Abstract: FR-PO592
Guanine Quadruplex DNA in Human PKD1 Reveals a Mechanism for Second-Hit Mutagenesis in ADPKD
Session Information
- Cystic Kidney Diseases: Mechanisms and Models
October 25, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1201 Genetic Diseases of the Kidneys: Cystic
Authors
- Larson, Erik D., Western Michigan University Homer Stryker MD School of Medicine, Kalamazoo, Michigan, United States
- Parsons, Agata M., Western Michigan University Homer Stryker MD School of Medicine, Kalamazoo, Michigan, United States
- Bouma, Gerrit J., Western Michigan University Homer Stryker MD School of Medicine, Kalamazoo, Michigan, United States
- Vanden Heuvel, Greg, Western Michigan University Homer Stryker MD School of Medicine, Kalamazoo, Michigan, United States
Background
Somatic inactivation of the PKD1 gene causes the cystogenesis characteristic of ADPKD, however the mechanisms of mutagenesis are unresolved. PKD1 is prone to mutation in humans, but not in mice. Both genes share similar functions, but mice do not faithfully recapitulate ADPKD. A notable difference between the genes is that human PKD1 is repetitive in sequence, with the potential to form four-stranded guanine-quadruplex (G4) DNAs. G4 DNA structures naturally occur in the genome and have regulatory roles, but if left unresolved they inhibit polymerases and cause DNA breaks and mutagenesis.
Methods
We asked if human or mouse PKD1 form G4 DNA structures in human HEK293T or mouse mIMCD3 cells using chromatin immunoprecipitation and immunofluorescence microscopy with antibodies specific for G4 DNA. DNA break formation at the PKD1 locus was measured by chromatin immunoprecipitation with an antibody specfiic to a chromatin marker for DNA breaks, gammaH2AX.
Results
We found abundant G4 sequences in human, but not mouse, PKD1. Immunoprecipitations with a G4-specific antibody enriched for G4-forming regions of human PKD1 and we observed nuclear G4 DNAs by immunofluorescence microscopy in ADPKD tissue. Antibodies to G4 also colocalizes with the PKD1 locus, which was identified using a tagged version of dCAS9 targeted to the 3’ end of the gene. Repeat regions in PKD1 form alternative DNA structures in vitro and stabilization of G4 DNA by culturing HEK cells with the G4 ligand Phen-DC3 resulted in phosphorylation of histone H2AX (gammaH2AX) at PKD1. Mouse Pkd1 did not form G4 DNA or G4-induced DNA breaks.
Conclusion
Our results connect G4 formation with promoting the precursor DNA lesions in human PKD1 that inactivate the gene, which provokes a pathway of cystogenesis in ADPKD individuals. This reveals a mechanism for second hit mutagenesis of human PKD1 where unresolved G4 DNAs increase the risk of DNA damage, which then leads to loss of heterozygosity and disruption of polycystin-1 activity.
Funding
- NIDDK Support