CRISPR/Cas9 Screening Identifies Key Transcription Factors Regulating Aqp2 Gene Transcription

Background: Aqp2, responsible for water reabsorption in kidney collecting duct cells, plays a crucial role in water balance. Prior translational studies demonstrated that dysregulation of Aqp2 transcription plays a central role in a variety of water balance disorders, yet the underlying regulation of Aqp2 transcription remains unclear. Methods: We engineered an mpkCCD cell line to express GFP under the control of the Aqp2 promoter and also stably express Cas9 and designed a lentiviral sgRNA library targeting over 1500 transcription factors (TFs) in the mouse genome. We then conducted CRISPR/Cas9 screens to identify TFs involved in regulation of Aqp2 transcription. Results: We demonstrated that the GFP provided a quantitative readout of Aqp2 and was sensitive to the stimulation of dDAVP and consequently could be used for the TF screen based on flow sorting. Two independent screens using the sgRNA TF library were carried out and the cells were sorted based on the GFP expression levels. The enrichment of sgRNA sequences was analyzed by MAGeCK. We hypothesize that sgRNAs targeting positive regulators of Aqp2 would be enriched in GFPnegative/GFPlow relative to GFPhigh, while those sgRNAs targeting negative regulators would show enrichment in GFPhigh relative to GFPnegative/GFPlow. The screens identify several known positive regulators for Aqp2 including Gata3, Nfat5, Hnf1b, Grhl2, Nr3c1, and Pax8. In addition, the analyses identify some novel positive regulators, among these are transcription factors that form DNA-binding heterodimers, transcription factors that are associated with histone modifications and chromatin remodelers, and several zinc finger TFs. For each of these candidates, cell lines with loss-of-function mutations were generated. Among the cell lines lacking the positive regulators, Gata3KO, Nfat5KO, Hnf1bKO, and Nr3c1KO cell lines showed no increase in the abundance of Aqp2 protein with dDAVP in contrast to control cell lines that showed large increases. Subsequent RNA-seq confirmed modulation of Aqp2 transcripts upon knockout of the candidate transcription factors. Further ATAC-seq analyses identified critical DNA elements involved in Aqp2 transcription. Conclusion: CRISPR/Cas9 identifies both positive and negative regulators for Aqp2 transcription, significantly expanding our understanding of Aqp2 gene regulation.