Key Points

CREB-family transcription factors (ATF1, cyclic AMP–responsive element-binding protein 1, and cAMP-responsive element modulator) were deleted in mpkCCD cells to assess their roles in Aqp2 gene transcription.Clustered regularly interspaced short palindromic repeats/CRISPR–associated protein 9 knockout of all three transcription factors together strongly reduced the ability of vasopressin to increase aquaporin 2 mRNA and protein.Re-expression of any of the three restored the vasopressin response, indicating overlapping roles of the three transcription factors.

Background

Water homeostasis is regulated by the peptide hormone arginine vasopressin, which promotes water reabsorption in the renal collecting duct. The regulation of aquaporin 2 (Aqp2) gene transcription is a key mechanism through which arginine vasopressin modulates water transport as disruption of this mechanism leads to water balance disorders. Therefore, an important goal is to understand the regulatory processes that control Aqp2 gene transcription. Although CREB (cyclic AMP–responsive element-binding protein 1 [CREB1]) has been proposed as the primary transcription factor responsible for Aqp2 transcription, recent evidence challenges this view, suggesting that other CREB-like transcription factors, including ATF1 and cAMP-responsive element modulator (CREM), may play a role.

Methods

We used the clustered regularly interspaced short palindromic repeats/CRISPR–associated protein 9 gene-editing system to delete Atf1, Creb1, and Crem in mpkCCD cells, an immortalized mouse collecting duct cell line. These cell lines were then exposed to the vasopressin analog, dDAVP, to assess the role of these transcription factors in regulating Aqp2 expression. AQP2 protein levels were measured by immunoblotting, and RNA-seq was used to analyze changes in Aqp2 mRNA abundance as well as other transcriptomic changes.

Results

Deletion of all three transcription factors (ATF1, CREB1, and CREM) together led to a significant reduction in the vasopressin-induced upregulation of AQP2 protein, confirming their role in regulating Aqp2 expression. RNA-seq data showed that Aqp2 mRNA levels mirrored changes in protein abundance, supporting the idea that these transcription factors affect Aqp2 transcription. Rescue experiments in triple knockout cells showed that expressing any of the three transcription factors restored the response to vasopressin. Additional RNA-seq analyses show similar transcriptomic changes when rescuing with ATF1, CREB1, or CREM.

Conclusions

Regulation of Aqp2 transcription was not dependent on CREB alone but instead occurred through any of the three CREB-family transcription factors (ATF1, CREB1, or CREM). RNA-seq studies show that these three transcription factors regulate highly overlapping sets of mRNA species.