Acetylation of the activated glucocorticoid receptor facilitates its proteasomal degradation thereby limiting hormonal signaling

The glucocorticoid (GC) receptor (GR) is a ligand-induced transcription factor regulating metabolism and inflammation. While homologous downregulation of ligand-bound GR limits the response, it also reduces hormone responsiveness resulting in glucocorticoid resistance. Regulatory mechanisms that refine this equilibrium are less understood. Here, we have identified seven lysine acetylation sites in the amino terminal domain of GR, lysine 154 within the regulatory AF-1 region being the dominant acetyl-acceptor. GR-Lys154 acetylation is mediated by p300/CBP in an exclusively agonist-induced manner and correlates with nuclear translocation and transcriptional activity. GR-Lys154 acetylation is removed by the NAD+ dependent deacetylase, SIRT1, indicating dynamic regulation of this mark. Notably, agonist-binding to both the wild type and acetylation-deficient mutant GR elicits similar short-term target gene expression. In contrast, upon extended treatment, the acetylation-deficient mutant shows impaired ubiquitination and higher protein stability with a concomitant increase in chromatin association and transactivation. Taken together, p300/CBP and SIRT1 fine-tune glucocorticoid response by siphoning acetylated GR for proteasomal degradation while GR deacetylation sustains the transcriptional cascade for prolonged periods. Overall design: To determine the impact of GR-Lys154 acetylation on Dex-dependent regulation of target gene expression, we performed RNA sequencing in cells expressing wildtype (WT), acetylation-deficient (K154R) or acetylation -mimetic (K154Q) GR. For this, we used CRISPR/Cas9 to knock out endogenous GR (gene: NR3C1) in HepG2 cells to generate HepG2 GR-/-.Subsequently, we transduced these cells with doxycycline-inducible psedotyped lentiviral constructs harboring cDNAs for Ty1-tagged WT-, K154R- and K154Q-GR respectively. Cells were induced with doxycycline to express the variants of GR followed by treatment with vehicle or 1 µM Dex for 6 hours before harvesting, RNA isolation, library preparation and sequencing. For each genotype, four biological replicates indicate two independent experiments with two independent transductions each.