repair of Oxidized Bases Maintains Fidelity in Mammalian Genomes: The Cowcatcher Role of NEIL1 DNA Glycosylase. Genes (Basel) 8:
Dutta, Arijit; Eckelmann, Bradley; Adhikari, Sanjay et al. (2017) Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex. Nucleic Acids Res 45:2585-2599
Yang, Chunying; Sengupta, Shiladitya; Hegde, Pavana M et al. (2017) Regulation of oxidized base damage repair by chromatin assembly factor 1 subunit A. Nucleic Acids Res 45:739-748
Hegde, Muralidhar L; Dutta, Arijit; Yang, Chunying et al. (2016) Scaffold attachment factor A (SAF-A) and Ku temporally regulate repair of radiation-induced clustered genome lesions. Oncotarget 7:54430-54444
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Hegde, Pavana M; Dutta, Arijit; Sengupta, Shiladitya et al. (2015) The C-terminal Domain (CTD) of Human DNA Glycosylase NEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome: DOMINANT NEGATIVE FUNCTION OF THE CTD. J Biol Chem 290:20919-33