Histone H1 Phosphorylation by Cdk2 Selectively Modulates Mouse Mammary Tumor Virus Transcription through Chromatin Remodeling

Abstract

Transcriptional activation of the mouse mammary tumor virus (MMTV) promoter by ligand-bound glucocorticoid receptor (GR) is transient. Previously, we demonstrated that prolonged hormone exposure results in displacement of the transcription factor nuclear factor 1 (NF1) and the basal transcription complex from the promoter, the dephosphorylation of histone H1, and the establishment of a repressive chromatin structure. We have explored the mechanistic link between histone H1 dephosphorylation and silencing of the MMTV promoter by describing the putative kinase responsible for H1 phosphorylation. Both in vitro kinase assays and in vivo protein expression studies suggest that in hormone-treated cells the ability of cdk2 to phosphorylate histone H1 is decreased and the cdk2 inhibitory p21 protein level is increased. To address the role of cdk2 and histone H1 dephosphorylation in the silencing of the MMTV promoter, we used potent cdk2 inhibitors, Roscovitine and CVT-313, to generate an MMTV promoter which is associated predominantly with the dephosphorylated form of histone H1. Both Roscovitine and CVT-313 block phosphorylation of histone H1 and, under these conditions, the GR is unable to remodel chromatin, recruit transcription factors to the promoter, or stimulate MMTV mRNA accumulation. These results suggest a model where cdk2-directed histone H1 phosphorylation is a necessary condition to permit GR-mediated chromatin remodeling and activation of the MMTV promoter in vivo.

ACKNOWLEDGMENTS

We thank Laurent Meijer (Roscoff) and Dov Schiffman (CV Therapeutics) for Roscovitine and CVT-313, respectively. We thank H. K Kinyamu for technical help. We are grateful to K. Brown, B. Deroo, H. K. Kinyamu, C. Weinberger, J. O'Bryan, and J. Cidlowski for critical review of the manuscript. We also thank Bonnie Deroo for help with the figures.

This work was supported in its initial stages by grants to T.K.A. from the National Cancer Institute of Canada and the Canadian Breast Cancer Research Initiative of Canada.