NIH Research Festival
–
–
Aqp2 plays a crucial role in water balance, yet the underlying regulation of Aqp2 transcription remains unclear. 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 the regulation of Aqp2 transcription. Two independent screens using the pooled sgRNA TF library were carried out and the cells were sorted based on GFPnegative, GFPlow, GFPmedium, and GFPhigh expression levels. The enrichment of sgRNA sequences in each population was analyzed by MAGeCK. 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. CRISPR/Cas9 identifies both positive and negative regulators for Aqp2 transcription, significantly expanding our understanding of Aqp2 gene regulation.
Scientific Focus Area: Systems Biology
This page was last updated on Tuesday, August 6, 2024