Abstract
AbstractLifelong kidney function relies on the complement of nephrons generated during mammalian development from a mesenchymal nephron progenitor cell (NPC) population. Low nephron endowment confers increased susceptibility to chronic kidney disease. We asked whether reduced nephron numbers in the popularSix2TGCtransgenic mouse line1was due to disruption of a regulatory gene at the integration site or to ectopic expression of a gene(s) contained within the transgene. Targeted locus amplification identified integration of theSix2TGCtransgene within an intron ofCntnap5aon chr1. We generated Hi-C datasets from NPCs isolated from theSix2TGCtg/+mice, theCited1CreERT2/+control mice, and theSix2TGCtg/+;Tsc1+/Flox,2mice that exhibited restored nephron number compared withSix2TGCtg/+mice, and mapped the precise integration ofSix2TGCandCited1CreERT2transgenes to chr1 and chr14, respectively. No changes in topology, accessibility, or expression were observed within the 50-megabase region centered onCntnap5ainSix2TGCtg/+mice compared with control mice. By contrast, we identified an aberrant regulatory interaction between aSix2distal enhancer and theSix3promoter contained within the transgene. Increasing theSix2TGCtgtoSix2locus ratio or removing oneSix2allele inSix2TGCtg/+mice, caused severe renal hypoplasia. Furthermore, CRISPR disruption ofSix3within the transgene (Six2TGCΔSix3CT) restored nephron endowment to wildtype levels and abolished the stoichiometric effect. Data from genetic and biochemical studies together suggest that inSix2TGC,SIX3 interferes with SIX2 function in NPC renewal through its C-terminal domain.SignificanceUsing high-resolution chromatin conformation and accessibility datasets we mapped the integration site of two popular transgenes used in studies of nephron progenitor cells and kidney development. Aberrant enhancer-promoter interactions drive ectopic expression ofSix3in theSix2TGCtgline which was correlated with disruption of nephrogenesis. Disruption ofSix3within the transgene restored nephron numbers to control levels; further genetic and biochemical studies suggest thatSix3interferes withSix2-mediated regulation of NPC renewal.
Publisher
Cold Spring Harbor Laboratory