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...

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-, H1.4 di-, and H1.3 and H1.2, only monophosphorylated. The phosphorylation is site-specific and occurs exclusively on serine residues of SP(K/A)K motifs. The phosphorylation sites of histone H1.5 from mitotically enriched cells were also examined. In contrast to the situation in interphase, at mitosis there were additional phosphorylations, exclusively at threonine residues. Whereas the tetraphosphorylated H1.5 arises from the triphosphosphorylated form by phosphorylation of one of two TPKK motifs in the C-terminal domain, namely Thr 137 and Thr 154 , the pentaphosphorylated H1.5 was the result of phosphorylation of one of the tetraphosphorylated forms at a novel nonconsensus motif at Thr 10 in the N-terminal tail. Despite the fact that histone H1.5 has five (S/T)P(K/A)K motifs, all of these motifs were never found to be phosphorylated simultaneously. Our data suggest that phosphorylation of human H1 variants occurs nonrandomly during both interphase and mitosis and that distinct serineor threonine-specific kinases are involved in different cell cycle phases. The order of increased phosphorylation and the position of modification might be necessary for regulated chromatin decondensation, thus facilitating processes of replication and transcription as well as of mitotic chromosome condensation.The nucleosome core, which consists of 146 bp of DNA wrapped 1.75 times around an octamer of core histones, represents the fundamental subunit of chromatin (for review, see Ref. 1). The H1 or linker histones are associated with the core histone-DNA complex and with the linker DNA between adjacent nucleosomes. Histone H1 is phosphorylated in a cell cycle-dependent manner: levels of H1 phosphorylation are usually lowest in the G 1 phase and rise continuously during S and G 2 . The M phase, where chromatin is highly condensed, shows the maximum number of phosphorylated sites. The individual H1 subtypes, however, differ in their degree of phosphorylation during the cell cycle (2, 3). A number of studies indicate that H1 phosphorylation is more likely involved in chromatin decondensation than in condensation (4). H1 phosphorylation seems to destabilize chromatin structure, thus weakening its binding to DNA. This decondensation of chromatin may give the DNA access to factors involved in transcription and replication in G 1 and S as well as to condensing factors active during mitosis (5). Recent studies demonstrate that H1 phosphorylation regulates specific gene expression in vivo and that it acts by mimicking the partial removal of H1 (6).The H1 histones consist of a globular central region flanked by short N...

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 trimethylation and H4 acetylation at induced (␤-major/␤-minor globin), repressed (c-myc), and silent (embryonic ␤-globin) genes during in vitro differentiation of mouse erythroleukemia cells. By using chromatin immunoprecipitation, we found that the ␤-major and ␤-minor promoter and the ␤-globin coding regions as well as the promoter and the transcribed exon 2 regions of the highly It has been proposed that distinct post-translational histone modifications act sequentially or in combination to form a "histone code" within chromatin (1). Acetylation and methylation of specific histone lysine residues can serve as a mark of either euchromatin or silent heterochromatin. Although methylation of H3-Lys 4 , H3-Lys 36 , and H3-Lys 79 has been linked to transcriptional activation and protection of euchromatin, H3-Lys 9 , H3-Lys 27 , and H4-Lys 20 methylation is generally thought to be associated with gene repression and heterochromatin formation (2-4). In this regard it must be noted that histone lysine residues can be mono-, di-, or trimethylated (5), thus extending the coding potential of a methylatable lysine position. Previous studies, however, focused only on detection of H3 (for review see Refs. 3 and 4) or H4 (6 -8) lysine methylation regardless of the methylation status. Recently, it was shown that a distinction between di-and trimethylation of various lysines of histone H3 is important for processes of transcriptional regulation or gene silencing (9 -11). Moreover, studies that focused on the in vivo distribution of mono-, di-, and trimethylated H3-Lys 9 and H3-Lys 27 demonstrate that mono-and dimethylated H3-Lys 9 and H3-Lys 27 are specifically localized to silent domains within euchromatin, whereas trimethylated H3-Lys 9 and monomethylated H3-Lys 27 were enriched at pericentric heterochromatin (12, 13). In contrast to findings suggesting a role for H4-Lys 20 methylation in regulating gene expression, a recently published study demonstrates that H4-Lys et al. (19), who found that the Drosophila epigenetic activator ASH-1, a histone methyltransferase, activates transcription by dimethylation of H3-Lys 4 , H3-Lys 9 , and H4-Lys 20 at the promoter of target genes. Significant differences in subnuclear localization of the mono-and trimethyl versions of histone H4-Lys

No abstract