Hormone-mediated Dephosphorylation of Specific Histone H1 Isoforms

Abstract

We have previously shown a connection between histone H1 phosphorylation and the transcriptional competence of the hormone inducible mouse mammary tumor virus (MMTV) promoter. Prolonged exposure of mouse cells to dexamethasone concurrently dephosphorylated histone H1 and rendered the MMTV promoter refractory to hormonal stimulation and, therefore, transcriptionally unresponsive. Using electrospray mass spectrometry, we demonstrate here that prolonged dexamethasone treatment differentially effects a subset of the six somatic H1 isoforms in mouse cells. H1 isoforms H1.0, H1.1, and H1.2 are non-responsive to hormone whereas prolonged dexamethasone treatment effectively dephosphorylated the H1.3, H1.4, and H1.5 isoforms. The protein kinase inhibitor staurosporine, shown to dephosphorylate histone H1 and down-regulate MMTV in cultured cells, appears only to completely dephosphorylate the H1.3 isoform. These results suggest that dephosphorylation of specific histone H1 isoforms may contribute to the previously observed decrease in transcriptional competence of the MMTV promoter through the modulation of chromatin structure. In a broader sense, this work advances the hypothesis that post-translational modifications of individual histone H1 isoforms directly influence the transcriptional activation/repression of specific genes.Eukaryotic DNA is efficiently organized into the highly ordered yet dynamic structure called chromatin (1, 2). The primary, repeating structural unit of chromatin is the nucleosome and is composed of 146 base pairs of DNA wrapped around an octamer of four core histone proteins: H2A, H2B, H3, and H4 (3). In addition, the linker histone or histone H1 protein binds to the linker DNA between nucleosomes and is thought to function primarily in the condensation of chromatin into higher ordered structures (4). While the core histones are highly conserved in eukaryotes, with H4 being nearly invariant, histone H1 is much more evolutionarily diverse, and in mammals, consists of six somatic H1 isoforms (H1.0 through H1.5, using the nomenclature of Doenecke and co-workers, Ref. 5) and one testis-specific isoform (H1.t). The H1 isoforms share a common protein structural fold with the N-and C-terminal "tail" regions extending from a central globular domain. The central globular domain is primarily responsible for binding to DNA, while the tail regions function to modulate protein interaction with DNA (4). It is generally accepted that histone H1 binds nucleosomes at the dyad axis and contacts duplex DNA as it enters and exits the nucleosome (6). However, in the specific case of the 5 S rRNA gene from Xenopus, there is evidence to suggest that histone H1 binds asymmetrically to the nucleosome on a position off the dyad axis and within a DNA gyre (6 -8).A growing body of evidence suggests that the linker histones function in part to modulate transcriptional activity through chromatin organization (reviewed in Ref. 9). Initially, it was thought that histone H1 would have a general role in global gene activity, as ...

Histone H1 and chromatin interactions in human fibroblast nuclei after H1 depletion and reconstitution with H1 subfractions

“…Histones were then purified from co-extracted proteins, in particular HMG proteins, by gel exclusion chromatography on a Bio Gel P 100 column (46,47) and separated into two fractions: (a) main H1 histones and (b) H1°. The purity of the fractions was confirmed by MALDI-TOF mass spectrometry using a Voyager-DE Pro (Applied Biosystems, Framingham, MA), which showed four major peaks with m/z values in the range of 21,186 to 22,496, indicating that the main H1 histone subfraction contained H1.2-H1.5 and a single peak at 20,781 for the H1°subfraction, in close agreement with previously published data (48).…”

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

“…19,20 From these studies, it was determined that specific mouse histone H1 isoforms showed differential changes in phosphorylation based on treatment. These data pointed specifically to the mouse H1.3, H1.4, and H1.5 isoforms as targets of CDK-specific phosphorylation.…”

“…Of these, ions were observed which correspond in mass to all of the nonphosphorylated form of the peptides, whereas only masses corresponding to phosphorylated TPVK (m/z obs = 524.28), SPK (m/z obs= 411. 19), and SPAK (m/z obs = 482.19) were observed. No ions were observed which would correspond in mass to phosphorylated ATGAATPK or TPK.…”

“…HPLC purifications were performed as previously described, 19,20 except gradients were performed using an Agilent (Palo Alto, CA) HP Series 1100 HPLC system consisting of an autoinjector with a 400 μL sample loop, a Series 1100 binary pump, a Series 1100 diode array detector, and a BioRad Model 2110 fraction collector (Hercules, CA). The H1 isoform purifications were performed at a flow rate of 1 mL/min using a Vydac C4 column and a gradient of 5% B for 10 min, 5-33% B for 10 min, then 33-45% B over 65 min.…”

“…19,20 Utilizing this approach, we investigated the phosphorylation state of the specific H1 isoforms before and after prolonged treatment with dexamethasone and after exposure of cells to specific kinase inhibitors. Specifically, our previous experiments showed that the relative phosphorylation levels of the histone H1.3, H1.4, and H1.5 isoforms decrease after prolonged hormone exposure.…”