Cellular tools for studying PKD1 regulation in autosomal dominant polycystic kidney disease - Project Summary Autosomal Dominant Polycystic Kidney Disease (ADPKD) is incurable, and cellular systems for modeling the genetics of cystogenesis are needed to understand the underlying pathways and to design preventatives. Mice do not faithfully recapitulate natural ADPKD, presenting a major limitation to cystogenesis research. In humans, cysts are formed when the PKD1 gene product, polycystin-1, is disrupted. Most cysts originate from a somatic “second hit” inactivation of one PKD1 allele, which joins the inherited pathogenic allele to result in biallelic inactivation. It happens hundreds of times in human ADPKD, but in mice Pkd1 heterozygotes do not form cysts and the gene escapes mutation. We discovered why, and will use that information here to create novel cellular tools for ADPKD research. We find that a defining characteristic of human PKD1 is an unusual capacity to form guanine-quadruplex (G4) DNA structures. G4 DNA is found throughout the genome where it helps regulate gene expression, but it is unusually abundant in human PKD1. G4 DNA is highly stable in the cell, concentrated in the 5’ region of genes, and is normally resolved by G4-specific helicases to prevent polymerase stalling and DNA breaks. G4-stabilizing ligands, reporter assays, and direct visualization in living cells has validated the contributions of G4 DNA to cell physiology as well as documented the propensity of the structure to increase locus-specific mutagenesis. Human PKD1 harbors ~10-times more G4 DNA than mouse Pkd1, and we observed G4 formation in human PKD1 by chromatin immunoprecipitation. Incubation of human cells with a G4-stabilizing ligand resulted in DNA breaks in PKD1, and lowered its expression. G4 DNA does not measurably form in mouse Pkd1 or cause DNA breaks in the gene. Since mutagenic inactivation of human PKD1 causes cysts and G4 DNA provokes breaks in the gene, G4 DNA regulates cystogenesis. Based on that premise, we will develop pluripotent stem cells altered for PKD1 G4 content, thereby creating a unique tool for cystogenesis research. In Aim 1 we will transfer two blocks of human PKD1 G4 repeats to mouse Pkd1 in induced pluripotent stem cells, thereby humanizing the murine ortholog in a cellular system capable of renal cell development. In Aim 2, we will delete two G4-rich regions (introns 21 and 22) from human PKD1 in pluripotent stem cells and in HEK293T, providing two novel cell-based reagents for studying the impact of G4 DNA on polycystin-1 activity in a genetic background amenable to cystogenesis studies. Following their validation as G4-modified PKD1 cell models, reagents will be made freely available for studies on ADPKD mechanisms and potential therapeutic interventions.
