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PubMed2003

Measurement of histone acetyltransferase and histone deacetylase activities and kinetics of histone acetylation.

Dynamic histone acetylation has a role in chromatin remodeling and in the regulation of transcription. Histone deacetylases (HDACs) and histone acetyltransferases (HATs) catalyze reversible histone acetylation. HATs and HDACs exist as multiprotein complexes that have coactivator and corepressor activities, respectively. The steady-state level of acetylation at a chromatin site is determined by the local net activities of these enzymes. Here we describe methods to isolate different subcellular fractions (cytosol, nuclei, tightly bound nuclear, loosely bound nuclear, immunoprecipitated multiprotein complexes, and nuclear matrix) to determine the subcellular distribution of HAT and HDAC activities. Procedures to assay the activities of these enzymes and to measure the kinetics of histone acetylation and deacetylation are presented.

Acetylation↗
PubMed2004

Age-dependent response of lymphocytes in the induction of the linker histone variant, H1 degrees and histone H4 acetylation after treatment with the histone deacetylase inhibitor, trichostatin A.

In the present study we investigated the age-related response of Phytohemaglutinin (PHA)-activated S phase human lymphocytes isolated from peripheral blood from donors of four different age groups, namely young (25-30 years), mid-aged (40-45 years), senior (60-65 years) and elderly (80-95 years) on the induction of the linker histone variant, H1 degrees and histone H4 acetylation after treatment with the very specific histone deacetylase (HDAC) inhibitor, trichostatin A (TSA). The cell system of peripheral blood lymphocytes is ideal for the study of H1 degrees induction since they do not synthesize this particular linker histone variant. Lymphocytes isolated from peripheral blood were activated with PHA (5 microg/10(6) cells/ml medium) and placed in culture for a duration of 72 h at which time cells are in the S phase. Forty-eight hours after inoculation, TSA (250 ng/10(6) cells/ml medium) was added to the cell cultures for a period of 24 h. Assays were performed 72 h after initiation of cultures. The results showed that the induction of H1 degrees after TSA treatment increased to a statistically significant degree in the elderly age group with respect to both the young and the mid-aged age groups. Moreover histone H4 acetylation was found to increase as a function of increasing donor age. A hyperacetylation pattern was observed even in the youngest age group analyzed. Specifically, the tetra-acetylated (H4.4) H4 form increased to a statistically significant degree with the concomitant decrease in the non-acetylated H4 for (H4.0) as a function of donor age. The other acetylated H4 forms (H4.1, H4.2, and H4.3) remained more or less constant, irrespective of donor age. These results show that the sensitivity of lymphocytes to TSA is enhanced with increasing donor age. Since to date, 11 class I and II HDACs have been isolated that have been found by other investigators to have differential responses to HDAC inhibitors, these findings may indicate that there is also a differential age-related response of certain HDACs or perhaps a senescent-specific HDAC. This line of research warrants further study.

Acetylation↗
PubMed1979

Histon-histone interactions within chromatin. Preliminary location of multiple contact sites between histones 2A, 2B, and 4.

The contact-site cross-linkers tetranitromethane, UV light, formaldehyde, and a monofunctional imido ester have been used to generate a collection of histone-histone dimers and trimers from nuclei and chromatin. Four different H2B-H4 dimers have been isolated. Preliminary CNBr peptide mapping has shown that all are cross-linked at different positions that are apparently clustered within the C-terminal regions of these histones. Similarily, two different H2A-H2B dimers and two different H2A-H2B-H4 trimers have been partially characterized. The data suggest a functional map for H2B in which the N-terminal third interacts with DNA, the middle third interacts with H2A, and the C-terminal third interacts with H4. We hope, by pursuing this type of analysis, to develop a detailed understanding of each histone-histone binding interaction through saturation cross-linking of the binding sites.

Animals↗
PubMed1978

Yeast inner histones and the evolutionary conservation of histone-histone interactions.

The inner histones of the yeast, Saccharomyces cerevisiae, have been isolated and identified by their amino acid compositions. H4 appears to be close to its calf and pea counterparts. H2a, H2b, and H3 have diverged. The isolation of the histones was accomplished by consecutive slab-gel fractionation, and a number of novel features of the method are described. These appear to be generally useful for preparing many types of protein. The binding pattern of the yeast inner histones is identical to the binding pattern for calf and for pea histones. Data on interspecies complexing indicate that the surfaces across which the histones interact are very highly conserved.

