RESUMEN
RNA helicase DHX9 is essential for genome stability by resolving aberrant R-loops. However, its regulatory mechanisms remain unclear. Here we show that SUMOylation at lysine 120 (K120) is crucial for DHX9 function. Preventing SUMOylation at K120 leads to R-loop dysregulation, increased DNA damage, and cell death. Cells expressing DHX9 K120R mutant which cannot be SUMOylated are more sensitive to genotoxic agents and this sensitivity is mitigated by RNase H overexpression. Unlike the mutant, wild-type DHX9 interacts with R-loop-associated proteins such as PARP1 and DDX21 via SUMO-interacting motifs. Fusion of SUMO2 to the DHX9 K120R mutant enhances its association with these proteins, reduces R-loop accumulation, and alleviates survival defects of DHX9 K120R. Our findings highlight the critical role of DHX9 SUMOylation in maintaining genome stability by regulating protein interactions necessary for R-loop balance.
Asunto(s)
ARN Helicasas DEAD-box , Inestabilidad Genómica , Estructuras R-Loop , Sumoilación , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Humanos , Células HEK293 , Daño del ADN , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Lisina/metabolismo , Mutación , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas de NeoplasiasRESUMEN
Chromatin organization and dynamics play important roles in governing the regulation of nuclear processes of biological cells. However, due to the constant diffusive motion of chromatin, examining chromatin nanostructures in living cells has been challenging. In this study, we introduce interferometric scattering correlation spectroscopy (iSCORS) to spatially map nanoscopic chromatin configurations within unlabeled live cell nuclei. This label-free technique captures time-varying linear scattering signals generated by the motion of native chromatin on a millisecond timescale, allowing us to deduce chromatin condensation states. Using iSCORS imaging, we quantitatively examine chromatin dynamics over extended periods, revealing spontaneous fluctuations in chromatin condensation and heterogeneous compaction levels in interphase cells, independent of cell phases. Moreover, we observe changes in iSCORS signals of chromatin upon transcription inhibition, indicating that iSCORS can probe nanoscopic chromatin structures and dynamics associated with transcriptional activities. Our scattering-based optical microscopy, which does not require labeling, serves as a powerful tool for visualizing dynamic chromatin nano-arrangements in live cells. This advancement holds promise for studying chromatin remodeling in various crucial cellular processes, such as stem cell differentiation, mechanotransduction, and DNA repair.
Asunto(s)
Cromatina , Cromatina/metabolismo , Cromatina/química , Humanos , Análisis Espectral/métodos , Interferometría/métodos , Ensamble y Desensamble de Cromatina , Núcleo Celular/metabolismoRESUMEN
Aberrant DNA/RNA hybrids (R-loops) formed during transcription and replication disturbances pose threats to genome stability. DHX9 is an RNA helicase involved in R-loop resolution, but how DHX9 is regulated in response to genotoxic stress remains unclear. Here we report that DHX9 is phosphorylated at S321 and S688, with S321 phosphorylation primarily induced by ATR after DNA damage. Phosphorylation of DHX9 at S321 promotes its interaction with γH2AX, BRCA1 and RPA, and is required for its association with R-loops under genotoxic stress. Inhibition of ATR or expression of the non-phosphorylatable DHX9S321A prevents DHX9 from interacting with RPA and R-loops, leading to the accumulation of stress-induced R-loops. Furthermore, depletion of RPA reduces the association between DHX9 and γH2AX, and in vitro binding analysis confirms a direct interaction between DHX9 and RPA. Notably, cells with the non-phosphorylatable DHX9S321A variant exhibit hypersensitivity to genotoxic stress, while those expressing the phosphomimetic DHX9S321D variant prevent R-loop accumulation and display resistance to DNA damage agents. In summary, we uncover a new mechanism by which ATR directly regulates DHX9 through phosphorylation to eliminate stress-induced R-loops.
Asunto(s)
Estructuras R-Loop , Serina , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Daño del ADN , Replicación del ADN , Fosforilación , ARN/metabolismo , Serina/metabolismo , HumanosRESUMEN
The genome consists of non-B-DNA structures such as G-quadruplexes (G4) that are involved in the regulation of genome stability and transcription. Telomeric-repeat containing RNA (TERRA) is capable of folding into G-quadruplex and interacting with chromatin remodeler ATRX. Here we show that TERRA modulates ATRX occupancy on repetitive sequences and over genes, and maintains DNA G-quadruplex structures at TERRA target and non-target sites in mouse embryonic stem cells. TERRA prevents ATRX from binding to subtelomeric regions and represses H3K9me3 formation. G4 ChIP-seq reveals that G4 abundance decreases at accessible chromatin regions, particularly at transcription start sites (TSS) after TERRA depletion; such G4 reduction at TSS is associated with elevated ATRX occupancy and differentially expressed genes. Loss of ATRX alleviates the effect of gene repression caused by TERRA depletion. Immunostaining analyses demonstrate that knockdown of TERRA diminishes DNA G4 signals, whereas silencing ATRX elevates G4 formation. Our results uncover an epigenetic regulation by TERRA that sequesters ATRX and preserves DNA G4 structures.
Asunto(s)
G-Cuádruplex , ARN Largo no Codificante , Animales , Ratones , Cromatina/genética , Epigénesis Genética , Telómero/genética , Telómero/metabolismo , ARN Largo no Codificante/metabolismo , ADN , Proteína Nuclear Ligada al Cromosoma X/genética , Proteína Nuclear Ligada al Cromosoma X/metabolismoRESUMEN
Alternative Lengthening of Telomeres (ALT) utilizes a recombination mechanism and break-induced DNA synthesis to maintain telomere length without telomerase, but it is unclear how cells initiate ALT. TERRA, telomeric repeat-containing RNA, forms RNA:DNA hybrids (R-loops) at ALT telomeres. We show that depleting TERRA using an RNA-targeting Cas9 system reduces ALT-associated PML bodies, telomere clustering, and telomere lengthening. TERRA interactome reveals that TERRA interacts with an extensive subset of DNA repair proteins in ALT cells. One of TERRA interacting proteins, the endonuclease XPF, is highly enriched at ALT telomeres and recruited by telomeric R-loops to induce DNA damage response (DDR) independent of CSB and SLX4, and thus triggers break-induced telomere synthesis and lengthening. The attraction of BRCA1 and RAD51 at telomeres requires XPF in FANCM-deficient cells that accumulate telomeric R-loops. Our results suggest that telomeric R-loops activate DDR via XPF to promote homologous recombination and telomere replication to drive ALT.