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. In the past, investigations concerning the biological significance of histone methylation were largely limited because of a lack of simple and sensitive analytical procedures for detecting this modification. The present work investigated the methylation pattern of histone H4 both in different mammalian organs of various ages and in cell lines by applying mass spectrometric analysis and a newly developed hydrophilic-interaction liquid chromatographic method enabling the simultaneous separation of methylated and acetylated forms, which obviates the need to work with radioactive materials. In rat kidney and liver the dimethylated lysine 20 was found to be the main methylation product, whereas the monomethyl derivative was present in much smaller amounts. In addition, for the first time a trimethylated form of lysine 20 of H4 was found in mammalian tissue. A significant increase in this trimethylated histone H4 was detected in organs of animals older than 30 days, whereas the amounts of mono-and dimethylated forms did not essentially change in organs from young (10 days old) or old animals (30 and 450 days old). Trimethylated H4 was also detected in transformed cells; although it was present in only trace amounts in logarithmically growing cells, we found an increase in trimethylated lysine 20 in cells in the stationary phase.In vivo methylation of the side chains of specific lysines, histidines, and arginines in proteins is a very common phenomenon in nature involving numerous classes of proteins in both prokaryotic and eukaryotic cells (1, 2). During the last several years, studies on the methylation of proteins have yielded many important observations. While these studies were under way, it was generally realized that protein methylation is far more complex and has more ramifications than originally assumed.Methylation is also a well known posttranslational modification reaction of histone proteins on lysine and/or arginine residues with a site selectivity for lysine methylation at specific positions in the N termini of histones H3 and H4. In combination with other posttranslational modifications, i.e. acetylation and phosphorylation, methylation seems to play a significant role in regulating nuclear functions. Thus, it has been suggested that distinct combinations of covalent histone modifications, also referred to as "histone code," provide a specific mark on the hydrophilic histone tails, which, when read by other proteins, cause specific downstream events finally inducing transi...

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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. Pulse-chase experiments show that phosphorylation during the S phase is a dynamic phosphorylation process with a limited phosphorylation maximum. In most H1 subtypes, phosphorylation occurs very rapidly at the G2/M transition with only small amounts of intermediate phosphorylated molecules. Phosphorylation of mouse H1c, however, occurs stepwise during this transition. Phosphorylated mouse histone subtypes from cells in mitosis contain four phosphate groups in the case of H1a, H1c, and H1e and five in the case of H1b and H1d. Comparison of the mouse phosphorylation pattern to that in rat C-6 glioma cells showed differences for H1e and H1d. By comparing the different phosphorylation patterns of the individual H1 variants during the cell cycle, we were able to classify the H1 histones into subtypes with low (H1a, H1c, H1(0)) and high (H1b, H1d, H1e) phosphorylation levels.

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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. HD1-A, HD1-B, and HD2 deacetylate all four core histones, although with different specificity. However, experiments with histones from different sources (hyperacetylated MELC and chicken histones) using antibodies specific for individually acetylated H4-lysine sites indicate that the enzymes recognize highly distinct acetylation patterns. Only RPD3-type deacetylase HD1-B is able to deacetylate the specific H4 di-acetylation pattern (position 12 and 5) introduced by the purified cytoplasmic histone acetyltransferase B after incubation with pure nonacetylated H4 subspecies. HD1-A and HD2 exist as phosphorylated forms. Dephosphorylation has dramatic, but opposite effects; whereas HD2 loses enzymatic activity upon dephosphorylation, HD1-A is activated with a change of specificity against acetylated H4 subspecies. The data suggest that different types of deacetylases interact with different and highly specific acetylation patterns on nucleosomes.

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 (1.3 kilobase pairs, 6 nucleosomes) at 270 -360 nM, corresponding to a molar ratio of six to eight H1 molecules per reconstituted nucleosome. To form aggregates with the histones H1t and H1b, however, greater amounts of protein were required. Furthermore, our results show that specific types of in vivo phosphorylation of the linker histone tails influence both the binding to mononucleosomes and the aggregation of polynucleosomes. S phase-specific phosphorylation with one to three phosphate groups at specific sites in the C terminus influences neither the binding to a mononucleosome nor the aggregation of polynucleosomes. In contrast, highly phosphorylated H1 histones with four to five phosphate groups in the C and N termini reveal a very high binding affinity to a mononucleosome but a low chromatin aggregation capability. These findings suggest that specific S phase or mitotic phosphorylation sites act independently and have distinct functional roles.H1 histones are a heterogeneous group of at least five subtypes with closely related but nonetheless different primary structures (1, 2). Two further H1 subtypes are known: the histone H1 0 , which is found in nonreplicative tissues (3, 4) and in rapidly proliferating cells (5), and the testis-specific histone variant H1t (6). The various linker histones containing a globular central region flanked by highly basic and hydrophilic C-and N-terminal tails (7,8) bind to the nucleosome and promote the organization of nucleosomes to a higher order structure (9, 10).There is evidence that histone H1 may interact differently with transcriptionally active and inactive regions of chromatin (11). Linker histones are also thought to modulate nucleosome position (12, 13) and to influence replication efficiency in vitro (14).The presence of this large number of various H1 histone subtypes and their possible posttranslational modifications, such as phosphorylation (15), make it very probable that H1 histones play numerous structural and functional roles in chromatin. Until now, no specific role for the various variants has been established although Kaludov et al. (16) showed that the mouse histone H1b binds preferentially to a regulatory sequence within a mouse H3.2 replication-dependent histone gene. Previous analysis of the structural role of H1 histones demonstrated that three subfractions of H1 histones differ in their effectiveness in condensing DNA fibers into ordered aggregates (17) and that histone subtype H1t, compared with other subtypes, dif...

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. H1 dephosphorylation is accompanied by chromatin condensation preceding the onset of typical chromatin oligonucleosomal fragmentation, whereas H2A.X hyperphosphorylation is strongly correlated to apoptotic chromatin fragmentation. Using various kinase inhibitors we were able to exclude some of the possible kinases which can be involved directly or indirectly in phosphorylation of histone H2A.X. Neither DNA-dependent protein kinase, protein kinase A, protein kinase G, nor the kinases driven by the mitogenactivated protein kinase (MAP) pathway appear to be responsible for H2A.X phosphorylation. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA), however, markedly reduced the induction of apoptosis in TNFa-treated cells with a simultaneous change in the phosphorylation pattern of histone H2A.X. Hyperphosphorylation of H2A.X in apoptotic cells depends indirectly on activation of caspases and nuclear scaffold proteases as shown in zVAD-(OMe)-fmkor zAPF-cmk-treated cells, whereas the dephosphorylation of H1 subtypes seems to be influenced solely by caspase inhibitors. Together, these results illustrate that H1 dephosphorylation and H2A.X hyperphosphorylation are necessary steps on the apoptotic pathway.