Effects of PAX8 rescue following PAX8 loss on gene expression in proximal tubule epithelial cells.

PAX2 and PAX8 are homologous DNA-binding proteins that orchestrate kidney development, regulate urine concentration in the kidney medulla, and contribute to sensitivity to and recovery from acute kidney injury. In some contexts, Pax proteins can recruit KMT2C/D histone methyl-transferase complexes that deposit histone H3K4 methylation marks to promote transcription or recruit the Polycomb Repressor Complex to silence gene expression. However, the spectrum of targets and functions of Pax proteins in kidney physiology remains poorly defined. This project aimed to identify genes regulated by PAX proteins in the proximal tubule. Proximal tubule epithelial cells were derived from mice with floxed Pax2 and Pax8 alleles and expressing a constitutive tamoxifen-response CreERT2 (mice described in PMID: 32381599). During propagation, Pax2 was spontaneously recombined, yielding a Pax2-null, Pax8-conditional cell line which was named PT-22. In this submission, the PT-22 line was transduced with a Tet-on expression system linked to a hemagglutinin (HA)-tagged Pax8a transgene and a mixed population of successfully transduced cells selected. PAX8 was depleted from these modified PT-22 cells with tamoxifen, then rescued by inducing re-expression of Pax8a transgene with doxycycline. Gene expression was measured to determine gene targets altered by PAX8 re-expression.