Preserving Polycystin-1 Function in ADPKD by Targeting G4 DNA in PKD1

Background: Individuals inheriting a pathogenic germline mutation in PKD1 form cysts upon somatic inactivation of the remaining allele, called second hit, or from dosage effects that reduce polycystin-1. Defining the molecular regulators of those processes is vital for designing strategies to inhibit cystogenesis. Methods: We have investigated the molecular characteristics of PKD1 using chromatin-IP and IF-microscopy with the goal of defining the mechanism of second hit PKD1 inactivation in human ADPKD. Results: We find that PKD1 in humans, but not mice, contains abundant and distributed guanine-quadruplex sequences that support the formation of four-stranded structures called G4 DNA. G4 DNA is well known to regulate gene expression at a small cost to gene stability, so their concentration in PKD1 reveals a pathway for gene regulation and second hit mutations. We find that G4 DNA forms within PKD1 in ADPKD and normal renal tissue, confirming that PKD1 naturally harbors these structures in the cell. Relevant to ADPKD, G4-stabilizing ligands both lowered polycystin-1 mRNA and caused DNA breaks in PKD1. Consistent with a model whereby G4 DNA formation lowers polycystin-1 levels, targeting G4 DNA at the PKD1 locus with G4-destabilizers enhanced mRNA levels and also prevented DNA breaks in the gene. Conclusion: Our results reveal a mechanism for second hit mutagenesis of human PKD1 where unresolved G4 DNAs lead to DNA breaks that then promote loss of heterozygosity and disruption of polycystin-1 activity. Preserving polycystin-1 function in individuals inheriting pathogenic PKD1 alleles is therefore an attractive strategy for blocking cystogenesis and reducing ADPKD severity. Since G4 DNA is known to be a binding platform for G4-specific activities in the cell, our results open new research fronts for clarifying the molecular dynamics of PKD1 gene structure and function. Funding: NIDDK Support