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1.
Su X Jacob NK Amunugama R Lucas DM Knapp AR Ren C Davis ME Marcucci G Parthun MR Byrd JC Fishel R Freitas MA 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2007,850(1-2):440-454
Here we describe the use of reverse-phase liquid chromatography mass spectrometry (RPLC-MS) to simultaneously characterize variants and post-translationally modified isoforms for each histone. The analysis of intact proteins significantly reduces the time of sample preparation and simplifies data interpretation. LC-MS analysis and peptide mass mapping have previously been applied to identify histone proteins and to characterize their post-translational modifications. However, these studies provided limited characterization of both linker histones and core histones. The current LC-MS analysis allows for the simultaneous observation of all histone PTMs and variants (both replacement and bulk histones) without further enrichment, which will be valuable in comparative studies. Protein identities were verified by the analysis of histone H2A species using RPLC fractionation, AU-PAGE separation and nano-LC-MS/MS. 相似文献
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Patterns of histone acetylation 总被引:9,自引:0,他引:9
A W Thorne D Kmiciek K Mitchelson P Sautiere C Crane-Robinson 《European journal of biochemistry》1990,193(3):701-713
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Simon Messner Matthias Altmeyer Hongtao Zhao Andrea Pozivil Bernd Roschitzki Peter Gehrig Dorothea Rutishauser Danzhi Huang Amedeo Caflisch Michael O. Hottiger 《Nucleic acids research》2010,38(19):6350-6362
The chromatin-associated enzyme PARP1 has previously been suggested to ADP-ribosylate histones, but the specific ADP-ribose acceptor sites have remained enigmatic. Here, we show that PARP1 covalently ADP-ribosylates the amino-terminal histone tails of all core histones. Using biochemical tools and novel electron transfer dissociation mass spectrometric protocols, we identify for the first time K13 of H2A, K30 of H2B, K27 and K37 of H3, as well as K16 of H4 as ADP-ribose acceptor sites. Multiple explicit water molecular dynamics simulations of the H4 tail peptide into the catalytic cleft of PARP1 indicate that two stable intermolecular salt bridges hold the peptide in an orientation that allows K16 ADP-ribosylation. Consistent with a functional cross-talk between ADP-ribosylation and other histone tail modifications, acetylation of H4K16 inhibits ADP-ribosylation by PARP1. Taken together, our computational and experimental results provide strong evidence that PARP1 modifies important regulatory lysines of the core histone tails. 相似文献
5.
Histories of the endosymbiont nucleus of the binucleate dinoflagellate Peridinium foliaceum Stein were prepared from isolated nuclei and analyzed by peptide mapping, ammo acid composition, and two-dimensional gel electrophoresis. Using these criteria, we identified the presence of two HI-like histories and the core histones H3, H2A, H2B, and H4. These histones are similar but not identical to those of the endosymbiont nucleus of the bi-nucleate dinoflagellate Peridinium balticum Levander. 相似文献
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Post-translational modifications of histones play crucial roles in the genetic and epigenetic regulation of gene expression from chromatin. Studies in mammals and yeast have found conserved modifications at some residues of histones as well as non-conserved modifications at some other sites. Although plants have been excellent systems to study epigenetic regulation, and histone modifications are known to play critical roles, the histone modification sites and patterns in plants are poorly defined. In the present study we have used mass spectrometry in combination with high performance liquid chromatography (HPLC) separation and phospho-peptide enrichment to identify histone modification sites in the reference plant, Arabidopsis thaliana. We found not only modifications at many sites that are conserved in mammalian and yeast cells, but also modifications at many sites that are unique to plants. These unique modifications include H4 K20 acetylation (in contrast to H4 K20 methylation in non-plant systems), H2B K6, K11, K27 and K32 acetylation, S15 phosphorylation and K143 ubiquitination, and H2A K144 acetylation and S129, S141 and S145 phosphorylation, and H2A.X S138 phosphorylation. In addition, we found that lysine 79 of H3 which is highly conserved and modified by methylation and plays important roles in telomeric silencing in non-plant systems, is not modified in Arabidopsis. These results suggest distinctive histone modification patterns in plants and provide an invaluable foundation for future studies on histone modifications in plants. 相似文献
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Here we report a novel two-dimensional liquid chromatography-mass spectrometry (2D LC-MS) method that combines offline hydroxyapatite (HA) chromatography with online reversed-phase liquid chromatography-mass spectrometry (HA/RP LC-MS). The 2D LC-MS method was used to enrich and characterize histones and their posttranslational modifications. The 2D HA/RP LC-MS approach separates histones based on their relative binding affinity to DNA and relative hydrophobicity. HA/RP separations showed improvement in the number of histone isoforms detected as compared with one-dimensional RP LC-MS of acid-extracted histones. The improved histone fractionation resulted in better detection of lower abundant histone variants as well as their posttranslationally modified isoforms. Histones eluted from the HA/RP in the following order: H1, H2A/H2B heterodimers followed by H3/H4 heterotetramers, as predicted from their spatial organization in nucleosomes for binding affinity to DNA. Comparison between HA-purified and acid-extracted histones revealed similar histone profiles with the exception that the HA fractions showed a greater number of H1 isoforms. Two elution conditions were also examined: batch elution and salt gradient elution. Although both elution techniques were able to fractionate the histones sufficiently, the salt gradient approach has the most potential for purification of selected histone isoforms. 相似文献
8.
