Genome-wide identifying of G-quadruplex structures directly by whole-genome resequencing
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE159307 | GEO | 2020/10/10
REPOSITORIES: GEO
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Project description:In the presence of monovalent alkali metal ions, G-rich DNA sequences containing four runs of contiguous guanines can fold into G-quadruplex (G4) structures. Recent studies showed that these structures are located in critical regions of the human genome and assume important functions in many essential DNA metabolic processes, including replication, transcription and repair. However, not all potential G4-forming sequences are actually folded into G4 structures in cells, where G4 structures are known to be dynamic and modulated by G4-binding proteins as well as helicases. It remains unclear whether there are other factors influencing the formation and stability of G4 structures in cells. Herein, we showed that DNA G4s can undergo phase separation in vitro. In addition, immunofluorescence microscopy and ChIP-Seq experiments with the use of BG4, a G4 structure-specific antibody, revealed that disruption of phase separation in cells could result in global destabilization of G4 structures. Together, our work revealed phase separation as a new determinant in regulating the formation and stability of G4 structures in human cells.
2023-07-05 | GSE225772 | GEO
Project description:The DNA Guanine quadruplexes (G4) plays important roles in multiple cellular processes, including regulation of DNA replication, transcription and genome stability. Here, we show that Yin Yang-1 (YY1), a ubiquitously expressed and multifunctional transcription factor, can bind with G4 structures directly. Fluorescence anisotropy experiments shows that YY1 binds towards G4 structures with Kd in low nanomolar range (<100 nM) through the C terminal zinc finger domains. ChIP-seq results show that 81% of the YY1 ChIP-seq peaks contain G4 forming sequences, which is highly overlapped with the G4 ChIP-seq peaks. YY1 facilitate the DNA-DNA interaction through its binding with G4 structures in vivo and in vitro, which can be disturbed by G4 stabilizer treatment. In all, our studies identify a novel G4 structure binding protein-YY1, and reveal that G4 structure functions in DNA-DNA interaction.
2020-08-27 | GSE128106 | GEO
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:G-rich DNA sequences can form four-stranded G-quadruplex (G4) secondary structures and are linked to fundamental biological processes such as transcription, replication and telomere maintenance. G4s are also implicated in promoting genome instability, cancer and other diseases. Here, we describe a detailed G4 ChIP-seq method that robustly enables the determination of G4 structure formation genome-wide in chromatin. This protocol adapts traditional ChIP-seq for the detection of DNA secondary structures through the use of a G4-structure-specific phage display antibody with refinements in chromatin immunoprecipitation followed by high-throughput sequencing. Beginning with chromatin isolation and antibody preparation the entire protocol can be completed in less than 1 week including computational analysis.
2017-11-15 | GSE99205 | GEO
Project description:G-quadruplex (or G4) structures are non-canonical DNA structures that form in guanine-rich sequences and threaten genome stability when not properly resolved. G4 unwinding occurs during S phase via an unknown mechanism. Using Xenopus egg extracts, we define a three-step G4 unwinding mechanism that acts during DNA replication. First, the replicative helicase (CMG) stalls at a leading strand G4 structure. Second, the DHX36 helicase mediates the bypass of the CMG past the intact G4 structure, which allows approach of the leading strand to the G4. Third, G4 structure unwinding by the FANCJ helicase enables the DNA polymerase to synthesize past the G4 motif. A G4 on the lagging strand template does not stall CMG, but still requires active DNA replication for unwinding. DHX36 and FANCJ have partially redundant roles, conferring robustness to this pathway. Our data reveal a novel genome maintenance pathway that promotes faithful G4 replication thereby avoiding genome instability.
