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...

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 monoacety- In eukaryotes, histone proteins associate with DNA to form nucleosomes that are folded into higher order chromatin structures. Differences in higher order chromatin structures, which are important prerequisites for numerous biological processes including cellular proliferation, differentiation, development, gene expression, genome stability, and cancer, are thought to be realized by a variety of posttranslational modifications of histone N termini, particularly of histones H3 and H4. Besides acetylation, histones are subjected to phosphorylation, methylation, ubiquitination, ADP-ribosylation, and deamidation (1, 2). Distinct combinations of covalent histone modifications including lysine acetylation, lysine and arginine methylation, and serine phosphorylation form the basis of the histone code hypothesis (3-5). This hypothesis proposes that a pre-existing modification affects subsequent modifications on histone tails and that these modifications generate unique surfaces for the binding of various proteins or protein complexes responsible for higher order chromatin organization and gene activation and inactivation. Some of the histone-modifying enzymes (e.g. lysine methyltransferases) are, when deregulated, considered to be involved in carcinogenesis (6).Histone H4 is typically acetylated at lysines 5, 8, 12, and 16, methylated at arginine 3 and lysine 20, and phosphorylated at serine 1 (3,7,8). Unlike the dynamic process of histone acetylation and phosphorylation, histone methylation is regarded as a relatively static long-term signal with a low turnover of the methyl group. Whereas arginine can be either mono-or dimethylated (the latter in symmetric or asymmetric form), lysine methylation can occur as a mono-, di-, or trimethylated derivative. In contrast to histone H3 methylation, H4-Lys 20 was long considered to be maximally dimethylated in mammals (9). Most recently, however, Sarg et al. (10) conducted a mass spectrometric analysis with a newly developed hydrophilic interaction chromatographic method enabling the simultaneous separation of methylated and acetylated forms and, for the first time, found in vivo evidence that H4-Lys 20 is also trimethylated in mammalian tissue. Moreover, in rat liver and kidney the proportion of trimethylated histone H4 increases during aging. In Raji and K562 cells the trimethylated form was ...

The replacement linker histones H1 0 and H5 are present in frog and chicken erythrocytes, respectively, and their accumulation coincides with cessation of proliferation and compaction of chromatin. These cells have been analyzed for the affinity of linker histones for chromatin with cytochemical and biochemical methods. Our results show a stronger association between linker histones and chromatin in chicken erythrocyte nuclei than in frog erythrocyte nuclei. Analyses of linker histones from chicken erythrocytes using capillary electrophoresis showed H5 to be the subtype strongest associated with chromatin. The corresponding analyses of frog erythrocyte linker histones using reverse-phase high performance liquid chromatography showed that H1 0 dissociated from chromatin at somewhat higher ionic strength than the three additional subtypes present in frog blood but at lower ionic strength than chicken H5. Which of the two H1 0 variants in frog is expressed in erythrocytes has thus far been unknown. Amino acid sequencing showed that H1 0 -2 is the only H1 0 subtype present in frog erythrocytes and that it is 100% acetylated at its N termini. In conclusion, our results show differences between frog and chicken linker histone affinity for chromatin probably caused by the specific subtype composition present in each cell type. Our data also indicate a lack of correlation between linker histone affinity and chromatin condensation.

Histone H1 is a family of nucleosomal proteins that exist in a number of subtypes. These subtypes can be modified after translation in various ways, above all by phosphorylation. Increasing levels of H1 phosphorylation has been correlated with cell cycle progression, while both phosphorylation and dephosphorylation of histone H1 have been linked to the apoptotic process. Such conflicting results may depend on which various apoptosis-inducing agents cause apoptosis via different apoptotic pathways and often interfere with cell proliferation. Therefore, we investigated the relation between apoptosis and H1 phosphorylation in Jurkat cells after apoptosis induction via both the extrinsic and intrinsic pathways and by taking cell cycle effects into account. After apoptosis induction by anti-Fas, no significant dephosphorylation, as measured by capillary electrophoresis, or cell cycle-specific effects were detected. In contrast, H1 subtypes were rapidly dephosphorylated when apoptosis was induced by camptothecin. We conclude that histone H1 dephosphorylation is not connected to apoptosis in general but may be coupled to apoptosis by the intrinsic pathway or to concomitant growth inhibitory signaling.