Amino Acids↗
PubMed1988

The reconstitution of a hybrid histone octamer containing avian 110Cys-des-thio-histone H3 and sea-urchin 73Cys-histone H4.

A hybrid histone octamer was reconstituted from erythrocyte H2A and H2B, avian [110 Cys-des-thio]histone H3 and the sea-urchin sperm [73Cys]H4 variant. [110Cys-Des-thio]histone H3 was prepared by reaction of natural H3 with Raney nickel. The ability of the hybrid octamer to crystallize to the same form as the natural octamer demonstrated that the chemical modification of cysteine to alanine in H3 and the mutation from threonine to cysteine in sperm H4 do not alter histone-histone interactions in the octamer. Since the sulfhydryl groups of both H4 molecules are fully accessible to 5,5'-dithiobis(2-nitrobenzoate) these residues provide suitable sites for the introduction of a single cysteine-specific label per H4 molecule in the octamer.

Amino Acids↗
PubMed1980

Influence of histone phosphorylation upon histone-histone interactions studied in vitro.

Histones H2b and H3, phosphorylated in vitro with the catalytic subunit of protein kinase I from rabbit skeletal muscle, were used to estimate the influence of histone phosphorylation upon histone-histone complex formation. Stoichiometry and interaction affinity of the complexes H2a-H2b, H4-H2b, and H4-H3 were determined by using the continuous variation method based on circular dichorism or fluorescence intensity. All complexes exhibit a 1:1 stoichiometry in sodium phosphate or sodium chloride solution of pH 7.0. The association constants of the complexes containing phosphorylated H2b were only slightly reduced, whereas that with phosphorylated H3 was strongly reduced relative to those of the nonphosphorylated species.

Animals↗
PubMed1975

The relationship between histones F 2al and F 2a2 and the ancestral histone A peptide. Further evidence for the common origin of histones F 2al, F 2a2 and F 3.

The relationship between histones F 2al and F 2a2 becomes much more apparent if the alignment is not made between the total sequences but between the ancestral A peptide, reconstructed earlier for histone F 2al (IV) and F 2a2. 46.5% of the latter's sequence can thus be clearly connected with F 2al through this ancestral dodecapeptide. A parallel development of histones F 2al, F 2a2 and F 3 from the A peptide is proposed.

Amino Acid Sequence↗
PubMed1978

High resolution proton magnetic resonance spectroscopy of histones and histone--histone complexes in aqueous solution.

Low molecular weight histone complexes of H2A (congruent to dimer), H2B (congruent to tetramer), H3--H4 (congruent to tetramer), H2A--H2B (congruent to dimer), and H2B--H4 (congruent to dimer) have been prepared in 2 M NaCl and neutral pH at 4 degrees C. These materials are free of nonspecific aggregate and are suitable for study by high resolution proton magnetic resonance spectroscopy. Such spectra have been recorded in aqueous solutions under conditions allowing a study of the exchangeable proton resonances of histone complexes for the first time and indicate that the structured regions are rich in hydrophobic amino acids, as well as arginine and some acidic amino acids. Most of the lysine and probably alanine residues remain in a motile, random coil-like state after formation of the complexes. It is suggested that arginine residues may be important in inter- and/or intra-subunit interactions in histone complexes.

Animals↗
PubMed1997

Antisera directed against anti-histone H4 antibodies recognize linker histones. Novel immunological probes to detect histone interactions.

We introduce a novel immunological approach to detect structural interactions between chromosomal proteins. Antigenically pure core histone H4 was prepared from chicken erythrocytes and used to produce anti-histone H4 antisera. IgG fractions were isolated from purified anti-H4 antibodies and used as antigens to produce "second generation" antisera. Epitopes cross-reacting with the second generation antisera were then identified within chromosomal proteins. These epitopes were presumed to mimic the complementary molecular surface of the original anti-H4 antibodies, and thus proteins containing these epitopes were putatively identified as specific ligands of H4 in chromatin. Surprisingly, we found this immunoreactivity was predominantly directed against H1 compared with H5 from chicken erythrocytes. Further, the immunoreactive epitopes were located within the C-terminal tail domain of the linker histones. These results suggest similar complementary interactions occur between H4 and the C-terminal tail domain of H1s in native chromatin. This could occur either within a single nucleosome as suggested by a previous report (Banères, J.-L., Essalouh, L., Jariel-Encontre, I., Mesnier, D., Garrod, S., and Parello, J. (1994) J. Mol. Biol., 243, 48-59) or between neighboring nucleosomes within the condensed chromatin fiber. The implications of these results with regard to the structure of the chromatin fiber and the future utility of this technique are discussed.

