Reduced nephron endowment in the common mouse line is due to misexpression by aberrant enhancer-promoter interactions in the transgene.

Unlabelled: Lifelong 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 popular transgenic mouse line was 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 the transgene within an intron of on chr1. We generated Hi-C datasets from NPCs isolated from the mice, the control mice, and the ; mice that exhibited restored nephron number compared with mice, and mapped the precise integration of and transgenes to chr1 and chr14, respectively. No changes in topology, accessibility, or expression were observed within the 50-megabase region centered on in mice compared with control mice. By contrast, we identified an aberrant regulatory interaction between a distal enhancer and the promoter contained within the transgene. Increasing the to locus ratio or removing one allele in mice, caused severe renal hypoplasia. Furthermore, CRISPR disruption of within the transgene ( ) restored nephron endowment to wildtype levels and abolished the stoichiometric effect. Data from genetic and biochemical studies together suggest that in SIX3 interferes with SIX2 function in NPC renewal through its C-terminal domain.

Significance: Using 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 of in the line which was correlated with disruption of nephrogenesis. Disruption of within the transgene restored nephron numbers to control levels; further genetic and biochemical studies suggest that interferes with -mediated regulation of NPC renewal.