Kidney Week

Abstract: FR-PO563

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

Session Information

• Fluid, Electrolyte, and Acid-Base Disorders: Basic
  October 25, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid, Electrolytes, and Acid-Base Disorders

• 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

• Chen, Lihe, National Institutes of Health, Bethesda, Maryland, United States

Lihe Chen, PhD

• Murillo-de-Ozores, Adrian Rafael, National Institutes of Health, Bethesda, Maryland, United States

Adrian Rafael Murillo-de-Ozores, PhD

• Park, Euijung, National Institutes of Health, Bethesda, Maryland, United States

Euijung Park, PhD

• Ou, Shuo-Ming, National Institutes of Health, Bethesda, Maryland, United States

Shuo-Ming Ou, MD, PhD

• Knepper, Mark A., National Institutes of Health, Bethesda, Maryland, United States

Mark A. Knepper, MD, PhD

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 GFP^negative/GFP^low relative to GFP^high, while those sgRNAs targeting negative regulators would show enrichment in GFP^high relative to GFP^negative/GFP^low. 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.