The pool of histones in the nucleosol and cytosol of proliferating Friend cells is small, uneven and chasable.
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This study examines the histones pools in the nucleosol and cytosol of proliferating Friend cells. By using the conventional approach, detectable amounts of these molecules were found in both compartments; however, only H3 and H2B were identified in nucleosol, and H3, H2B and H4 in cytosol. The authenticity of each of these histones was verified by two independent methods, migration in SDS/polyacrylamide gels and peptide mapping. When the sensitivity of the approach was increased by radiolabelling with 125I, two additional proteins, migrating as H2A and H4, were observed in nucleosol. Even by this approach, however, H1 was not detected. Direct quantitative measurements of the histones in both compartments indicated that these pools are uneven and small. This was found also in experiments involving inhibition of protein synthesis by cycloheximide. Considered together, our data do not support the idea of the existence of preformed histone heterocomplexes or octamers. Instead the assembly of nucleosomes during replication occurs by a successive deposition of individual core histones. 相似文献
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Posttranslational modifications of histones are involved in regulation of chromatin structure and gene activity. Whereas the modifications of the core histones H2A, H2B, H3, and H4 have been extensively studied, our knowledge of H1 modifications remained mainly limited to its phosphorylation. Here we analyzed the composition of histone H1 variants and their modifications in two human cell lines and nine mouse tissues. Use of a hybrid linear ion trap-orbitrap mass spectrometer facilitated assignment of modifications by high resolution and low ppm mass accuracy for both the precursor and product mass spectra. Across different tissues we identified a range of phosphorylation, acetylation, and methylation sites. We also mapped sites of ubiquitination and report identification of formylated lysine residues. Interestingly many of the mapped modifications are located within the globular domain of the histones at sites that are thought to be involved in binding to nucleosomal DNA. Investigation of mouse tissue in addition to cell lines uncovered a number of interesting differences. For example, whereas methylation sites are frequent in tissues, this type of modification was much less abundant in cultured cells and escaped detection. Our study significantly extends the known spectrum of linker histone variability. 相似文献
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The histones of the endosymbiont nucleus of the binucleate dinoflagellate Peridinium balticum were characterized by amino acid analysis and peptide mapping, and compared to calf thymus histones. Using these and various other criteria we have identified two H1-like histones as well as the highly conserved histones H3 and H4. A 13,000 dalton component in sodium dodecyl sulphate (SDS) gels can be separated into two components in Triton-containing gels. We suggest that these histones (HPb1 and HPb2) correspond to the vertebrate histones H2A and H2B, respectively. 相似文献
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L. I. Fedoreyeva T. A. Smirnova G. Ya. Kolomijtseva V. Kh. Khavinson B. F. Vanyushin 《Biochemistry. Biokhimii?a》2013,78(2):166-175
Judging from fluorescence modulation (quenching), short peptides (Ala-Glu-Asp-Gly, Glu-Asp-Arg, Ala-Glu-Asp-Leu, Lys-Glu-Asp-Gly, Ala-Glu-Asp-Arg, and Lys-Glu-Asp-Trp) bind with FITC-labeled wheat histones H1, H2в, H3, and H4. This results from the interaction of the peptides with the N-terminal histone regions that contain respective and seemingly homologous peptide-binding motifs. Because homologous amino acid sequences in wheat core histones were not found, the peptides seem to bind with some core histone regions having specific conformational structure. Peptide binding with histones and histone-deoxyribooligonucleotide complexes depends on the nature of the histone and the primary structures of the peptides and oligonucleotides; thus, it is site specific. Histones H1 bind