Characterization of post-translational modifications of the linker histones H1 and H5 from chicken erythrocytes using mass spectrometry

Histone linker proteins H1 and H5 were purified from chicken erythrocyte cell nuclei under nondenaturing conditions. The purified linker histones were analyzed using in-solution enzymatic digestions followed by nanoflow reverse-phase high-performance liquid chromatography tandem mass spectrometry. We have identified all six major isoforms of the chicken histone H1 (H101, H102, H103, H110, H11R and H11L) and, in addition, the specialist avian isoform H5. In all the histone variants, both the acetylated and nonacetylated N (alpha)-terminal peptides were identified. Mass spectrometry analysis also enabled the identification of a wide range of post-translational modifications including acetylation, methylation, phosphorylation and deamidation. Furthermore, a number of amino acids were identified that were modified with both acetylation and methylation. These results highlight the extensive modifications that are present on the linker histone proteins, indicating that, similar to the core histones, post-translational modifications of the linker histones may play a role in chromatin remodelling and gene regulation.     

Extraction of histones H2A, H3 and H4 from yeast nuclei. Measurement of the extent of yeast histone acetylation following one-dimensional gel electrophoresis

Yeast histones H2A, H3 and H4 were specifically extracted from purified nuclei using a 2% NaCl/75% ethanol solution. The extraction resulted in the complete removal of H2A, H3 and H4 from the nuclear pellet, as monitored by SDS-polyacrylamide gel electrophoresis of the protein. The relative absence of nonhistone proteins from this histone subset simplifies the determination of the extent of histone modification in yeast. Levels of H4 acetylation were measured directly on Coomassie blue-stained Triton acid-urea gels and the levels verified by gel fluorography of the [3H]acetate-labeled histone.     

Pathways mediating the nuclear import of histones H3 and H4 in yeast.

The correct assembly of chromatin is necessary for the maintenance of genomic stability in eukaryotic cells. A critical step in the assembly of new chromatin is the cell cycle-regulated synthesis and nuclear import of core histones. Here we demonstrate that the nuclear import pathway of histones H3 and H4 is mediated by at least two karyopherins/importins, Kap123p and Kap121p. Cytosolic H4 is found associated with Kap123p and H3. Kap121p is also present in the H4-PrA-associated fractions, albeit in lesser amounts than Kap123p, suggesting that this Kap serves as an additional import receptor. We further demonstrate that cytosolic Kap123p is associated with acetylated H3 and H4. H3 and H4 each contain a nuclear localization signal (NLS) in their amino-terminal domains. These amino-terminal domains were found to be essential for the nuclear accumulation of H3 and H4-green fluorescent protein reporters. Each NLS mediated direct binding to Kap123p and Kap121p, and decreased nuclear accumulation of H3 and H4 NLS-green fluorescent protein reporters was observed in specific kap mutant strains. H3 and H4 are the first histones to be assembled onto DNA, and these results show that their import is mediated by at least two import pathways.     

Extraction of histones H2A,H3 and H4 from yeast nuclei: Measurement of the extent of yeast histone acetylation following one-dimensional gel electrophoresis

Yeast histones H2A, H3 and H4 were specifically extracted from purified nuclei using a 2% NaCl/75% ethanol solution. The extraction resulted in the complete removal of H2A, H3 and H4 from the nuclear pellet, as monitored by SDS-polyacrylamide gel electrophoresis of the protein. The relative absence of nonhistone proteins from this histone subset simplifies the determination of the extent of histone modification in yeast. Levels of H4 acetylation were measured directly on Coomassie blue-stained Triton acid-urea gels and the levels verified by gel fluorography of the [³H]acetate-labeled histone.     

