Role of H1 phosphorylation in rapid GR exchange and function at the MMTV promoter

Abstract

Photobleaching technology has demonstrated in live cells that the glucocorticoid receptor (GR) exchanges rapidly at the mouse mammary tumor virus (MMTV) promoter. GR rapid exchange at MMTV depends on chaperone and proteasome activity, and as suggested by several in vitro and in vivo biochemical approaches, may also involve chromatin remodeling activity. Inhibition of H1 phosphorylation, chromatin remodeling and transcription from MMTV can be accomplished by long-term blocking of Cdk2 protein kinase activity. We find that Cdk2 is recruited by a tandem array of MMTV promoters, strengthening the model that this kinase has a specific role in MMTV transcription. We also demonstrate that following a brief Cdk2 inhibition by a selective cyclin-dependent kinase inhibitor (Roscovitine), transcription from MMTV drops and GR exchange at MMTV becomes slower, with a fraction of GR molecules now tightly bound at the promoter. This immobile fraction is absent elsewhere in the nucleus, suggesting a specific effect of Cdk2 inhibition on GR-MMTV interactions. These are the first live cell data suggesting a role for H1 phosphorylation, and by implication chromatin remodeling, in rapid exchange of GR at MMTV.