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.DNA in eukaryotic nuclei is highly packaged into chromatin by two molecules each of the core histone proteins H2A, H2B, H3, and H4 and one molecule of linker histone H1 (44). In addition, to the intrinsic stearic considerations of wrapping DNA around the histone octamer, the posttranslation modification of the core histones have come under increased scrutiny (22,44). Numerous studies support a strong link between transcriptional regulation and the remodeling of chromatin structure through the acetylation of core histones H3 and H4 (20, 40, 46). The acetylation of core histones in vivo is presumed to play a role in increasing the accessibility of transcription factors to the promoters of target genes (23). More recently, the Mi-2 ATPase complex, which contains chromatin remodeling activity, has been linked to both DNA methylation and histone deacetylation (39, 47).The role of histone H1 in the regulation of transcription is less clear, but there is evidence that histone H1 interacts differentially with transcriptionally active and inactive regions of chromatin (29). Indeed, studies in Xenopus and Tetrahymena thermophila have ruled out an exclusive role for histone H1 phosphorylation in chromatin condensation (31,36). However, other studies in mammals and T. thermophila have found a correlation between transcriptional activation and decreased amounts of histone H1 (9,12,14). Thus, it is plausible, given the role of histone H1 in the packaging of the nucleosome, that posttranslational modifications of this protein may also be involved in transcript...

Expression of thymidylate synthase in human cells is an early G1 event regulated by CDK4 and p16INK4A but not E2F

“…CDK2 Inhibitor III and CDK4 inhibitor have been previously described (Bhattacharjee et al, 2001;Zhu et al, 2003). Roscovitine, CDK2 Inhibitor III and CDK4 inhibitor were purchased from Calbiochem (Darmstadt, Germany) and dissolved in DMSO.…”

From Roscovitine to CYC202 to Seliciclib – from bench to bedside: discovery and development

“…CDK1 and CDK2 are also capable of phosphorylating the CTD of RNA Pol II so have potential roles in transcriptional control [232][233][234][235]. Roscovitine may also inhibit viral transcription via its effects on chromatin structure [236].…”

Global Changes in and Characterization of Specific Sites of Phosphorylation in Mouse and Human Histone H1 Isoforms upon CDK Inhibitor Treatment Using Mass Spectrometry

“…23 The doses used in these experiments are in the effective concentration range reported for these drugs. 16,22,24,25 In addition, cells were subjected to prolonged treatment (48 h) with 10 nM dexamethasone. Cells were harvested by trypsinization, collected into PBS, and fixed in cold 70% ethanol overnight at 4 °C.…”

“…For this work, we are using a combination of enzymatic proteolysis and peptide sequencing by mass spectrometry. We have previously reported 16 that the CDK2 inhibitor, CVT313, blocked H1 phosphorylation in mouse 1471.1 cells and so are most interested in determining those sites of phosphorylation. After these phosphorylation sites were determined, we generated an antibody that is phospho-site specific for H1.3/H1.4 isoforms.…”

“…For a number of years, the Archer laboratory has been investigating the mechanisms of glucocorticoid-mediated transcription activation. [15][16][17][18] In these studies, the hormone inducible mouse mammary tumor virus (MMTV) promoter has been utilized. It was reported that when mouse cells were exposed to prolonged treatment with dexamethasone, a significant decrease in the level of histone H1 phosphorylation was observed.…”