Glucocorticoid-induced phosphorylation by CDK9 specifies the coactivator functions of transcriptional cofactor GRIP1 in macrophages

Glucocorticoid Receptor (GR) suppresses inflammation by activating anti-inflammatory and repressing pro-inflammatory genes. GR-interacting protein (GRIP)1 of the p160 family has emerged as a unique GR corepressor in macrophages (MF), however, whether GRIP1 contributes to GR-activated transcription, and what dictates its context-specific coactivator vs. corepressor properties is unknown. We report that loss of GRIP1 in human and mouse MF attenuated GR-mediated induction of several anti-inflammatory targets, revealing a non-redundant function of GRIP1 in coactivation. Moreover, glucocorticoid treatment of quiescent MF globally directed GRIP1 toward GR-bound genomic sites dominated by classic palindromic GREs, suggesting its dedicated role as a GR coactivator. Further, GRIP1 N-terminal region was phosphorylated at a serine cluster by Cyclin-Dependent Kinase (CDK)9, which was recruited into GC-induced GR:GRIP1:CDK9 ternary complexes, producing distinct GRIP1 phospho-isoforms at different GREs even associated with the same gene. Functionally, phosphorylation potentiated GRIP1 coactivator properties by facilitating its recruitment and/or creating novel protein:protein interaction surfaces in a GRE-specific manner. Strikingly, GRIP1 function as a GR corepressor was phosphorylation-independent; consistently, no phospho-GRIP1 or CDK9 was detected at GR transrepression sites near pro-inflammatory genes. Thus, liganded GR in MF restricts actions of its own coregulator via CDK9-mediated phosphorylation to a subset of complexes driving anti-inflammatory gene transcription. Overall design: 1. Analysis of Glucocorticoid receptor (NR3C1) cistromes in differentiated THP1 cells (human) and mouse bone marrow derived macrophages treated with dexamethasone in comparasion to untreated control, 2. Analysis of GRIP1 (Ncoa2) cistromes in differentiated THP1 cells (human) and mouse bone marrow derived macrophages treated with dexamethasone in comparasion to untreated control, 3) Analysis of phospho-S469 GRIP1(Ncoa2) cistrome in differentiated THP1 cells (human) treated with dexamethasone in comparasion to untreated control