DNA damage and genome instability by G-quadruplex ligands are mediated by R-loops in human cancer cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE115957 | GEO | 2018/12/27
REPOSITORIES: GEO
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Project description:G-quadruplexes (G4s) are noncanonical DNA secondary structures formed through the self-association of guanines, and they are distributed widely across the genome. G4 participates in multiple biological processes including gene transcription, and G4-targeted ligands serve as potential therapeutic agents for DNA-targeted therapies. However, genome-wide studies of the exact roles of G4s in transcriptional regulation are still lacking. We found that drug-induced promoter-proximal RNA polymerase II pausing promotes nearby G4 formation, and oppositely, G4 stabilization by G4-targeted ligands globally reduces RNA polymerase II occupancy at gene promoters as well as nascent RNA synthesis. To study the underlying mechanisms by which native G4 affects transcriptional regulation, we annealed the biotin-labeled core promoter DNA to form G4s and performed pull-down assays with nuclear extraction proteins in the presence or absence of TMPyP4. Mass spectrometry analysis was performed to identify the interacting proteins with G4-forming core promoter DNA.
2021-07-19 | PXD026830 | Pride
Project description:Single-stranded genomic DNA can fold into G-quadruplex (G4) structures or form DNA:RNA hybrids (R loops). Recent evidence suggests that such non-canonical DNA structures affect gene expression, DNA methylation, replication fork progression and genome stability. When and how G4 structures form and are resolved remains unclear. Here we report the use of Cleavage Under Targets and Tagmentation (CUT&Tag) for mapping native G4 in mammalian cell lines at high resolution and low background. Mild native conditions used for the procedure retain more G4 structures and provide a higher signal-to-noise ratio than ChIP-based methods. We determine the G4 landscape of mouse embryonic stem cells (mESC), discovering G4 formation at active and poised promoters and enhancers. We discover that the presence of G4 motifs and G4 structures distinguishes active and primed enhancers in mESCs. Further performing R-loop CUT&Tag, we demonstrate the widespread co-occurence of single-stranded DNA, G4s and R loops, suggesting an intricate interrelation of non-canonical DNA structures, transcription and the formation and turnover of G4s.
2021-04-27 | GSE173103 | GEO
Project description:DNA G-quadruplexes (G4s) have been identified as important biological targets for transcriptional, translational, and epigenetic regulation. The stabilisation of G4s with small molecule ligands has emerged as a technique to regulate gene expression and as a potential therapeutic approach for human diseases. Here, we demonstrate that ligand stabilisation of G4s causes altered chromatin accessibility dependant on the targeting specificity of the molecule. In particular, stabilisation of a target G4 using the highly specific GTC365 ligand resulted in differential accessibility of 61 genomic regions, while the broad targeted GQC-05 ligand stabilised many G4s and induced a global shift towards increased accessibility of gene promoter regions. Interestingly, while we observed distinct effects of each ligand on RNA expression levels and the induction of DNA double-stranded breaks, both ligands modified DNA damage response pathways. Our work represents the dual possibility of G4-stabilising ligands for specific or global chromatin modulation via unique targeting characteristics.
2023-06-28 | GSE235953 | GEO
Project description:DNA G-quadruplexes (G4s) have been identified as important biological targets for transcriptional, translational, and epigenetic regulation. The stabilisation of G4s with small molecule ligands has emerged as a technique to regulate gene expression and as a potential therapeutic approach for human diseases. Here, we demonstrate that ligand stabilisation of G4s causes altered chromatin accessibility dependant on the targeting specificity of the molecule. In particular, stabilisation of a target G4 using the highly specific GTC365 ligand resulted in differential accessibility of 61 genomic regions, while the broad targeted GQC-05 ligand stabilised many G4s and induced a global shift towards increased accessibility of gene promoter regions. Interestingly, while we observed distinct effects of each ligand on RNA expression levels and the induction of DNA double-stranded breaks, both ligands modified DNA damage response pathways. Our work represents the dual possibility of G4-stabilising ligands for specific or global chromatin modulation via unique targeting characteristics.
2023-06-28 | GSE235952 | GEO
Project description:R-loops are prevalent in mammalian genomes and involved in many fundamental cellular processes. Depletion of BRCA2 leads to aberrant R-loop accumulation, contributing to genome instability. Here, we show that ZFP281 cooperates with BRCA2 in preventing R-loop accumulation to facilitate DNA replication in embryonic stem cells. Mechanistically, we demonstrate that ZFP281 can interact with BRCA2, and that BRCA2 is enriched at G/C-rich promoters and requires both ZFP281 and PRC2 for its proper recruitment to the bivalent chromatin at the genome-wide scale. Furthermore, depletion of ZFP281 or BRCA2 leads to accumulation of R-loops over the bivalent regions, and compromises activation of the developmental genes by retinoic acid during stem cell differentiation. In summary, our results reveal that ZFP281 recruits BRCA2 to the bivalent chromatin regions to ensure proper progression of DNA replication through preventing persistent R-loops.