2022-02-17 | PXD027623 | Pride
Project description:DNA sequences of high guanine (G) content have the potential to fold into G quadruplex (G4) structures. DNA G4 is known to play important roles in various cellular processes including DNA replication, transcriptional regulation, and maintenance of genomic integrity. A more complete understanding about the biological functions of G4 DNA requires the investigation about how these structures are recognized by cellular proteins. Here, we conducted exhaustive quantitative proteomic experiments to profile the interaction proteomes of three well-defined G4 structures derived from the human telomere and the promoters of cMYC and cKIT genes. Our results led to the identification of a number of candidate G4-interacting proteins; some were previously reported, e.g., FUS, TOP1, and PARP1, and many others were discovered here for the first time. These included three proteins that can bind to all three DNA G4 structures and many proteins that can bind specifically to selected DNA G4 structure(s). We also validated that GRSF1 can bind directly and selectively toward G4 DNA structure derived from the cMYC promoter. Taken together, we uncovered a number of cellular proteins that exhibit general and selective recognitions of G4 folding patterns, which underscore the complexity of G4 DNA in biology and the importance in understanding fully the G4-interaction proteome.
2021-09-29 | PXD026794 | Pride
Project description:DNA structure can regulate genome function. Four-stranded DNA G-quadruplex (G4) structures have been implicated in transcriptional regulation; however, previous studies have not directly addressed the role of an individual G4 within its endogenous cellular context. Using CRISPR to genetically abrogate endogenous G4 structure folding, we directly interrogate the G4 found within the upstream regulatory region of the critical human MYC oncogene. G4 loss leads to suppression of MYC transcription from the P1 promoter that is mediated by the deposition of a de novo nucleosome alongside alterations in RNA polymerase recruitment. We also show that replacement of the endogenous MYC G4 with a different G4 structure from the KRAS oncogene restores G4 folding and MYC transcription. Moreover, we demonstrate that the MYC G4 structure itself, rather than its sequence, recruits transcription factors and histone modifiers. Overall, our work establishes that G4 structures are important features of transcriptional regulation that coordinate recruitment of key chromatin proteins and the transcriptional machinery through interactions with DNA secondary structure, rather than primary sequence.
2024-02-06 | GSE223370 | GEO
Project description:High-order chromatin organization plays an important role in biological processes and disease development. Previous studies revealed a widespread occurrence of guanine quadruplex (G4) structures in the human genome, with enrichment in gene regulatory regions, especially in promoters. However, it remains unclear if G4 structures contribute to RNA polymerase II (RNAPII)-mediated long-range DNA interactions and transcription activity. In this study, we conducted an intuitive overlapping analysis of previously published data of RNAPII ChIA-PET (chromatin interaction analysis with paired-end tag) and BG4 ChIP-seq (chromatin immunoprecipitation followed by sequencing with the use of a G4 structure-specific antibody). We observed a strong positive correlation between RNAPII-mediated DNA loops and G4 structures in chromatin. Additionally, our RNAPII HiChIP-seq (in situ Hi-C followed by ChIP-seq) results showed that treatment of HepG2 cells with pyridostatin (PDS), a small-molecule G4-stabilization ligand, could diminish RNAPII-linked long-range DNA contacts, with more pronounced decreases being observed for those contacts involving G4 structure loci.
2023-09-13 | GSE222206 | GEO
Project description:G-quadruplexes (G4s) are alternative DNA secondary structures of the human genome with key roles in transcription, replication and genome stability. Structure-specific small molecules and antibodies, such as BG4, have facilitated the detection of G4s in cells and chromatin. The availability of an alternative probe to BG4 would help to address some of its potential limitations in mapping G4 structures and would independently confirm and/or extend our earlier findings. Herein, we describe the development of SG4, a camelid single-chain only derived nanobody raised against the human MycG4 promoter structure. SG4 is small, easily expressed and demonstrates low nanomolar affinity for folded G4 structures in vitro. Clues to SG4 binding were obtained experimentally by mutating single amino acids. Moreover, AlphaFold2 protein predictions combined with molecular dynamics/docking simulations generated a de novo binding model of SG4-G4 interactions that identifies key amino acids with significant contributions to G4 binding. Using SG4 in G4 ChIP-seq, we also demonstrate the detection of G4 structures in two cancer cell lines. SG4 thus provides an alternative tool for G4 genome mapping and independently verifies our earlier findings.
2023-03-06 | GSE207567 | GEO
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