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Genome-wide mapping of i-Motifs reveals their association with transcription regulation in live human cells [RNA-seq]

ABSTRACT: i-Motifs (iMs) are four-stranded DNA structures that form at cytosine (C)-rich sequences and in acidic conditions in vitro. However, their formation and distribution in cells is still under debate. Here we performed CUT&Tag sequencing using the anti-iM antibody iMab and showed that iMs form within the human genome in live cells. We mapped native iMs in two different human cell lines and recovered C-rich sequences that were confirmed to fold into iMs in vitro. We found that iMs in cells are mainly present at actively transcribing gene promoters and that their abundance and distribution are specific to each cell type. iMs with both long and short C-tracts were recovered, further extending the relevance of iMs throughout the genome. By simultaneously mapping G-quadruplexes (G4s), four-stranded structures that form at guanine-rich regions, and comparing the results with iMs, we proved that the two structures can form in independent genomic regions; however, when both iMs and G4s are present in the same genomic tract, their formation is favored. Our findings support the in vivo formation of iM structures and provide new insights into their interplay with G4s as new regulatory elements in the human genome.

ORGANISM(S): Homo sapiens

PROVIDER: GSE220881 | GEO | 2023/08/02

REPOSITORIES: GEO

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Genome-wide mapping of i-Motifs reveals their association with transcription regulation in live human cells [CUT&Tag]

Project description:i-Motifs (iMs) are four-stranded DNA structures that form at cytosine (C)-rich sequences and in acidic conditions in vitro. However, their formation and distribution in cells is still under debate. Here we performed CUT&Tag sequencing using the anti-iM antibody iMab and showed that iMs form within the human genome in live cells. We mapped native iMs in two different human cell lines and recovered C-rich sequences that were confirmed to fold into iMs in vitro. We found that iMs in cells are mainly present at actively transcribing gene promoters and that their abundance and distribution are specific to each cell type. iMs with both long and short C-tracts were recovered, further extending the relevance of iMs throughout the genome. By simultaneously mapping G-quadruplexes (G4s), four-stranded structures that form at guanine-rich regions, and comparing the results with iMs, we proved that the two structures can form in independent genomic regions; however, when both iMs and G4s are present in the same genomic tract, their formation is favored. Our findings support the in vivo formation of iM structures and provide new insights into their interplay with G4s as new regulatory elements in the human genome.

2023-08-02 | GSE220878 | GEO

iMbody - a nanobody for i-Motif research

Project description:i-Motifs (iMs) are four-stranded structures formed by certain cytosine-rich nucleic acids. They may be involved in regulation of cellular processes such as transcription. In addition, the pH dependency of iMs makes them suitable for certain nanobiotechnological applications. Probes that recognize these structures with high specificity and affinity can significantly advance the iM research field. Here we report the generation of an iM specific nanobody (iMbody) and describe its applications for the in vitro characterization of a potential iM-forming oligonucleotide, strand-specific pull-down of iMs present in the human genome at near physiological pH and temperature followed by sequencing, and visualization of iMs in the nuclei of human cells. Utilization of iMbody could be beyond above-mentioned applications and it can be used to investigate iMs in other organisms. The plasmid encoding iMbody for the bacterial expression is available via Addgene (Plasmid #184496).

2022-12-06 | GSE212765 | GEO

Differential impacts of DNA methylation on pH dependent i-motifs formation in rice [ChIP-seq]

Project description:I-motifs (iMs) are four-stranded non-B DNA structures containing C-rich DNA sequences, which can be formed under various pH conditions. DNA methylation exhibits complex impacts on iM formation in vitro. However, how DNA methylation differentially affects pH dependent iM formation on a genome wide scale is completely uncharacterized in both humans and plants. To this end, we conducted iM-IP-seq under pH 5.5 and 7.0 conditions using CK and hyper/hypomethylated DNA in combination with BS-seq in rice. We found that pH 7.0 and 5.5 biased iMs had distinct genomic features and differential DNA methylation levels, the former having higher DNA methylation levels than the latter. Moreover, DNA demethylation and hyper methylation had more impacts on a subset of iM formation under pH 7.0 and 5.5, respectively, indicating that DNA de/hyper-methylation exhibits pH dependent impacts on iM formation. Importantly, we found that CG hypo-DMRs and CHH hyper-DMRs alone or coordinated with CG/CHG hyper-DMRs may play determinant roles in the regulation of iM formation under pH 5.5 and 7.0. Thus, our study shows that intrinsic DNA sequences alone or in combination with DNA methylation play vital roles in determining pH-dependent formation of iMs. It will contribute to in-depth understanding of DNA methylation in the modulation of pH dependent dynamics of iM conformation, therefore broadening its biological implications and practical application ranges.

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Differential impacts of DNA methylation on pH dependent i-motifs formation in rice [Bisulfite-seq]

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Genome-wide mapping of G-quadruplex structures with CUT&Tag

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.

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Control of cellular translation by endogenous RNA G-quadruplex structures

Project description:Four-stranded G-quadruplex (G4) structures form guanine-rich tracts, but their role in RNA biology remains poorly defined. Herein, we first delineate the presence of endogenous RNA G4s in the human cellular transcriptome by proxy of their binding to interacting proteins, DHX36, GRSF1 and DDX3X. We then demonstrate that a sub-population of these RNA G4s are reliably detected as folded structures in cross-linked cellular lysates using the G4 structure-specific antibody BG4. The 5' UTRs of protein-coding mRNAs show significant enrichment in such folded RNA G4s, particularly within mRNA for ribosomal proteins. As G4s in ribosomal mRNA are evolutionarily conserved in higher vertebrates this points to a common mode for translational co-regulation mediated by RNA G4 structures. Supporting this we find that G4-stabilising small molecules inhibit the translation of ribosomal protein mRNA and significantly down-regulate cellular translation.

2021-11-10 | GSE154570 | GEO

Genome-wide mapping of native G-loops in living cells

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).

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Chromatin immunoprecipitation for genome-wide mapping of endogenous G-quadruplex DNA structures

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

Custom DNA microarrays reveal diverse binding preferences of proteins and small molecules to thousands of G-quadruplexes [design 2]

Project description:Single-stranded DNA (ssDNA) containing guanine repeats can form G-quadruplex (G4) structures. While cellular proteins and small molecules can bind G4s, it has been difficult to broadly assess their sequence specificity. Here, we use custom DNA microarrays to examine the binding specificities of proteins, small molecules, and antibodies across ~15,000 G4 structures. Molecules used include fluorescently labeled pyridostatin (Cy5-PDS, a small molecule), BG4 (Cy5-BG4, a G4-specific antibody), and eight proteins (GST-tagged nucleolin, IGF2, CNBP, FANCJ, PIF1, BLM, DHX36, and WRN). Cy5-PDS and Cy5-BG4 selectively bind sequences known to form G4s, confirming their formation on the microarrays. Cy5-PDS binding decreased when G4 formation was inhibited using lithium or when ssDNA features on the microarray were made double-stranded. Similar conditions inhibited the binding of all other molecules except for CNBP and PIF1. We report that proteins have different G4 binding preferences suggesting unique cellular functions. Finally, competition experiments are used to assess the binding of an unlabeled small molecule, revealing the structural features in the G4 required to achieve selectivity. These data demonstrate that the microarray platform can be used to assess the binding preferences of molecules to G4s on a broad scale, helping to understand the properties that govern molecular recognition.

2020-03-27 | GSE133366 | GEO

Custom DNA microarrays reveal diverse binding preferences of proteins and small molecules to thousands of G-quadruplexes [design 1]

2020-03-27 | GSE133365 | GEO