The glucocorticoid receptor inhibits NFκB by interfering with serine-2 phosphorylation of the RNA polymerase II carboxy-terminal domain

• Published in: Genes & development Vol. 14; no. 18; pp. 2314 - 2329
• Main Authors: Nissen, Robert M., Yamamoto, Keith R.
• Format: Journal Article
• Language: English
• Published: Cold Spring Harbor Laboratory Press 15-09-2000
• Subjects: intracellular cell adhesion molecule-1; NF-kB protein; Research Paper
• ISSN: 0890-9369; 1549-5477
• DOI: 10.1101/gad.827900

Glucocorticoids repress NFκB-mediated activation of proinflammatory genes such as interleukin-8 (IL-8) and ICAM-1. Our experiments suggest that the glucocorticoid receptor (GR) confers this effect by associating through protein–protein interactions with NFκB bound at each of these genes. That is, we show that the GR zinc binding region (ZBR), which includes the DNA binding and dimerization functions of the receptor, binds directly to the dimerization domain of the RelA subunit of NFκB in vitro and that the ZBR is sufficient to associate with RelA bound at NFκB response elements in vivo. Moreover, we demonstrate in vivo and in vitro that GR does not disrupt DNA binding by NFκB. In transient transfections, we found that the GR ligand binding domain is essential for repression of NFκB but not for association with it and that GR can repress an NFκB derivative bearing a heterologous activation domain. We used chromatin immunoprecipitation assays in untransfected A549 cells to infer the mechanism by which the tethered GR represses NFκB-activated transcription. As expected, we found that the inflammatory signal TNFα stimulated preinitiation complex (PIC) assembly at the IL-8 and ICAM-1 promoters and that the largest subunit of RNA polymerase II (pol II) in those complexes became phosphorylated at serines 2 and 5 in its carboxy-terminal domain (CTD) heptapeptide repeats (YSPTSPS); these modifications are required for transcription initiation. Remarkably, GR did not inhibit PIC assembly under repressing conditions, but rather interfered with phosphorylation of serine 2 of the pol II CTD.