Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA387558 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR5586990.fastq.gz | Fastqsanger.gz | |||
| SRR5586987.fastq.gz | Fastqsanger.gz | |||
| SRR5586988.fastq.gz | Fastqsanger.gz | |||
| SRR5586989.fastq.gz | Fastqsanger.gz |
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Project description:The G-quadruplex is an alternative DNA structural motif that is considered to be functionally important in the mammalian genome. Herein, we address the hypothesis that G-quadruplex structures can exist within double-stranded genomic DNA using a G-quadruplex-specific probe. An engineered antibody is employed to enrich for DNA containing G-quadruplex structures, followed by deep sequencing to detect and map G-quadruplexes at high resolution in genomic DNA from human breast adenocarcinoma cells. Our high sensitivity structure-based pull-down strategy enables the isolation of genomic DNA fragments bearing a single as well as multiple G-quadruplex structures. Stable G-quadruplex structures are found in sub-telomeres, gene bodies and gene regulatory regions. For a sample of identified target genes, we show that G-quadruplex stabilizing ligands can modulate transcription. These results confirm the existence of G-quadruplex structures and their persistence in human genomic DNA. Four independent libraries have been enriched in DNA G-quadruplex structures using a G-quadruplex-specific probe. One genomic input library was sequenced as control. Deep-sequencing of these libraries allowed the mapping of G-quadruplexes on the genome.
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Project description:Rif1 regulates replication timing and repair of double-stranded DNA breaks. Using Chromatin-immunoprecipitation-Sequencing method, we have identified 35 high-affinity Rif1 binding sites in fission yeast chromosomes. Binding sites, preferentially located to the vicinity of dormant origins, tended to contain at least two copies of a conserved motif, CNWWGTGGGGG, and base substitution within these motifs resulted in complete loss of Rif1 binding and activation of late-firing or dormant origins located as far as 50 kb away. We show that Rif1 binding sites adopt G-quadruplex-like structures in vitro in a manner dependent on the conserved sequence as well as on other G-tracts, and that the purified Rif1 preferentially binds to this structure. These results suggest that Rif1 recognizes and binds to G-quadruplex-like structures at selected intergenic regions to generate local chromatin structures that may exert a long-range suppressive effects on origin firing. ChIP-Seq profiles of Rif1 and DNA replicaiton (BrdU-incorporation) vs Input in wildt type, rap1∆, taz1∆, Rif1BS mutants and rif1∆
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Project description:G-quadruplex (G4) structural motifs have been linked to transcription, replication and genome instability and are implicated in cancer and other diseases. However, it is crucial to demonstrate the bona fide formation of G4 structures within an endogenous chromatin context. Herein we address this through the development of G4 ChIP-seq, an antibody-based G4 chromatin immunoprecipitation and high-throughput sequencing approach. We find ∼10,000 G4 structures in human chromatin, predominantly in regulatory, nucleosome-depleted regions. G4 structures are enriched in the promoters and 5' UTRs of highly transcribed genes, particularly in genes related to cancer and in somatic copy number amplifications, such as MYC. Strikingly, de novo and enhanced G4 formation are associated with increased transcriptional activity, as shown by HDAC inhibitor-induced chromatin relaxation and observed in immortalized as compared to normal cellular states. Our findings show that regulatory, nucleosome-depleted chromatin and elevated transcription shape the endogenous human G4 DNA landscape.
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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.
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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.
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Project description:High-throughput sequencing of DNA G-quadruplex structures in the human genome
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