Histone H1 phosphorylation occurs site-specifically during interphase and mitosis: identification of a novel phosphorylation site on histone H1.

H1 histones, isolated from logarithmically growing and mitotically enriched human lymphoblastic T-cells (CCRF-CEM), were fractionated by reversed phase and hydrophilic interaction liquid chromatography, subjected to enzymatic digestion, and analyzed by amino acid sequencing and mass spectrometry. During interphase the four H1 subtypes present in these cells differ in their maximum phosphorylation levels: histone H1.5 is tri-, H1.

Histone H4-lysine 20 monomethylation is increased in promoter and coding regions of active genes and correlates with hyperacetylation.

Methylation and acetylation of position-specific lysine residues in the N-terminal tail of histones H3 and H4 play an important role in regulating chromatin structure and function. In the case of H3-Lys(4), H3-Lys(9), H3-Lys(27), and H4-Lys(20), the degree of methylation was variable from the mono- to the di- or trimethylated state, each of which was presumed to be involved in the organization of chromatin and the activation or repression of genes. Here we investigated the interplay between histone H4-Lys(20) mono- and trim-ethylation and H4 acetylation at induced (beta-major/beta-minor glo-bin), repressed (c-myc), and silent (embryonic beta-globin) genes during in vitro differentiation of mouse erythroleukemia cells.

Characterization of sequence variations in human histone H1.2 and H1.4 subtypes.

In humans, eight types of histone H1 exist (H1.1-H1.5, H1 degrees , H1t and H1oo), all consisting of a highly conserved globular domain and less conserved N- and C-terminal tails.

Histone H4 hyperacetylation precludes histone H4 lysine 20 trimethylation.

Posttranslational modification of histones is a common means of regulating chromatin structure and thus diverse nuclear processes. Using a hydrophilic interaction liquid chromatographic separation method in combination with mass spectrometric analysis, the present study investigated the alterations in histone H4 methylation/acetylation status and the interplay between H4 methylation and acetylation during in vitro differentiation of mouse erythroleukemia cells and how these modifications affect the chromatin structure. Independently of the type of inducer used (dimethyl sulfoxide, hexamethylenebisacetamide, butyrate, and trichostatin A), we observed a strong increase in non- and monoacetylated H4 lysine 20 (H4-Lys(20)) trimethylation.

Capillary electrophoresis analysis of histones, histone variants, and their post-translationally modified forms: a review.

Histones are the most intensively studied group of basic nuclear proteins and are of great importance with regard to the organization of chromatin structure and control of gene activity. The complexity of this protein family places very high demands on the analytical methods applied. The advent of high-performance capillary electrophoresis provided a promising new tool for their separation, which is an essential prerequisite for studying the biological role of this protein family.

Postsynthetic trimethylation of histone H4 at lysine 20 in mammalian tissues is associated with aging.

Methylation of the N-terminal region of histones was first described more than 35 years ago, but its biological significance has remained unclear. Proposed functions range from transcriptional regulation to the higher order packing of chromatin in progress of mitotic condensation. Primarily because of the recent discovery of the SET domain-depending H3-specific histone methyltransferases SUV39H1 and Suv39h1, which selectively methylate lysine 9 of the H3 N terminus, this posttranslational modification has regained scientific interest.

Hyperphosphorylation of histone H2A.X and dephosphorylation of histone H1 subtypes in the course of apoptosis.

Chromatin condensation paralleled by DNA fragmentation is one of the most important nuclear events occurring during apoptosis. Histone modifications, and in particular phosphorylation, have been suggested to affect chromatin function and structure during both cell cycle and cell death. We report here that phosphate incorporation into all H1 subtypes decreased rapidly after induction of apoptosis, evidently causing a strong reduction in phosphorylated forms of main H1 histone subtypes.

Age-dependent deamidation of asparagine residues in proteins.

Nonenzymatic deamidation of peptides and proteins represents an important degradation reaction occurring in vitro in the course of isolation or storage and in vivo during development and/or aging of cells. This review first presents a synopsis of the influence of structure on deamidation reaction proceeding via a five-membered succinimide intermediate, followed by an outline of procedures for separation and detection of deamidated forms. Selected examples for in vitro and in vivo deamidation are reviewed including the possible biological consequences of this protein degradation.

The N-terminally acetylated form of mammalian histone H1(o), but not that of avian histone H5, increases with age.

We report here on the HPCE separation of two chicken H5 histones, which do not show the heterogeneity (Gln/Arg) at residue 15 first found by Greenaway and Murray [Greenaway and Murray (1971) Nat. New Biol. 229, 233-238].

Different types of maize histone deacetylases are distinguished by a highly complex substrate and site specificity.

Enzymes involved in histone acetylation have been identified as important transcriptional regulators. Maize embryos contain three histone deacetylase families: RPD3-type deacetylases (HD1-B), nucleolar phosphoproteins of the HD2 family, and a third form unrelated to RPD3 and HD2 (HD1-A). Here we first report on the specificity of deacetylases for core histones, acetylated histone H4 subspecies, and acetylated H4-lysine residues.

Age-dependent deamidation of H1(0) histones in chromatin of mammalian tissues.

The composition of the H1(o) histone subfractions was examined in different rat and mouse tissues. Using reverse-phase HPLC and hydrophilic-interaction liquid chromatography we have found that the relative proportions of all four forms of H1(o) differ from tissue to tissue and from species to species. In principle, we observed an age-dependent increase in the amount of both the N-terminally acetylated (H1(o)a Asn-3 and H1(o)a Asp-3) and the deamidated forms of H1(o) (H1(o)a Asp-3 and H1(o)b Asp-3).

In vitro binding of H1 histone subtypes to nucleosomal organized mouse mammary tumor virus long terminal repeat promotor.

