R-loop associated G-quadruplexes reinforce CTCF interactions
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE208145 | GEO | 2023/06/26
REPOSITORIES: GEO
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Project description:The identification of DNA G-quadruplexes (G4s) in the genome is important to study different biological processes in which these structures play a role, such as genome rearrangement, transcriptional regulation and DNA replication. G4-seq allowed the high-throughput experimental mapping of G-quadruplexes in the human genome. We developed here an improved version of this method, named G4-seq2, which we applied to generate G-quadruplexes genomic maps for 12 species, selected as important models organism to study development or as pathogens of clinical relevance. Those multi-species maps, publicly available for the community, will allow to further understand the design principle of G-quadruplex formation in genomic context, to study G-quadruplex biology in those model organisms, to predict ligand targeting for therapeutic usage and to design G-quadruplex computational predictors based on genome-wide experimental measurements.
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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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Project description:G-quadruplex (G4) sites in the human genome frequently colocalize with CCCTC-binding factor (CTCF)-bound sites within topologically associating domains (TADs) or at TAD boundaries. We investigated three mechanisms by which G4s may contribute to CTCF recruitment. One involved direct interactions between CTCF and G4s that persisted in the G0/G1 phase of the cell cycle. Synthetic G4s from CpG islands (CGIs) formed complexes with CTCF in vitro, and CTCF occupancy at the respective sites in the genome was modulated by a G4-stabilizing ligand. Another possible mechanism was through G4 interference with DNA methyltransferase activity in CGIs. Bioinformatics analysis confirmed that G4s underlie the association between CTCF and CGIs, but did not support a critical role for methylation in CTCF recruitment to G4-harboring CGIs. The third mechanism was through attracting additional protein factors. We found that G4s are recognized by the nucleosome density modulating high-mobility group (HMG) proteins and the cohesin-interacting protein additional sex combs-like 1. The affinity for these proteins is the basis for indirect G4 contributions to CTCF positioning and TAD demarcation.
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Project description:We investigated herein the interaction between nucleolin (NCL) and a set of G4 sequences derived from the CEB25 human minisatellite which adopt a parallel topology while differing by the length of the central loop (from 9nt to1nt). It is revealed that NCL strongly binds to long-loop (9-5 nt) G4 whilst interacting weakly with the shorter variants (loop < 3nt). Photocrosslinking experiments using 5-bromouracil (BrdU) modified sequences further confirmed the loop-length dependency thereby indicating that the CEB25-WT (9nt) is the best G4 substrate. Quantitative proteomic analysis (LC-MS/MS) of the photocrosslinking product(s) obtained with NCL bound to this sequence enabled the identification of one contact site within the 9nt loop. The protein fragment identified is located in the helix of the RBD2 domain of NCL, shedding light on the role of this structural element in the G4-loop recognition. Then, the ability of a panel of benchmark G4 ligands to prevent the NCL/G4 interaction was explored. It was found that only the most potent ligand PhenDC3 is able to inhibit NCL binding, thereby suggesting that the terminal guanine quartet is also a strong determinant of G4 recognition, putatively through interaction with the RGG domain. This study puts forward the molecular mechanism by which NCL recognizes G4-containing long loops and leads to propose a model implying a concerted action of RBD2 and RGG domains to achieve specific G4 recognition via a dual loop-quartet interaction.
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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.
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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.
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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.
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