TSC22D, WNK and NRBP gene families exhibit functional buffering and evolved with Metazoa for cell volume regulation

The ability to sense and respond to osmotic fluctuations is critical for the maintenance of cellular integrity. We used gene co-essentiality analysis to identify an unappreciated relationship between TSC22D2, WNK1 and NRBP1 in regulating cell volume homeostasis. Each of these genes have paralogs and are functionally buffered for osmo-sensing and cell volume control. Within seconds of hyperosmotic stress, TSC22D, WNK and NRBP family members physically associate into biomolecular condensates, a process that is dependent on intrinsically disordered regions (IDRs). A close examination of these protein families across metazoans revealed that TSC22D genes evolved alongside a domain in NRBPs that specifically binds to TSC22D proteins, which we have termed NbrT (NRBP binding region with TSC22D), and this co-evolution is accompanied by rapid IDR length expansion in WNK family kinases. Our study reveals that TSC22D, WNK and NRBP genes evolved in metazoans to co-regulate rapid cell volume changes in response to osmolarity. Genome-wide CRISPR gene knockout screens were performed in human HAP1 cells across TWN body component knockout queries. Genetic interactions were identified by comparison of each query's fitness profile to screens in HAP1 WT cells documented from Aregger et. al. 2020. WNK463 drug screen were performed in human HAP1 cells and treated with 2.5uM WNK463. The drug screen is compared with DMSO vehicle control to look for sensitizers and maskers.