Animals↗
PubMed1987

High energy radiation effects in single histones. I. Preparation of histones and irradiation of histone H2B.

Histone H2B from calf thymus was irradiated with 50 or 100 ns pulses of 16 MeV electrons in N2O-saturated aqueous solution at pH 9 in the presence of NaN3. All tyrosine moieties in the histone were found to be freely accessible to the attack of .N3 radicals (formed by the reaction .OH + N3(-)----OH- + .N3). At sufficiently high concentrations of H2B, tyrosyl radicals were formed with G(TyrO.) = 5.4/100 eV and dityrosine groups with G(dityr) = 1.6/100 eV, indicating that about 60 per cent of tyrosyl radicals formed bisphenolic products. There is no polymer effect with respect to G(dityr) as inferred from comparison with other authors' data obtained with low molecular weight compounds. Kinetic measurements revealed that tyrosyl radicals reacted in two modes, a fast one with a value of tau 1/2 of about several milliseconds and a slow second order process also in the millisecond range. The fast process is assigned to intramolecular reactions of tyrosyl radicals generated in close proximity to each other and the slow process to intermolecular self reactions of isolated tyrosyl radicals distributed statistically in the solution. There is a polymer effect with respect to the rate constant of the slow process: 2k8 = 4.8 X 10(7) dm3 mol-1 s-1 (H2B) and 2k8 = 4 X 10(8) dm3 mol-1 s-1 (Lys-Tyr-Lys, Prütz et al. (1983)). The five histones contained in calf thymus were isolated chromatographically with the aid of two gels, Bio-Gel P-60 (BioRad) and Sephadex G100 (Pharmacia).

Animals↗
PubMed1978

Histone-DNA interactions within chromatin. Isolation of histones from DNA-histone adducts induced in nuclei by UV light.

We have developed a method by which to isolate histones that have been crosslinked to DNA following irradiation of calf thymus nuclei by UV light. The procedure involves separation of protein-DNA adducts from uncrosslinked protein by Sepharose 4B chromatography under dissociating conditions. Histones which are crosslinked to DNA are released by chemical hydrolysis of the DNA and identified by SDS gel electrophoresis. The results indicate that, of the histones, H1 and H3 become crosslinked to the DNA most readily under our irradiation conditions.

Animals↗
PubMed1977

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.

Animals↗
PubMed1976

Histone-histone interactions. II. Structural stability of the histone H3-H4 complex.

The stability of the histone H3-H4 complex toward urea, changes in pH and ionic strength, and certain chemical modifications have been examined by gel electrophoresis anc circular dichronism. When uncomplexed, the two cysteine residues of histone H3 become rapidly oxidized, forming an intramolecular disulfide bridge which apparently blocks complex formation on return to complexing conditions. The complex was found to be unstable toward low values of pH and ionic strength, concentrations of urea exceeding 1 M, modifications of the cysteine residues, and fragmention in which the C terminal portions of either H3 or H4 are removed. A possible structure for this complex is proposed.

Animals↗
PubMed1973

Addition of histones to histone-depleted nuclei: effect on template activity toward DNA and RNA polymerases.

