RNA-seq analysis of wild-type HEK293, HEK293 treated with DMSO, ML216 or NSC617145, wild-type mESCs and dhx9KO mESCs
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ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens
PROVIDER: GSE254763 | GEO | 2024/07/25
REPOSITORIES: GEO
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Project description:Four-stranded G-quadruplexes (G4s) are prevalent secondary structural features of the human genome. They are formed through stacking of Hoogsteen hydrogen-bonded G-tetrads connected by loops of variable length and sequence. G4 structures are found widely in genome control elements including gene promoters and are tightly linked to active gene expression and the recruitment of transcription factors and chromatin remodellers. To systematically investigate how G4 structures modulate transcription, we have developed a strategy in which different G4 sequences are engineered into the promoter of a synthetic reporter gene and ectopically into the same endogenous position in the genome of human cells. Here, we show that G4 sequences introduced into the host genome can fold into an G4 structure. Crucially, we demonstrate that G4 formation within a gene promoter stimulates transcription compared to G4-negative promoters and that this is not due to inherent G-richness. Finally, we provide robust evidence that transcriptional levels are directly related to the degree of thermodynamic stability of the G4 structure. Taken together, our results provide compelling evidence that G4 structures have a direct and positive regulatory role in gene transcription.
2024-02-21 | GSE241008 | GEO
Project description:G4 are noncanonical secondary structures consist in stacked tetrads of Hoogsteen hydrogen-bonded guanines bases. An essential feature of G-quadruplexes is their intrinsic polymorphic nature. Indeed, depending on the length and the composition of the sequence, as well as the environmental conditions (including the nature and concentration of metal cations, and local molecular crowding), a G-quadruplex-forming sequence can adopt different topologies in which the strands are in parallel or antiparallel conformations, with the co-existence of different types of loops (lateral, diagonal or propeller) with variable lengths. The impact of G4 on cellular metabolism is associated with protein or enzymatic factors that promote, inhibits or resolve these structures. Thus, the major impact of G4 on the human cell metabolism is associated with genetic defects affecting proteins that counteract their formation. Although a large number of proteins able to bind and/or "to resolve" G-quadruplex structures have been identified in vitro, less is known about their mode of interaction with G-quadruplex, their specificity versus the topology and finally their specificity for G4 structures relatively to G-rich single-stranded sequences. In this study we aimed to identify and characterize human proteins interacting with locked G4 structures.
2021-07-16 | PXD021003 | Pride
Project description:G-quadruplex (G4) are four‑stranded DNA secondary structures that form in guanine‑rich regions of the genome, which can enhance or repress gene expression. An R-loop is a special triple-stranded nucleic acid structure formed when nascent RNA invades double-stranded DNA (dsDNA) during transcription. G-loops are constituted by one or more DNA G4 on one strand and a stable RNA/DNA hybrid on the other. We developed the HepG4-seq for mapping the native G4s and the HBD-seq for mapping native R-loops. We combined the HepG4-seq and HBD-seq to profile the genomic native G-loops, which are regions co-occupied by both native G4s and R-loops, in both HEK293 cells and mouse embryonic stem cells (mESCs).
2024-07-25 | GSE254764 | GEO
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-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:Background G-quadruplexes (G4s) are stable non-canonical DNA secondary structures consisting of stacked arrays of four guanines, each held together by Hoogsteen hydrogen bonds. Sequences with the ability to form these structures in vitro, G4 motifs, are found throughout bacterial and eukaryotic genomes. The budding yeast Pif1 DNA helicase, as well as several bacterial Pif1 family helicases, unwind G4 structures robustly in vitro and suppress G4-induced DNA damage in S. cerevisiae in vivo. Results We determined the genomic distribution and evolutionary conservation of G4 motifs in four fission yeast species and investigated the relationship between G4 motifs and Pfh1, the sole S. pombe Pif1 family helicase. Using chromatin immunoprecipitation combined with deep sequencing, we found that many G4 motifs in the S. pombe genome were associated with Pfh1. Cells depleted of Pfh1 had increased fork pausing and DNA damage near G4 motifs, as indicated by high DNA polymerase occupancy and phosphorylated histone H2A, respectively. In general, G4 motifs were underrepresented in genes. However, Pfh1-associated G4 motifs were located on the transcribed strand of highly transcribed genes significantly more often than expected, suggesting that Pfh1 has a function in replication or transcription at these sites. Conclusions In the absence of functional Pfh1, unresolved G4 structures cause fork pausing and DNA damage of the sort associated with human tumors.
2014-12-03 | GSE59178 | GEO
Project description:G-quadruplexes (G4s) and R-loops are non-canonical DNA structures that can play regulatory functions of basic nuclear processes and can trigger DNA damage and genome instability. We here show that specific G4 ligands can stabilize G4s and simultaneously increase R-loop levels in human cancer cells likely by spreading DNA:RNA heteroduplexes to adjacent regions containing G4 structures. DNA cleavage and DNA damage response induced by G4 ligands were rescued by overexpression of exogenous RNaseH1 in cancer cells independently of BRCA2 status. The data thus show that R-loops are involved in the induction of DNA damage by chemical G4 stabilization. In addition, G4 ligands trigger the formation of micronuclei, particularly in BRCA2-silenced cancer cells, in an R-loop dependent manner. Our results uncover the mechanism of genome instability caused by G4 ligands and can open to the development of unexpected anticancer strategies using G4-targeted agents.
2018-12-27 | GSE115957 | 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