preferentially with single-stranded oligonucleotides by homologous sites in the C-terminal region of the protein. Unlike histone H1, the core histones bind pre-dominantly with double-stranded methylated oligonucleotides and methylated DNA. Stern-Volmer constants of interaction of histone H1 and core histones with double-stranded hemimethylated oligonucleotides are higher compared with that of binding with unmethylated ones. DNA or deoxyribooligonucleotides in a complex with histones can enhance or inhibit peptide binding. It is suggested that site-specific interactions of short biologically active peptides with histone tails can serve in chromatin as control epigenetic mechanisms of regulation of gene activity and cellular differentiation. 相似文献
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Purification and initial characterization of a protein which facilitates assembly of nucleosome-like structure from mammalian cells 总被引:17,自引:0,他引:17
Y Ishimi J Hirosumi W Sato K Sugasawa S Yokota F Hanaoka M Yamada 《European journal of biochemistry》1984,142(3):431-439
A protein, which facilitates assembly of a nucleosome-like structure in vitro, was previously partially purified from mouse FM3A cells [Ishimi, Y. et al. (1983) J. Biochem. (Tokyo) 94, 735-744]. The protein has been purified to approximately 80% from FM3A cells by using histone-Sepharose column chromatography. It sedimented at 4.6 S and had a molecular mass of 53kDa. A preincubation of core histones with the 53-kDa peptide before DNA addition was necessary for the nucleosome assembly. The 53-kDa peptide bound to core histones and formed a 12-S complex. This complex contained stoichiometrical amounts of the 53-kDa peptide and core histones, and the core histones in this complex were composed of equal amounts of H2A, H2B, H3 and H4 histones. The nucleosomes were assembled by adding pBR322 DNA to the 12-S complex. When mononucleosome DNA and core histones were mixed in the presence of the 53-kDa peptide, formation of a 10.5-S complex was observed. The complex contained DNA and core histones in equal amounts, while no 53-kDa peptide was detected in the complex. From above results it is suggested that the 53-kDa peptide facilitates nucleosome assembly by mediating formation of histone octamer and transferring it to DNA. Rat antibody against the 53-kDa peptide did not bind to nucleoplasmin from Xenopus eggs. The relationship between the 53-kDa peptide and nucleoplasmin is discussed. 相似文献
14.
Interplay of various covalent modifications of histone tails has an essential role in regulation of chromatin function. Peptidylarginine deiminase (PADI) 4 deiminates protein arginine to citrulline in a Ca(2+)-dependent manner and is present in the nucleus of granulocyte-differentiated HL-60 cells. When these cells are treated with the calcium ionophore A23187, core histone deimination occurs. To determine the deimination sites of histones, histone species were purified by reverse-phase high-performance liquid chromatography (RP-HPLC) from the cells. Immunoblotting using antimodified citrulline antibody indicated that histones H2A, H3, and H4 but not H2B were deiminated. H2A and H4 were digested with Staphylococcus aureus V8 protease, and the digests were separated by RP-HPLC. Immuno dot-blotting and mass spectrometry showed that the deiminated residues were present in H2A (1-56) and H4 (1-52) regions but not in other regions. The H2A peptide (1-56) was digested with alpha-chymotrypsin, and the deiminated peptide was separated from the corresponding nondeiminated peptide by RP-HPLC. The deiminated residue was found to be limited to residues 1-23. Similarly, digestion of the H4 peptide (1-52) with endoproteinase Asp-N and separation of the deiminated peptide from the nondeiminated peptide indicated that the deiminated residue was limited to residues 1-23. Mass spectrometry of lysylendopeptidase digests of the H2A (1-23) and H4 (1-23) peptides showed that deimination occurred at arginine 3 of the N-terminal sequence Ac-SGRGK common to H2A and H4. These results suggest that PADI4 deiminates only a restricted site of target proteins in cells. Deimination of histones is discussed in relation to chromatin structure and function. 相似文献
15.