2022-05-31 | GSE127262 | GEO
Project description:R-loops and guanine quadruplexes (G4s) are secondary structures of nucleic acids that are ubiquitously present in cells, and are enriched in promoter regions of genes. By employing a bioinformatic approach based on overlap analysis of transcription factor ChIP-seq datasets, we found that many splicing factors, including U2AF1 whose recognition of 3¢ splicing site is crucial for pre-mRNA splicing, exhibit pronounced enrichment at endogenous R-loop- and DNA G4-structure loci in promoter regions of human genes. We revealed that U2AF1 can bind directly to R-loops and DNA G4 structures at low nM binding affinity. Additionally, we showed the ability of U2AF1 to undergo phase separation, which could be stimulated by binding with R-loops, but not duplex DNA, RNA/DNA hybrid, DNA G4, or single-stranded RNA. We also demonstrated that U2AF1 binds to promoter R-loops in human cells, and this binding competes with U2AF1’s interaction with 3¢-splicing site, thereby modulating pre-mRNA splicing. Together, we uncovered a group of candidate proteins that can bind to both R-loops and DNA G4s, revealed the direct and strong interaction of U2AF1 with these nucleic acid structures, and unveiled new functions of this interaction, i.e., promotion of phase separation of U2AF1 and modulation of mRNA splicing. Our work also established, for the first time, a biochemical basis for U2AF1’s occupancy in gene promoters.
2023-10-12 | GSE230254 | GEO
Project description:DNA G-quadruplex (G4) is a non-canonical four-stranded nucleic acid structure functioning in various biological processes in mammals. G4 ChIP-seq assay, In this study, we developed a robust BG4-DNA ChIP-seq for global identification of DNA G4 in rice..We found that G4s harbored some functional motifs for binding of certain trans-factors, were co-localized with R-loops and DHSs in the rice genome. Importantly, G4s exhibited differential effects on DNA methylation between TE and non-TE genes in rice. Especially, G4 intensity-dependent enrichment occurred between DNA 5mC and 6mA.These DNA-related features suggest potential regulatory roles of G4 in rice. Thus, our study will pave the way for prompting intensive characterization towards identifying novel functions of G4 in plants, especially for agronomic important crops.
2021-07-01 | GSE132775 | GEO
Project description:G-quadruplexes (G4s) are four-stranded nucleic acid structures abundant at gene promoters. They can adopt several distinctive conformations. G4s have been shown to form in the herpes simplex virus-1 (HSV-1) genome during its viral cycle. Here by cross-linking/pull-down assay we identified ICP4, the major HSV-1 transcription factor, as the protein that most efficiently interacts with viral G4s during infection. ICP4 specific and direct binding and unfolding of parallel G4s, including those present in HSV-1 immediate early gene promoters, induced transcription in vitro and in infected cells. This mechanism was also exploited by ICP4 to promote its own transcription. Proximity ligation assay allowed visualization of G4-protein interaction at the single selected G4 in cells. G4 ligands inhibited ICP4 binding to G4s. Our results indicate the existence of a well-defined G4-viral protein network that regulates the productive HSV-1 cycle. They also point to G4s as elements that recruit transcription factors to activate transcription in cells.
2021-09-10 | PXD024743 | Pride
Project description:DNA secondary structures are important for fundamental genome functions such as transcription and replication1. The G-quadruplex (G4) structural motif has been linked to gene regulation2,3 and genome instability4,5 and may be important to cancer development and other diseases6-8. Recently, ~700,000 discrete G4s have been observed in naked human single-stranded genomic DNA using G4-seq, a high-throughput sequencing technique that detects structural features in vitro.9 It is of vital importance to investigate G4 structures within an endogenous chromatin context, which until now remained elusive10,11. Herein, we address this via the development of G4 ChIP-seq, an antibody-based G4 chromatin immunoprecipitation and high-throughput sequencing approach. We identified ~10,000 endogenous G4 structures and show that G4s are predominantly seen in regulatory, nucleosome-depleted, chromatin regions. G4s were enriched in the promoters and 5âUTR regions of highly transcribed genes, particularly in genes related to cancer and in somatic copy number amplifications, such as MYC. Reorganization of the chromatin landscape using a histone deacetylase inhibitor, resulted in de novo G4 formation in new and more prominent regulatory, nucleosome-depleted regions associated with increased transcriptional output. Our findings suggest a striking relationship between promoter nucleosome-depleted regions, G4 formation and elevated transcriptional activity. Comparison between normal human epidermal keratinocytes and their immortalized counterparts revealed a ï¾7-fold greater G4 abundance in immortalized cells, of which 80 % were found in regulatory, nucleosome-depleted regions common to both cell types. Consequently, cells exhibiting more G4s displayed significantly increased transcriptional output and were more sensitive to growth inhibition by a small molecule G4 ligand. Overall, our results provide new mechanistic insights into where and when DNA adopts G4 structure in vivo. Our findings show for the first time that regulatory, nucleosome-depleted chromatin and transcriptional states predominantly shape the endogenous G4 DNA landscape. Two cell lines, treated with entinostat or untreated, analyzed to detect gene expression differences, presence of G-Qudruplexes and chromatin state. Each combination of conditions replicated in duplicates or triplicates.
2016-08-05 | E-GEOD-76688 | biostudies-arrayexpress
Project description:RNAPII is mainly responsible for mRNA transcription and plays a vital role in gene expression. In light of the above results showing that cellular G4s are highly correlated with RNAPII-mediated DNA loops, we performed the RNA-seq to evaluate how G4-dependent long-range DNA interactions modulate gene expression. We revealed that PDS treatment modulates the expression of not only genes with G4 in their promoters, but also those with promoters being connected with distal G4 through RNAPII-linked long-range DNA interactions.
2023-09-13 | GSE222207 | GEO