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Topoisomerase 1 inhibits MYC promoter activity by inducing G-quadruplex formation.

ABSTRACT: We have investigated the function of human topoisomerase 1 (TOP1) in regulation of G-quadruplex (G4) formation in the Pu27 region of the MYC P1 promoter. Pu27 is among the best characterized G4 forming sequences in the human genome and it is well known that promoter activity is inhibited upon G4 formation in this region. We found that TOP1 downregulation stimulated transcription from a promoter with wildtype Pu27 but not if the G4 motif in Pu27 was interrupted by mutation(s). The effect was not specific to the MYC promoter and similar results were obtained for the G4 forming promoter element WT21. The other major DNA topoisomerases with relaxation activity, topoisomerases 2α and β, on the other hand, did not affect G4 dependent promoter activity. The cellular studies were supported by in vitro investigations demonstrating a high affinity of TOP1 for wildtype Pu27 but not for mutant sequences unable to form G4. Moreover, TOP1 was able to induce G4 formation in Pu27 inserted in double stranded plasmid DNA in vitro. This is the first time TOP1 has been demonstrated capable of inducing G4 formation in double stranded DNA and of influencing G4 formation in cells.

SUBMITTER: Keller JG

PROVIDER: S-EPMC9226537 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Indenoisoquinoline Topoisomerase Inhibitors Strongly Bind and Stabilize the <i>MYC</i> Promoter G-Quadruplex and Downregulate <i>MYC</i>.

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Project description:This SuperSeries is composed of the SubSeries listed below.

2024-01-01 | GSE239694 | GEO

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Structural Analysis of G-Quadruplex Formation at the Human MEST Promoter.

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A Thermodynamic Perspective on Potential G-Quadruplex Structures as Silencer Elements in the MYC Promoter.

Project description:Multiple G-tracts within the promoter region of the c-myc oncogene may fold into various G-quadruplexes with the recruitment of different tracts and guanosine residues for the G-core assembly. Thermodynamic profiles for the folding of wild-type and representative truncated as well as mutated sequences were extracted by comprehensive DSC experiments. The unique G-quadruplex involving consecutive G-tracts II-V with formation of two one-nucleotide and one central two-nucleotide propeller loop, previously proposed to be the biologically most relevant species, was found to be the most stable fold in terms of its Gibbs free energy of formation at ambient temperatures. Its stability derives from its short propeller loops but also from the favorable type of loop residues. Whereas quadruplex folds with long propeller loops are significantly disfavored, a snap-back loop structure formed by incorporating a 3'-terminal guanosine into the empty position of a tetrad seems highly competitive based on its thermodynamic stability. However, its destabilization by extending the 3'-terminus questions the significance of such a species under in vivo conditions.

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Significant structural change in human c-Myc promoter G-quadruplex upon peptide binding in potassium.

Project description:We selected the G-quadruplex motif located in the nuclease-hypersensitive elements (NHE) III1 region of the c-Myc promoter and for the first time performed its interaction studies with a designed peptide (QW10). Our CD results showed that the peptide bound to the c-Myc G-quadruplex and induced a significant blue shift in the positive peak of 20 nm in KCl alone or with 40wt% PEG200 or 20wt% PEG8000 in comparison to NaCl. Our Native Gel results confirmed that peptide binding destabilized the duplex and stabilized the unimolecular G-quadruplex and not binding to i-motif. UV thermal results confirmed destabilization of bimolecular structure and stabilization of unimolecular G-quadruplex. QW10 showed preferential binding towards c-MYC promoter G4 with binding constant (K b) values of the order of 0.05 ± 0.2 μM, 0.12 ± 0.1 μM and 0.05 ± 0.3 μM for complexes in K+ alone or 40wt% PEG 200 or 20wt% PEG 8000 respectively. QW10 showed preferential cytotoxicity with IC50 values of 11.10 μM and 6.44 μM after 72 and 96 hours' incubation on Human Breast Carcinoma MDA-MB 231 cells and was found to be non-toxic with Human Embryonic Kidney (HEK-1) cells. Interestingly, we observed reduction of c-Myc gene expression by 2.5 fold due to QW10 binding and stabilizing c-MYC G4. Our study for the first time provides an expanded overview of significant structural change in human c-Myc promoter G-quadruplex upon peptide binding in potassium.

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