Deposition of newly synthesized histones: new histones H2A and H2B do not deposit in the same nucleosome with new histones H3 and H4

We have developed procedures to study histone-histone interactions during the deposition of histones in replicating cells. Cells are labeled for 60 min with dense amino acids, and subsequently, the histones within the nucleosomes are cross-linked into an octameric complex with formaldehyde. These complexes are sedimented to equilibrium in density gradients and octamer and dioctamer complexes separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With reversal of the cross-link, the distribution of the individual density-labeled histones in the octamer is determined. Newly synthesized H3 and H4 deposit as a tetramer and are associated with old H2A and H2B. Newly synthesized H2A and H2B deposit as a dimer associated with old H2A, H2B, H3, and H4. The significance of these results with respect to the dynamics of histone interactions in the nucleus is discussed. Control experiments are presented to test for artifactual formation of these complexes during preparative procedures. In addition, reconstitution experiments were performed to demonstrate that the composition of these octameric complexes can be determined from their distribution on density gradients.     

Distribution of the core histones H2A.H2B.H3 and H4 during cell replication.

The distribution of newly synthesized core histones H2A, H2B, H3 and H4 relative to the DNA strand synthesized in the same generation has been examined in replicating Chinese Hamster ovary cells. Cells are grown for one generation in [14C]-lysine and thymidine, and then for one generation in [3H]-lysine and 5-bromodeoxyuridine (BrUdRib) and a further generation in unlabeled lysine and thymidine. This protocol produces equal amounts of unifilarly substituted and unsubstituted DNA. Monomer nucleosomes isolated from chromatin containing these two types of DNA can be distinguished by crosslinking with formaldehyde and banding to equilibrium in CsCl density gradients. The results indicate that the core histones are equally distributed between the two types of DNA. These findings are discussed in terms of current models for chromatin replication; they do not support any long term association of newly replicated histones with either the leading or lagging side of the replication fork.     

Phylogenetic analysis of the core histones H2A, H2B, H3, and H4.

Despite the ubiquity of histones in eukaryotes and their important role in determining the structure and function of chromatin, no detailed studies of the evolution of the histones have been reported. We have constructed phylogenetic trees for the core histones H2A, H2B, H3, and H4. Histones which form dimers (H2A/H2B and H3/H4) have very similar trees and appear to have co-evolved, with the exception of the divergent sea urchin testis H2Bs, for which no corresponding divergent H2As have been identified. The trees for H2A and H2B also support the theory that animals and fungi have a common ancestor. H3 and H4 are 10-fold less divergent than H2A and H2B. Three evolutionary histories are observed for histone variants. H2A.F/Z-type variants arose once early in evolution, while H2A.X variants arose separately, during the evolution of multicellular animals. H3.3-type variants have arisen in multiple independent events.     

Global loss of Set1-mediated H3 Lys4 trimethylation is associated with silencing defects in Saccharomyces cerevisiae

Post-translational histone modifications, such as acetylation, phosphorylation, ubiquitination, and methylation, have been correlated with regulation of gene expression. In Saccharomyces cerevisiae, Set1 has been identified as the sole histone methyltransferase required for histone H3 lysine 4 (Lys(4)) methylation. Yeast cells that do not express Set1 have several apparent phenotypes, including slow growth and defects in telomere, HML, and rDNA silencing. However, the mechanism by which the Set1 methyltransferase mediates differential histone H3 methylation (mono-, di-, and tri-) is still not understood, and the involvement of domains or regions in Set1 contributing to H3 Lys(4) methylation has not been well characterized. In this study, the N terminus of Set1 was shown to be important for global and gene specific histone H3 trimethylation. We show that Set1 trimethyl-defective mutants can rescue a set1Delta slow growth defect. In contrast, Set1 trimethyl mutants were defective in telomere, rDNA, HML, and HMR silencing. Taken together, these data suggest that histone H3 Lys(4) trimethylation is required for proper silencing, while mono- and/or dimethylation is sufficient for cell growth.     

