CREB-Family Transcription Factors and Vasopressin-Mediated Regulation of Aqp2 Gene Transcription

Background Water homeostasis is regulated by the peptide hormone arginine vasopressin (AVP), which promotes water reabsorption in the renal collecting duct. The regulation of Aqp2 gene transcription is a key mechanism through which AVP 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. While CREB (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 CREM, may play a role. Methods We employed the CRISPR/Cas9 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 analysis show similar transcriptomic changes when rescuing with either ATF1, CREB1 or CREM. Conclusions Regulation of Aqp2 transcription is not dependent on CREB alone, but instead can occur via 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.