Effect of reduced glucocorticoid receptor-RNA affinity on dexamethasone treatment

The RNA binding ability of the glucocorticoid receptor (GR) remains an understudied area of GR regulation. Through in vitro binding assays, we identified a separation-of-function mutation (SoF) in the DNA-binding domain that greatly reduces RNA affinity with a slight reduction in DNA affinity. To study the effects of RNA binding on GR function, we first generated stable U2OS cells expressing wild-type GR-HaloTag, the SoF mutant, or a control (Ctrl) mutant that mimics the SoF's reduction in DNA affinity without impairing RNA affinity. We then treated the cells with dexamethsone every hour over three hours with 4-thiouridine labeling, isolated the labeled RNAs, and used deep sequencing to measure changes in the dexamethasone-driven transcriptome. 4sU-seq to measure differences in the transcriptome between glucocorticoid receptor constructs in U2OS over 3 hours of 100 nM dexamethasone treatment.