Combinatorial modification of human histone H4 quantitated by two-dimensional liquid chromatography coupled with top down mass spectrometry

Quantitative proteomics has focused heavily on correlating protein abundances, ratios, and dynamics by developing methods that are protein expression-centric (e.g. isotope coded affinity tag, isobaric tag for relative and absolute quantification, etc.). These methods effectively detect changes in protein abundance but fail to provide a comprehensive perspective of the diversity of proteins such as histones, which are regulated by post-translational modifications. Here, we report the characterization of modified forms of HeLa cell histone H4 with a dynamic range >10(4) using a strictly Top Down mass spectrometric approach coupled with two dimensions of liquid chromatography. This enhanced dynamic range enabled the precise characterization and quantitation of 42 forms uniquely modified by combinations of methylation and acetylation, including those with trimethylated Lys-20, monomethylated Arg-3, and the novel dimethylated Arg-3 (each <1% of all H4 forms). Quantitative analyses revealed distinct trends in acetylation site occupancy depending on Lys-20 methylation state. Because both modifications are dynamically regulated through the cell cycle, we simultaneously investigated acetylation and methylation kinetics through three cell cycle phases and used these data to statistically assess the robustness of our quantitative analysis. This work represents the most comprehensive analysis of histone H4 forms present in human cells reported to date.     

An octamer of core histones in solution: central role of the H3-H4 tetramer in the self-assembly.

The association of histones H2A, H2B, H3, and H4 in solution has been studied. In 2 M NaCl and at neutral pH they can assemble in a complex in which each histone is present in equimolar amounts. The complex has a weight average molecular weight of 98,000 (+/- 3700) and a sedimentation coefficient (so20,w) of 4.8. The value of the weight average molecular weight and the histone stoichiometry indicate that the complex is an octamer. The pairs of histones H2A,H2B and H3,H4 studied separately under identical conditions only associated as equimolar complexes consistent with dimeric and tetrameric structures, respectively. The stability of the core histone octamer is a function of the ionic strength, pH, and concentration of protein. The octamer dissociates by losing dimers of H2A,H2B until the main complexes existing in solution are the H3.H4 tetramer and the H2A.H2B dimer. This process is reversible upon reestablishing the original conditions.     

An octamer of histones H3 and H4 forms a compact complex with DNA of nucleosome size.

Equimolar mixtures of histones H3 and H4 have been reconstituted onto DNA of nucleosome core size. Two distinct complexes are formed in a relative abundance that depends on the starting ratio of H3 + H4 to DNA. One of these complexes contains two H3-H4 dimers for each DNA molecule, and has a sedimentation coefficient of 7.5S. The other complex contains an octamer consisting of four H3-H4 dimers, and has a sedimentation coefficient of 10.4S. On the basis of these measurements, we conclude that the octamer complex (but not the tetramer complex) is a fully folded, compact structure resembling the nucleosome.     

Identification of peptide superagonists for a self-K-ras-reactive CD4+ T cell clone using combinatorial peptide libraries and mass spectrometry.

The proliferative responses of a human CD4+ T cell clone 29.15.2, reactive with a self-K-ras-derived peptide (3EYKLVVVGAGGVGKSALT20), were tested using a set of X9 combinatorial peptide libraries containing the flanking residues (EYKLVXXXXXXXXXSALT, where X indicates random amino acids). Certain peptide libraries, such as EYKLVXXXXXXM XXSALT and EYKLVXXXXXXXH XSALT, stimulated a marked proliferation of 29.15.2. However, no combinations of substitutions tested, such as EYKLVXXXXXXMH XSALT, exhibited additive effects. We subsequently synthesized peptides with degenerate sequences (a mixture of 480 species), where each position is composed of the wild-type (wt) residue or of amino acids that induced the proliferation of 29.15.2, in positional scanning. Interestingly, one fraction of degenerate peptides, separated by reverse-phase HPLC, stimulated much higher proliferation than did the wt; in addition, the retention time of this fraction was distinct from that of the wt. Mass spectrometry analysis of this fraction and flanking fractions identified five peptide species that exhibit strong signals in a manner that parallels the antigenic activity. Finally, 17 candidate peptide sequences were deduced from mass spectrometry and hydrophobicity scoring results, of which two peptides (EYKLVVVGAGGML KSALT and EYKLVVVGAGGMI KSALT) did induce 52- and 61-fold stronger proliferation, respectively, compared with the wt. These findings indicate that: 1) synthetic peptides that carry "the best" residue substitution at each position of combinatorial peptide libraries do not always exhibit superagonism, and 2) such a drawback can be overcome with the use of mass spectrometry. This approach provides new perspectives for the accurate and efficient identification of peptide superagonists.     

