Raphael Kopan

Key Points Aberrant enhancer–promoter interactions detected by Hi-C drive ectopic expression of Six3 in the Six2TGCtg line. Disruption of Six3 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 Six2TGC 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 Six2TGC transgene. Genome-wide chromatin conformation capture (Hi-C) datasets were generated from nephron progenitor cells isolated from the Six2TGC +/tg mice, the Cited1 CreERT2/+ control mice, and the Six2TGC +/tg ; Tsc1 +/Flox mice that exhibited restored nephron number compared with Six2TGC +/tg mice. Modified transgenic mice lacking the C-terminal domain of Six3 were used to evaluate the mechanism of nephron number reduction in the Six2TGC +/tg mouse line. Results Targeted locus amplification revealed integration of the Six2TGC transgene within an intron of Cntnap5a on chr1, and Hi-C analysis mapped the precise integration of Six2TGC and Cited1 CreERT2 transgenes to chr1 and chr14, respectively. No changes in topology, accessibility, or expression were observed within the 50-megabase region centered on Cntnap5a in Six2TGC +/tg mice compared with control mice. By contrast, we identified an aberran