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PreprintCited by 10Renal and related cancersDevelopmental Biology and Gene RegulationGenomics and Chromatin Dynamics

The DOI matches its Crossref record; 1 of the 1 references it cites were found in a registry or an index.

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

Open access
Perl, Alison J., Liu, Han, Hass, Matthew, Adhikari, Nirpesh, Chaturvedi, Praneet, Hu, Yueh-Chiang, Jiang, Rulang, Liu, Yaping, Kopan, Raphael·2023·Journal of the American Society of Nephrology
DOI 10.1101/2023.10.06.561202PMC PMC10592608

Preprint — published as 10.1681/asn.0000000000000324 · Paper page

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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 line 1 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 Six2TGC tg/+ mice, the Cited1 CreERT2/+ control mice, and the Six2TGC tg/+ ; Tsc1 +/Flox,2 mice that exhibited restored nephron number compared with Six2TGC tg/+ mice, and 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, CRISPR disruption of Six3 within the transgene ( Six2TGC Δ 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. 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 Six3 in the Six2TGC tg line 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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Metadata

Authors
Perl, A. J., Liu, H., Hass, M., Adhikari, N., Chaturvedi, P., Hu, Y.-C., Jiang, R., Liu, Y., & Kopan, R.
Affiliations
Cincinnati Children's Hospital Medical Center (US)
Corresponding author
Kopan, Raphael
ORCID
Venue
Journal of the American Society of Nephrology
Publisher
openRxiv
Published
2023-10-09
Volume
35
Issue
5
Pages
566-577
ISSN
1046-6673 (ISSN-L)
DOI
10.1101/2023.10.06.561202
PMC
PMC10592608
OpenAlex
W4392756755
Cited by
10
References
1
Citations by year
2026: 4 · 2025: 4 · 2024: 1 · 2023: 1
FWCI
1.23
Type
journal article
Language
en
Open access
Locations
  • Journal of the American Society of Nephrology (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed Central (submittedVersion)
Funders
  • Cincinnati Children's Hospital Medical Center
  • National Institutes of Health (NIH-R01, 1s10od023410-01)
  • National Institute of Diabetes and Digestive and Kidney Diseases (F30DK123841, DK106225, NIH R01, DK123841, R01DK106225)
Topics
  • Health Sciences › Medicine › Oncology › Renal and related cancers
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
Keywords
nephron endowment, Six2, enhancer-promoter interactions, chromatin looping, nephron progenitor cells, transgene integration site, renal hypoplasia, TSC1

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