The interaction of histone H3 with histone H4 and with other histones studied by 19F nuclear magnetic resonance.

The behaviour, upon variations in ionic strength, pH and temperature of 19F nuclear nuclear magnetic resonance signals of the trifluoroacetonylated derivative of histone H3 is compared with those of the H3-H4 complex and of the Hv fraction (an equimolar mixture of H2A, H2B, H3 and h4). The line width of the 19F-labelled histone H3 signals increases with ionic strength or pH, an effect consistent with aggregation of the protein. In the case of H3-H4 complex or Hv the line width decreases at intermediate ionic strengths (0.1-0.25 M NaCl). This effect is interpreted as the consequence of the formation of a well defined structure with ionic strength. At high salt concentrations the line width increases as a consequence of the final rigid quaternary structure or of the formation of higher aggregates.     

Association of nucleosome core particle DNA with different histone oligomers. Transfer of histones between DNA-(H2A,H2B) and DNA-(H3,H4) complexes

In non-denaturing low ionic strength gels, the titration of core DNA with H2A,H2B produces five well-defined bands. Quantitative densitometry and cross-linking experiments indicate that these bands are due to the successive binding of H2A,H2B dimers to core DNA. Only two bands are obtained with DNA-(H3,H4) samples. The slower of these bands is broad and presumably corresponds to two complexes containing one and two H3,H4 tetramers, respectively. In gels of higher ionic strength, DNA-(H2A,H2B) samples produce an ill-defined band, suggesting that the lifetime of the complexes containing H2A,H2B is relatively short. However, the low intensity of the free DNA band observed in these gels indicates that most of the DNA is associated with H2A,H2B. In agreement with this, our results obtained using different techniques (sedimentation, cross-linking, trypsin and nuclease digestions, and thermal denaturation) demonstrate that the association of H2A,H2B with core DNA occurs in free solution in both the absence and presence of NaCl (0.1 to 0.2 M). The low mobilities of DNA-(H2A,H2B) complexes, together with sedimentation and DNase I digestion results, indicate that the DNA in these complexes is not folded into the compact structure found in the core particle. Furthermore, non-denaturing gels have been used to study the dynamic properties of DNA-(H2A,H2B) and DNA-(H3,H4) complexes in 0.2 M-NaCl. Our results show that: (1) H2A,H2B and H3,H4 can associate, respectively, with DNA-(H3,H4) and DNA-(H2A,H2B) to produce complexes containing the four core histones; (2) DNA-(H2A,H2B) and DNA-(H3,H4) are able to transfer histones to free core DNA; (3) an exchange of histone pairs takes place between DNA-(H2A,H2B) and DNA-(H3,H4) and produces complexes with the same histone composition as that of the normal nucleosome core particle; and (4) although both histone pairs can exchange, histones H2A,H2B show a higher tendency than H3,H4 to migrate from one incomplete core particle to another. The complexes produced in these reactions have the same compact structure as reconstituted core particles containing the four core histones. Our kinetic results are consistent with a reaction mechanism in which the transfer of histones involves direct contacts between the reacting complexes. The possible participation of these spontaneous reactions on the mechanism of nucleosome assembly is discussed.     