DNase I site mapping and micrococcal nuclease digestion of pachytene chromatin reveal novel structural features 总被引:1,自引:0,他引:1
A comparison of the DNase I digestion products of the 32P-5'-end-labeled pachytene nucleosome core particles (containing histones H2A, TH2A, X2, H2B, TH2B, H3, and H4) and liver nucleosome core particles (containing somatic histones H2A, H2B, H3, and H4) revealed that the cleavage sites that are 30, 40, and 110 nucleotides away from the 5'-end are significantly more accessible in the pachytene core particles than in the liver core particles. These cleavage sites correspond to the region wherein H2B interacts with the nucleosome core DNA. These results, therefore, suggest that the histone-DNA interaction at these sites in the pachytene core particles is weaker, possibly because of the presence of the histone variant TH2B interacting at similar topological positions in the nucleosome core as that of its somatic counterpart H2B. Such a loosened structure may also be maintained even in the native pachytene chromatin since micrococcal nuclease digestion of pachytene nuclei resulted in a higher ratio of subnucleosomes (SN4 + SN7) to mononucleosomes than that observed in liver chromatin. 相似文献
16.
The acidic protein prothymosin alpha (ProTalpha), with a broad presence in mammalian cells, has been widely considered to have a role in cell division, through an unrevealed mechanism in which histones may be involved in view of their ability to interact with ProTalpha in vitro. Results of co-immunoprecipitation experiments presented here demonstrate that ProTalpha interacts in vivo with core histones in proliferating B-lymphocytes (NC-37 cells). This interaction occurs with histones H3, H2A, H2B and H4 located free in the nucleoplasm, whereas no interaction was detected with histone H1, mono-nucleosome particles or chromatin. Moreover, the core histones form part of a nuclear multiprotein complex of about 700 kDa separated by ProTalpha-Sepharose affinity, with components including H3 and H4 acetyltranferases, H3 methyltransferases, hnRNP isotypes A3, A2/B1 and R, ATP-dependent and independent DNA helicases II, beta-actin and vimentin, all co-purifying by gel filtration. This indicates that the interaction of ProTalpha with core histones in the nucleus may be related to the structural modification of histones H3 and H4, and hence to chromatin activity, raising the possibility that the other proteins in the nuclear complex may play a role in this process. 相似文献
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A comparative study of the amino acid composition of histone fractions P4b from slime mold Physarum polycephalum and H2B from calf thymus was carried out using peptide mapping. It was shown that 75% of peptides are common for both proteins. The slime mold histones contain two fractions (P4B and P3), which are homologous to the H2B histone fraction of calf thymus. The data of amino acid analysis, peptide mapping and some physico-chemical properties of the histones revealed the following correlation of the two types of histone fractions: P1--H1, P4a--H3, P4b and P3--H2B, P5-H2A, P6--H4. 相似文献
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Chew YC Camporeale G Kothapalli N Sarath G Zempleni J 《The Journal of nutritional biochemistry》2006,17(4):225-233
In eukaryotic cell nuclei, DNA associates with the core histones H2A, H2B, H3 and H4 to form nucleosomal core particles. DNA binding to histones is regulated by posttranslational modifications of N-terminal tails (e.g., acetylation and methylation of histones). These modifications play important roles in the epigenetic control of chromatin structure. Recently, evidence that biotinidase and holocarboxylase synthetase (HCS) catalyze the covalent binding of biotin to histones has been provided. The primary aim of this study was to identify biotinylation sites in histone H2A and its variant H2AX. Secondary aims were to determine whether acetylation and methylation of histone H2A affect subsequent biotinylation and whether biotinidase and HCS localize to the nucleus in human cells. Biotinylation sites were identified using synthetic peptides as substrates for biotinidase. These studies provided evidence that K9 and K13 in the N-terminus of human histones H2A and H2AX are targets for biotinylation and that K125, K127 and K129 in the C-terminus of histone H2A are targets for biotinylation. Biotinylation of lysine residues was decreased by acetylation of adjacent lysines but was increased by dimethylation of adjacent arginines. The existence of biotinylated histone H2A in vivo was confirmed by using modification-specific antibodies. Antibodies to biotinidase and HCS localized primarily to the nuclear compartment, consistent with a role for these enzymes in regulating chromatin structure. Collectively, these studies have identified five novel biotinylation sites in human histones; histone H2A is unique among histones in that its biotinylation sites include amino acid residues from the C-terminus. 相似文献
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Calf thymus histones are separated into the five classical components H1, H2A, H2B, H3, and H4 using reversed-phase high-performance liquid chromatography. This method is fast and sensitive; a single run takes 80 min and protein quantities ranging from 3 μg up to 1 mg can be separated. The primary structure of the proteins is not affected, as demonstrated by peptide mapping and gel electrophoresis. Yeast, nematode, and calf thymus histones are compared to each other and have similar retention times for individual peaks, thus demonstrating the evolutionary stability of these proteins by using this different approach. 相似文献