The ability of histones to block the accessibility of DNA in chromatin to DNA and RNA polymerases was measured by addition of lysine-rich or arginine-rich histones to nuclei selectively depleted of these histones. By this procedure nuclei were obtained in which all of the original lysine-rich histone in the chromatin was replaced by arginine-rich histone. Conversely in other nuclei, additional lysine-rich histone replaced some of the endogenous arginine-rich histone. Lysine-rich histone was much more effective than arginine-rich histone in blocking accessibility to DNA polymerase. Both classes of histone inhibited template activity toward RNA polymerase to a similar extent. In addition to lysine-rich histone and total arginine-rich histone, phosphorylated lysine-rich histone, two fragments of lysine-rich histone produced by cleavage with N-bromosuccinamide, and fractions IIB and IV of arginine-rich histone were added to histone-depleted nuclei. With both DNA and RNA polymerases as probes, no differences in inhibition of template activity were found when native lysine-rich histone was compared to phosphorylated lysine-rich histone. Similarly, fractions IIB and IV were indistinguishable from total arginine-rich histone. On a molar basis, the carboxyl fragment of lysine-rich histone was as effective as intact lysine-rich histone only when the amino fragment was added to it. By itself, the amino portion of lysine-rich histone was without inhibitory effect in the RNA polymerase assay and resulted in only slight inhibition of template activity toward DNA polymerase.

Arginine↗
PubMed2004

Characterization of yeast histone H3-specific type B histone acetyltransferases identifies an ADA2-independent Gcn5p activity.

BACKGROUND: The acetylation of the core histone NH2-terminal tails is catalyzed by histone acetyltransferases. Histone acetyltransferases can be classified into two distinct groups (type A and B) on the basis of cellular localization and substrate specificity. Type B histone acetyltransferases, originally defined as cytoplasmic enzymes that acetylate free histones, have been proposed to play a role in the assembly of chromatin through the acetylation of newly synthesized histones H3 and H4. To date, the only type B histone acetyltransferase activities identified are specific for histone H4. RESULTS: To better understand the role of histone acetylation in the assembly of chromatin structure, we have identified additional type B histone acetyltransferase activities specific for histone H3. One such activity, termed HatB3.1, acetylated histone H3 with a strong preference for free histones relative to chromatin substrates. Deletion of the GCN5 and ADA3 genes resulted in the loss of HatB3.1 activity while deletion of ADA2 had no effect. In addition, Gcn5p and Ada3p co-fractionated with partially purified HatB3.1 activity while Ada2p did not. CONCLUSIONS: Yeast extracts contain several histone acetyltransferase activities that show a strong preference for free histone H3. One such activity, termed HatB3.1, appears to be a novel Gcn5p-containing complex which does not depend on the presence of Ada2p.

Acetyltransferases↗
PubMed1980

Points of contact between histone H1 and the histone octamer.

The topography of the interaction between histone H1 and the histone octamer has been investigated. Bovine thymus nuclei or enzymatically fragmented chromatin were treated 1-ethyl-3(3-dimethylaminopropyl)carbodiimide, which catalyzes the formation of covalent bonds between residues of proteins in electrostatic contact. Histone H1-core histone dimers were identified and the segments of molecules participating in crosslinking were elucidated. The results demonstrate that the major histone H1-core histone dimer generated upon carbodiimide crosslinking of intact nuclei, chromatin, or mononucleosomes consists of the segment of histone H1 containing amino acids 74-106 crosslinked to the segment of histone H2A containing amino acids 58-129. Thus, the central globular region of histone H1 intimately contacts the histone octamer. Besides histone H1-H2 dimers, two other histone H1-containing crosslinked products were detected. In these instances, the segments of histone H1 molecules containing amino acids 1-72 were shown to participate in crosslinking. The histone H1 contact points defined here all occur within mononucleosomes and not between nucleosomes. These results permit the formulation of a testable model for the arrangement of histone H1 along polynucleosome chains.

Amino Acid Sequence↗
PubMed1976

Histone dimers: a fundamental unit in histone assembly.

Histone interactions which occur, at moderate ionic strengths, when several types of purified, renatured histones are mixed at equimolar ratios have been studied. The four histones H2A,H2B,H3 and H4 complex and form dimers. Histone H1 does not interact with the other four histone types and does not form dimers. Mixing of single histone species with preformed histone pairs as well as mixing of two different types of histone pairs, leads to exchange of histones among the pairs and formation of dimers. No trimers are formed. The dimers are in equilibrium with high-molecular weight histone structures. The results indicate that histone dimers may serve as a stable intermediate in histone assembly. Because each histone type (except H1) can interact with itself as well as with each of the other three histone types we suggest that each histone type should be considered as an interchangeable subunit of a multichain protein in which the dimer species is the most stable structure.

Animals↗