H3 Cys-110 is in close proximity to the C-terminal regions of H2B and H4 in a nucleosome core with an altered internal arrangement of histones

A particle obtained by nuclease digestion of nucleohistone complexes prepared by direct mixing of histones with DNA in 0.15 M NaCl was indistinguishable by composition and physical properties from nucleosome cores prepared under the same conditions from nucleohistone preannealed in 0.6 M NaCl. We show here that different photo-cross-links form when these particles are prepared from H3 labeled with photoaffinity reagents on the unique histone H3 cysteine. H3-H3 histone dimers were dominant when the particles were prepared by dilution of the nucleohistone from 0.6 M NaCl while H3-H2B and H3-H4 histone dimers were prominent if the nucleohistone complex was prepared directly in 0.15 M NaCl. Peptide mapping of the novel H3-H4 and H3-H2B dimers showed that Cys-110 of histone H3 is cross-linked to the 18 amino acid C-terminal end of H4 or to the 66 amino acid C-terminal half of H2B.     

Quantitative analysis of phospholipids in functionally important membrane domains from RBL-2H3 mast cells using tandem high-resolution mass spectrometry.

We recently showed that ligand-mediated cross-linking of FcepsilonRI, the high-affinity receptor for immunoglobulin E, on RBL-2H3 mast cells results in its co-isolation with detergent-resistant membranes (DRM) and its consequent tyrosine phosphorylation by the co-localized tyrosine kinase Lyn that is a critical early event in signaling by this receptor [Field et al. (1997) J. Biol. Chem. 272, 4276-4280]. As part of efforts to determine the structural bases for these interactions, we examined the phospholipid composition of DRM vesicles isolated from RBL-2H3 cells under conditions that preserve FcepsilonRI association. We used positive and negative mode electrospray Fourier transform ion cyclotron resonance mass spectrometry to compare quantitatively the phospholipid composition of isolated DRM to that of total cell lipids and to a plasma membrane preparation. From these analyses, over 90 different phospholipid species were spectrally resolved and unambiguously identified; more than two-thirds of these were determined with a precision of +/-0.5% (absolute) or less. Quantitative characterization of lipid profiles shows that isolated DRM are substantially enriched in sphingomyelin and in glycerophospholipids with a higher degree of saturation as compared to total cellular lipids. Plasma membrane vesicles isolated from RBL-2H3 cells by chemically induced blebbing exhibit a degree of phospholipid saturation that is intermediate between DRM and total cellular lipids, and significant differences in the headgroup distribution between DRM and plasma membranes vesicles are observed. DRM from cells with cross-linked FcepsilonRI exhibit a larger ratio of polyunsaturated to saturated and monounsaturated phospholipids than those from unstimulated cells. Our results support and strengthen results from previous studies suggesting that DRM have a lipid composition that promotes liquid-ordered structure. Furthermore, they demonstrate the potential of mass spectrometry for examining the role of membrane structure in receptor signaling and other cellular processes.     

Determination of the antioxidant 3-tert.-butyl-4-hydroxyanisole in rat plasma using high-resolution gas chromatography—mass spectrometry

A method is described for the determination of the antioxidant 3-tert.-butyl-4-hydroxyanisole in rat plasma using high-resolution capillary gas chromatography—mass spectrometry with selective ion monitoring. Following the addition of the isomer 2-tert.-butyl-4-hydroxyanisole, used as an internal standard, extraction was made with n-hexane and the extract derivatized with heptafluorobutyric anhydride.The gas chromatographic separation was carried out on a SE-52 fused silica capillary column and the derivatized 3-tert.-butyl-4-hydroxyanisole and its isomer detected by recording the intensities of their common fragment ion at m/e 361. The sensitivity of the method allowed the antioxidant to be measured in 0.1-ml rat plasma samples down to a level of 10 ng/ml with a high degree of specificity and accuracy. The method has been applied to a preliminary pharmacokinetic study in rats after oral dosage.