The functional significance of mono-, di-, and trimethylation of lysine residues within histone proteins remains unclear. Antibodies developed to selectively recognize each of these methylated states at histone H3 lysine 9 (H3 Lys9) demonstrated that mono- and dimethylation localized specifically to silent domains within euchromatin. In contrast, trimethylated H3 Lys9 was enriched at pericentric heterochromatin. Enzymes known to methylate H3 Lys9 displayed remarkably different enzymatic properties in vivo. G9a was responsible for all detectable H3 Lys9 dimethylation and a significant amount of monomethylation within silent euchromatin. In contrast, Suv39h1 and Suv39h2 directed H3 Lys9 trimethylation specifically at pericentric heterochromatin. Thus, different methylated states of H3 Lys9 are directed by specific histone methyltransferases to "mark" distinct domains of silent chromatin.

Histone H1 isoforms isolated from asynchronously grown HeLa cells were subjected to enzymatic digestion and analyzed by nano-flow reversed-phase high performance liquid chromatography (RP-HPLC) tandem mass spectrometry (MS/MS) on both quadrupole ion trap and linear quadrupole ion trap-Fourier transform ion cyclotron resonance mass spectrometers. We have observed all five major isoforms of histone H1 (H1.1, H1.2, H1.3, H1.4, and H1.5) as well as a lesser studied H1, isoform H1.X. MS/MS experiments confirmed N-terminal acetylation on all isoforms plus a single internal acetylation site. Immobilized metal affinity chromatography in combination with tandem mass spectrometry was utilized to identify 19 phosphorylation sites on the five major H1 isoforms plus H1.X. Fourteen of these phosphorylation sites were located on peptides containing the cyclin dependent kinase (CDK) consensus motif (S/T)-P-X-Z (where X is any amino acid and Z is a basic amino acid). Five phosphorylation sites were identified in regions that did not fit the consensus CDK motif. One of these phosphorylation sites was found on the serine residue on the H1.4 peptide KARKSAGAAKR. The adjacent lysine residue to the phosphoserine was also shown to be methylated. This finding raises the question of whether the hypothesized "methyl/phos" switch could be extended to linker histones, and not exclusive to core histones.

Histone phosphorylation has long been associated with condensed mitotic chromatin; however, the functional roles of these modifications are not yet understood. Histones H1 and H3 are highly phosphorylated from late G2 through telophase in many organisms, and have been implicated in chromatin condensation and sister chromatid segregation. However, mutational analyses in yeast and biochemical experiments with Xenopus extracts have demonstrated that phosphorylation of H1 and H3 is not essential for such processes. In this study, we investigated additional histone phosphorylation events that may have redundant functions to H1 and H3 phosphorylation during mitosis. We developed an antibody to H4 and H2A that are phosphorylated at their respective serine 1 (S1) residues and found that H4S1/H2AS1 are highly phosphorylated in the mitotic chromatin of worm, fly, and mammals. Mitotic H4/H2A phosphorylation has similar timing and localization as H3 phosphorylation, and closely correlates with the chromatin condensation events during mitosis. We also detected a lower level of H4/H2A phosphorylation in 5-bromo-2-deoxyuridine-positive S-phase cells, which corroborates earlier studies that identified H4S1 phosphorylation on newly synthesized histones during S-phase. The evolutionarily conserved phosphorylation of H4/H2A during the cell cycle suggests that they may have a dual purpose in chromatin condensation during mitosis and histone deposition during S-phase.

Endothelial cell nitric oxide synthase (ECNOS) is a membrane-associated enzyme that generates endothelium-derived relaxing factor/nitric oxide (EDRF/NO) from L-arginine. We have suggested, from the cloning of the bovine ECNOS cDNA, that the presence of an N-myristoylation consensus sequence may impart its membrane localization since cytosolic forms of NOS do not contain such domains. To test the hypothesis that N-myristoylation is necessary for particulate ECNOS, we performed site-directed mutagenesis of the myristic acid acceptor site, Gly-2, and changed the glycine codon to alanine by a single nucleotide substitution. Expression of wild-type ECNOS in COS cells resulted in greater than 95% of the enzymatic activity in crude membrane fractions (as measured by the conversion of [3H]L-arginine to [3H]L-citrulline). In contrast, expression of the Gly-2 to Ala-2 mutant (G2A) demonstrated 8% ECNOS activity in membranes and 92% in the cytosol. The back mutation (from Ala-2 to Gly-2, A2G) restored ECNOS activity to the particulate fraction as seen with the wild type. Both wild-type membrane ECNOS and cytosolic G2A ECNOS activities were dependent on NADPH and calcium and were inhibited to the same extent by NG-monomethyl L-arginine (L-NMMA) and NG-nitro-L-arginine methyl ester (L-NAME). Moreover, kinetic analysis of these enzymes revealed similar Kms for L-arginine (2-4 microM, n = 3), demonstrating that the mutation did not affect ECNOS function. Thus, N-myristoylation is necessary for the membrane localization of ECNOS and may be of special significance for the basal or flow-induced production of NO by the endothelium.