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Detection of genomic G-quadruplexes in living cells using a small artificial protein

ABSTRACT: Detection of genomic G-quadruplexes in living cells using a small artificial protein

PROVIDER: PRJNA551290 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR9603960_1.fastq.gz	Fastqsanger.gz
	SRR9603960_2.fastq.gz	Fastqsanger.gz
	SRR9603970_1.fastq.gz	Fastqsanger.gz
	SRR9603970_2.fastq.gz	Fastqsanger.gz
	SRR9603961_1.fastq.gz	Fastqsanger.gz

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Detection of genomic G-quadruplexes in living cells using a small artificial protein

Project description:G-quadruplex (G4) structures formed by guanine-rich nucleic acids are implicated in essential physiological and pathological processes and serve as important drug targets. The genome-wide detection of G4s in living cells is important for exploring the functional role of G4s but has not yet been achieved due to the lack of a suitable G4 probe. Here we report an artificial 6.7 kDa G4 probe (G4P) protein that binds G4s with high affinity and specificity. We used it to capture G4s in living human, mouse, and chicken cells with the ChIP-Seq technique, yielding genome-wide landscape as well as details on the positions, frequencies, and sequence identities of G4 formation in these cells. Our results indicate that transcription is accompanied by a robust formation of G4s in genes. In human cells, we detected up to >123,000 G4P peaks, of which >1/3 had a fold increase of ≥5 and were present in >60% promoters and ~70% genes. Being much smaller than a scFv antibody (27 kDa) or even a nanobody (12-15 kDa), we expect that the G4P may find diverse applications in biology, medicine, and molecular devices as a G4 affinity agent.

2020-10-13 | GSE133379 | GEO

Detection of genomic G-quadruplexes in living cells using a small artificial protein.

Project description:G-quadruplex (G4) structures formed by guanine-rich nucleic acids are implicated in essential physiological and pathological processes and serve as important drug targets. The genome-wide detection of G4s in living cells is important for exploring the functional role of G4s but has not yet been achieved due to the lack of a suitable G4 probe. Here we report an artificial 6.7 kDa G4 probe (G4P) protein that binds G4s with high affinity and specificity. We used it to capture G4s in living human, mouse, and chicken cells with the ChIP-Seq technique, yielding genome-wide landscape as well as details on the positions, frequencies, and sequence identities of G4 formation in these cells. Our results indicate that transcription is accompanied by a robust formation of G4s in genes. In human cells, we detected up to >123 000 G4P peaks, of which >1/3 had a fold increase of ≥5 and were present in >60% promoters and ∼70% genes. Being much smaller than a scFv antibody (27 kDa) or even a nanobody (12-15 kDa), we expect that the G4P may find diverse applications in biology, medicine, and molecular devices as a G4 affinity agent.

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Genome-wide profiling of DNA G-quadruplexes in living neurons

Project description:We report a genome-wide mapping of DNA G-quadruplexes and histone modifications in living neurons. We used primary cultured neurons from the fetus mouse cortex. Cultured cells were treated with 2 µM Ara-C at DIV3 to prevent the proliferation of mitotic cells and were harvested at DIV10. For iG4 CUT&Tag, a BG4-expressing plasmid was transfected by electroporation at DIV0.

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Genome-wide profiling of DNA G-quadruplexes in living neurons

Project description:Genome-wide profiling of DNA G-quadruplexes in living neurons

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Project description:In vitro, some RNAs can form stable four-stranded structures known as G-quadruplexes. Although RNA G-quadruplex structures have been implicated in post-transcriptional gene regulation and diseases, direct evidence for quadruplex formation in cells has been lacking. Here, we developed a suite of methods that identify RNAs with quadruplex-forming ability and measure their folding state in living cells. Applying these methods to mammalian cell lines, the budding yeast S. cerevisiae and several bacteria, we characterized the folding landscapes of RNA G-quadruplexes in these species.

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Identification of G-quadruplex-interacting proteins in living cells using an artificial G4-targeting biotin ligase

Project description:G-quadruplexes (G4s) are noncanonical nucleic acid structures pivotal to cellular processes and disease pathways. Deciphering G4-interacting proteins is imperative for unraveling G4's biological significance. In this study, we developed a G4-targeting biotin ligase named G4PID, meticulously assessing its binding affinity and specificity both in vitro and in vivo. Capitalizing on G4PID, we devised a tailored approach termed G-quadruplex-interacting proteins specific biotin-ligation procedure (PLGPB) to precisely profile G4-interacting proteins.

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Project description:G-quadruplexes (G4s) are noncanonical DNA secondary structures formed through the self-association of guanines. They are distributed genome-widely and participate in multiple biological processes including gene transcription, and quadruplex-targeted ligands serve as potential therapeutic agents for DNA-targeted therapies. However, the roles of G-quadruplexes in transcriptional regulation remains elusive. Here, we establish a sensitive G4-CUT&Tag method for genome-wide profiling of native G-quadruplexes with high resolution and specificity. We find that native G-quadruplex signals are cell-type specific and are associated with transcriptional regulatory elements with active epigenetic modifications. Promoter-proximal RNA polymerase II pausing promotes native G-quadruplex formation, oppositely, G-quadruplex stabilization by quadruplex-targeted ligands globally reduces RNA polymerase II occupancy at gene promoters as well as nascent RNA synthesis. Moreover, G-quadruplex stabilization modulates chromatin states and impedes transcription initiation via inhibiting the loading of general transcription factors to promoters.Together, these studies reveal a reciprocal regulation between native G-quadruplex dynamics and gene transcription in the genome, which will deepen our knowledge of G-quadruplex biology towards considering therapeutically targeting G-quadruplexes in human diseases.

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Identification of G-quadruplex-interacting Proteins in Living Cells using an artificial G4-targeting Biotin ligase

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