ABSTRACT
Oxidative genome damage is an unavoidable consequence of cellular metabolism. It arises at gene regulatory elements by epigenetic demethylation during transcriptional activation1,2. Here we show that promoters are protected from oxidative damage via a process mediated by the nuclear mitotic apparatus protein NuMA (also known as NUMA1). NuMA exhibits genomic occupancy approximately 100 bp around transcription start sites. It binds the initiating form of RNA polymerase II, pause-release factors and single-strand break repair (SSBR) components such as TDP1. The binding is increased on chromatin following oxidative damage, and TDP1 enrichment at damaged chromatin is facilitated by NuMA. Depletion of NuMA increases oxidative damage at promoters. NuMA promotes transcription by limiting the polyADP-ribosylation of RNA polymerase II, increasing its availability and release from pausing at promoters. Metabolic labelling of nascent RNA identifies genes that depend on NuMA for transcription including immediate-early response genes. Complementation of NuMA-deficient cells with a mutant that mediates binding to SSBR, or a mitotic separation-of-function mutant, restores SSBR defects. These findings underscore the importance of oxidative DNA damage repair at gene regulatory elements and describe a process that fulfils this function.
Subject(s)
Cell Cycle Proteins , DNA Damage , DNA Repair , Oxidative Stress , Promoter Regions, Genetic , Cell Cycle Proteins/metabolism , Chromatin/genetics , Genes , Genetic Complementation Test , Mitosis , Mutation , Oxidative Stress/genetics , Phosphoric Diester Hydrolases/metabolism , Poly ADP Ribosylation , Promoter Regions, Genetic/genetics , RNA/biosynthesis , RNA/genetics , RNA Polymerase II/metabolism , Spindle Apparatus/metabolism , Transcription Initiation SiteABSTRACT
R-loops are by-products of transcription that must be tightly regulated to maintain genomic stability and gene expression. Here, we describe a mechanism for the regulation of the R-loop-specific helicase, senataxin (SETX), and identify the ubiquitin specific peptidase 11 (USP11) as an R-loop regulator. USP11 de-ubiquitinates SETX and its depletion increases SETX K48-ubiquitination and protein turnover. Loss of USP11 decreases SETX steady-state levels and reduces R-loop dissolution. Ageing of USP11 knockout cells restores SETX levels via compensatory transcriptional downregulation of the E3 ubiquitin ligase, KEAP1. Loss of USP11 reduces SETX enrichment at KEAP1 promoter, leading to R-loop accumulation, enrichment of the endonuclease XPF and formation of double-strand breaks. Overexpression of KEAP1 increases SETX K48-ubiquitination, promotes its degradation and R-loop accumulation. These data define a ubiquitination-dependent mechanism for SETX regulation, which is controlled by the opposing activities of USP11 and KEAP1 with broad applications for cancer and neurological disease.
Subject(s)
DNA Helicases/genetics , DNA/genetics , Kelch-Like ECH-Associated Protein 1/genetics , Multifunctional Enzymes/genetics , Protein Processing, Post-Translational , Proteostasis/genetics , RNA Helicases/genetics , Thiolester Hydrolases/genetics , Cell Line , Cellular Senescence/genetics , DNA/chemistry , DNA/metabolism , DNA Helicases/antagonists & inhibitors , DNA Helicases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , HEK293 Cells , Humans , Kelch-Like ECH-Associated Protein 1/antagonists & inhibitors , Kelch-Like ECH-Associated Protein 1/metabolism , Multifunctional Enzymes/antagonists & inhibitors , Multifunctional Enzymes/metabolism , Nucleic Acid Conformation , Promoter Regions, Genetic , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Stability , Proteolysis , RNA Helicases/antagonists & inhibitors , RNA Helicases/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Thiolester Hydrolases/antagonists & inhibitors , Thiolester Hydrolases/metabolism , UbiquitinationABSTRACT
As we age, our bodies accrue damage in the form of DNA mutations. These mutations lead to the generation of sub-optimal proteins, resulting in inadequate cellular homeostasis and senescence. The build-up of senescent cells negatively affects the local cellular micro-environment and drives ageing associated disease, including neurodegeneration. Therefore, limiting the accumulation of DNA damage is essential for healthy neuronal populations. The naked mole rats (NMR) are from eastern Africa and can live for over three decades in chronically hypoxic environments. Despite their long lifespan, NMRs show little to no biological decline, neurodegeneration, or senescence. Here, we discuss molecular pathways and adaptations that NMRs employ to maintain genome integrity and combat the physiological and pathological decline in organismal function.
Subject(s)
Adaptation, Physiological/genetics , Cellular Senescence/genetics , DNA Damage/genetics , Oxidative Stress/genetics , Aging/genetics , Animals , DNA/genetics , Homeostasis , Mole Rats/genetics , Oxidative Stress/physiologyABSTRACT
Hexanucleotide repeat expansions represent the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, though the mechanisms by which such expansions cause neurodegeneration are poorly understood. We report elevated levels of DNA-RNA hybrids (R-loops) and double strand breaks in rat neurons, human cells and C9orf72 ALS patient spinal cord tissues. Accumulation of endogenous DNA damage is concomitant with defective ATM-mediated DNA repair signaling and accumulation of protein-linked DNA breaks. We reveal that defective ATM-mediated DNA repair is a consequence of P62 accumulation, which impairs H2A ubiquitylation and perturbs ATM signaling. Virus-mediated expression of C9orf72-related RNA and dipeptide repeats in the mouse central nervous system increases double strand breaks and ATM defects and triggers neurodegeneration. These findings identify R-loops, double strand breaks and defective ATM-mediated repair as pathological consequences of C9orf72 expansions and suggest that C9orf72-linked neurodegeneration is driven at least partly by genomic instability.
