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 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 aberrant regulatory interaction between a Six2 distal enhancer and the Six3 promoter contained within the transgene. Increasing the Six2TGC(tg) to Six2 locus ratio or removing one Six2 allele in Six2TGC(+/tg) mice caused severe renal hypoplasia. Furthermore, clustered regularly interspaced short palindromic repeats disruption of Six3 within the transgene (Six2TGC(∆Six3CT)) 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 Six2TGC kidneys, SIX3 interferes with SIX2 function in nephron progenitor cell renewal through its C-terminal domain.