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bioRxiv · 10.1101/2023.10.06.561202

Reduced nephron endowment in the common Six2-TGCtg mouse line is due to Six3 misexpression by aberrant enhancer-promoter interactions in the transgene

Perl, A. J.·Liu, H.·Hass, M.·Adhikari, N.·Chaturvedi, P.·Hu, Y.-C.·Jiang, R.·Liu, Y.·Kopan, R.
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Abstract

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 Six2TGC transgenic mouse line1 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 Six2TGC transgene within an intron of Cntnap5a on chr1. We generated Hi-C datasets from NPCs isolated from the Six2TGCtg/+ mice, the Cited1CreERT2/+control mice, and the Six2TGCtg/+;Tsc1+/Flox,2 mice that exhibited restored nephron number compared with Six2TGCtg/+mice, and mapped the precise integration of Six2TGC and Cited1CreERT2transgenes to chr1 and chr14, respectively. No changes in topology, accessibility, or expression were observed within the 50-megabase region centered on Cntnap5a in Six2TGCtg/+ 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 Six2TGCtg to Six2 locus ratio or removing one Six2 allele in Six2TGCtg/+ mice, caused severe renal hypoplasia. Furthermore, CRISPR disruption of Six3 within the transgene (Six2TGC{Delta}Six3CT) restored nephron endowment to wildtype levels and abolished the stoichiometric effect. Data from genetic and biochemical studies together suggest that in Six2TGC, 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 of Six3 in the Six2TGCtgline which was correlated with disruption of nephrogenesis. Disruption of Six3 within the transgene restored nephron numbers to control levels; further genetic and biochemical studies suggest that Six3 interferes with Six2-mediated regulation of NPC renewal.

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Perl, A. J., Liu, H., Hass, M., Adhikari, N., Chaturvedi, P., Hu, Y.-C., Jiang, R., Liu, Y., Kopan, R.. 2023-10-09. Reduced nephron endowment in the common Six2-TGCtg mouse line is due to Six3 misexpression by aberrant enhancer-promoter interactions in the transgene. https://doi.org/10.1101/2023.10.06.561202

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