The binding of all known linker histones, named H1a through H1e, including H1(0) and H1t, to a model chromatin complex based on a DNA fragment containing the mouse mammary tumor virus long terminal repeat promotor was systematically studied. As for the histone subtype H1b, we found a dissociation constant of 8-16 nM to a single mononucleosome (210 base pairs), whereas the binding constant of all other subtypes varied between 2 and 4 nM. Most of the H1 histones, namely H1a, H1c, H1d/e, and H1(0), completely aggregate polynucleosomes (1.

The microheterogeneity of the mammalian H1(0) histone. Evidence for an age-dependent deamidation.

Histone H1(0) is known to consist of two subfractions named H1(0)a and H1(0)b. The present work was performed with the aim of elucidating the nature of these two subfractions. By using reversed-phase high performance liquid chromatography in combination with hydrophilic interaction liquid chromatography, we fractionated human histone H1(0) into even four subfractions.

Application of hydrophilic-interaction liquid chromatography to the separation of phosphorylated H1 histones.

A new two-step high-performance liquid chromatography (HPLC) procedure has been developed to separate modified histone H1 subtypes. Reversed-phase (RP) HPLC followed by hydrophilic-interaction liquid chromatography (HILIC) was used for analytical and semi-preparative scale fractionation of multi-phosphorylated H1 histone subtypes into their non-phosphorylated and distinct phosphorylated forms. The HILIC system utilizes the weak cation-exchange column PolyCAT A and an increasing sodium perchlorate gradient in a methanephosphonic acid-triethylamine buffer (pH 3.

Separation of acetylated core histones by hydrophilic-interaction liquid chromatography.

Hydrophilic-interaction liquid chromatography (HILIC) has recently been introduced as a highly efficient chromatographic technique for the separation of a wide range of solutes. The present work was performed with the aim of evaluating the potential utility of HILIC for the separation of postranslationally acetylated histones. The protein fractionations were generally achieved by using a weak cation-exchange column and an increasing sodium perchlorate gradient system in the presence of acetonitrile (70%, v/v) at pH 3.

In vivo phosphorylation of histone H1 variants during the cell cycle.

In vivo phosphorylation of the five histone H1 variants H1a-H1e including H1(0) in NIH 3T3 mouse fibroblasts was examined during the cell cycle by using a combination of HPLC techniques and conventional AU gel electrophoresis. Phosphorylation starts during the late G1 phase and increases throughout the S phase. In the late S phase, the H1 variants exist as a combination of molecules containing 0 or 1 (H1a, H1c), 0-2 (H1d), or 0-3 (H1b, H1e) phosphate groups with a share of unphosphorylated protein ranging between 35% and 75%, according to the particular subtype.

Effect of buffer composition on the migration order and separation of histone H1 subtypes.

The effects of different buffer concentrations and compositions on the elution order and separation of H1 histone subtypes and their phosphorylated modifications isolated from several species was studied using high-performance capillary electrophoresis (CE). Various cations and anions were tested in an untreated silica capillary and low pH buffers, in the presence of the dynamic coating agent hydroxypropylmethyl cellulose. It was found that the cations and anions of buffers have a remarkable influence on both the efficiency and the selectivity of protein separations.

Application of high-performance capillary electrophoresis to the analysis of H1 histones.

High-performance capillary electrophoresis for the separation of rat testis H1 histone variants and their phosphorylated modifications is described. The influence of buffer pH, hydroxypropylmethyl cellulose, and buffer concentration has been investigated. Under optimized conditions (500 mM phosphate buffer, pH 2, 0.

G1- and S-phase synthesis of histone H1 subtypes from mouse NIH fibroblasts and rat C6 glioma cells.

The rates of synthesis of histone H1 subtypes in synchronized mouse NIH 3T3 fibroblasts were compared with those of rat C6 glioma cells during the G0, G1, and S phases by using a combination of HPLC techniques and conventional gel electrophoresis. In the mouse cell line, all H1 subtypes, H1a-H1e including histone H1(0), were detectable. In the rat cell line, however, no histone H1a was found.

Alteration in proportions of histone H1 variants during the differentiation of murine erythroleukaemic cells.

We have investigated the changes in the relative amounts of histone H1 zero and all five H1 variants during the differentiation in vitro of Friend erythroleukaemic cells. Three different agents were used as inducers of differentiation: dimethyl sulphoxide, hexamethylenebisacetamide and sodium butyrate. By applying a combination of reverse-phase h.

Separation of phosphorylated histone H1 variants by high-performance capillary electrophoresis.

High-performance capillary electrophoresis (HPCE) was used to separate successfully distinct phosphorylated derivatives of individual histone H1 variants. With an untreated capillary (50 cm x 75 microns I.D.

High-performance capillary electrophoresis of core histones and their acetylated modified derivatives.

By using high-performance capillary electrophoresis, we have successfully separated rat liver core histones into several subfractions. Inconvenient interactions of the highly basic proteins with the capillary wall were eliminated by a phosphate buffer system containing 0.03% hydroxyprophylmethylcellulose.

Separation of rat tissue histone H1 subtypes by reverse-phase h.p.l.c. Identification and assignment to a standard H1 nomenclature.

H1 histones from rat liver and rat testis were separated by reverse-phase h.p.l.

Identification of ADP-ribosylated histones by the combined use of high-performance liquid chromatography and electrophoresis.

Reversed-phase high-performance liquid chromatography (HPLC) was employed for analysing mono- and oligo(ADP-ribosyl)ated histones. Under the chromatographic conditions described, the ADP-ribosylated histones showed similar retention times to the unmodified histones, although the molecular weight and the charge of the proteins are significantly altered by their modification. The simultaneous elution of unmodified and labelled modified histones was detected by two types of gel electrophoresis and by autoradiography.