Reduced Nephron Endowment in Six2-TGCtg Mice Is Due to Six3 Misexpression by Aberrant Enhancer-Promoter Interactions in the Transgene.

Key Points: Aberrant enhancer–promoter interactions detected by Hi-C drive ectopic expression of in the Six2TGCtg line. Disruption of in the Six2TGCtg line restores nephron number, implicating SIX3 interference with SIX2 function in nephron progenitor cell renewal.

Background: Lifelong kidney function relies on the complement of nephrons generated during mammalian development from a mesenchymal nephron progenitor cell population. Low nephron endowment confers increased susceptibility to CKD. Reduced nephron numbers in the popular transgenic mouse line may be due to disruption of a regulatory gene at the integration site and/or ectopic expression of a gene(s) contained within the transgene.

Methods: Targeted locus amplification was performed to identify the integration site of the transgene. Genome-wide chromatin conformation capture (Hi-C) datasets were generated from nephron progenitor cells isolated from the mice, the control mice, and the ; mice that exhibited restored nephron number compared with mice. Modified transgenic mice lacking the C-terminal domain of were used to evaluate the mechanism of nephron number reduction in the mouse line.

Results: Targeted locus amplification revealed integration of the transgene within an intron of on chr1, and Hi-C analysis 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, clustered regularly interspaced short palindromic repeats disruption of within the transgene () restored nephron endowment to wild-type levels and abolished the stoichiometric effect.

Conclusions: These findings broadly demonstrate the utility of Hi-C data in mapping transgene integration sites and architecture. Data from genetic and biochemical studies together suggest that in kidneys, SIX3 interferes with SIX2 function in nephron progenitor cell renewal through its C-terminal domain.