Histone Subunits Research Articles

Identification of Stimulators and Inhibitors of Cdc7 Kinase in Vitro

Cdc7 is a serine-threonine kinase that regulates initiation and progression of DNA replication. The activity of purified Cdc7 kinase is significantly stimulated by polyamines such as spermine or spermidine. Positively charged polymers of lysine or arginine also stimulate its kinase activity, whereas the negatively charged substances such as polyglutamate or nucleic acids significantly inhibit the kinase activity. Spermine affects both the K(m) and V(max) of Cdc7 kinase for a minichromosome maintenance (MCM) substrate. We also found that histones, lysine- and arginine-rich basic proteins, can stimulate Cdc7 kinase activity, and a MCM complex in association with histone is a more efficient substrate of Cdc7 than the free MCM complex. These results identify potential cellular inhibitors and stimulators of Cdc7 kinase and suggest that Cdc7 may be another target of cellular polyamines and that histones may stimulate Cdc7-mediated phosphorylation of chromatin-bound substrates. Ectopic expression of an antizyme, known to reduce the cellular polyamine levels, resulted in reduction of Cdc7-mediated phosphorylation of MCM4 protein, suggesting physiological roles of polyamines in regulation of Cdc7 kinase activity in the cells.

Acetylation-dependent ADP-ribosylation by Trypanosoma brucei Sir2

Sirtuins are a highly conserved family of proteins implicated in diverse cellular processes such as gene silencing, aging, and metabolic regulation. Although many sirtuins catalyze a well characterized protein/histone deacetylation reaction, there are a number of reports that suggest protein ADP-ribosyltransferase activity. Here we explored the mechanisms of ADP-ribosylation using the Trypanosoma brucei Sir2 homologue TbSIR2rp1 as a model for sirtuins that reportedly display both activities. Steady-state kinetic analysis revealed a highly active histone deacetylase (k cat = 0.1 s(-1), with Km values of 42 microm and for NAD+ and 65 microm for acetylated substrate). A series of biochemical assays revealed that TbSIR2rp1 ADP-ribosylation of protein/histone requires an acetylated substrate. The data are consistent with two distinct ADP-ribosylation pathways that involve an acetylated substrate, NAD+ and TbSIR2rp1 as follows: 1) a noncatalytic reaction between the deacetylation product O-acetyl-ADP-ribose (or its hydrolysis product ADP-ribose) and histones, and 2) a more efficient mechanism involving interception of an ADP-ribose-acetylpeptide-enzyme intermediate by a side-chain nucleophile from bound histone. However, the sum of both ADP-ribosylation reactions was approximately 5 orders of magnitude slower than histone deacetylation under identical conditions. The biological implications of these results are discussed.

Histone chaperones regulate histone exchange during transcription

Transcription by RNA polymerase II is accompanied by dynamic changes in chromatin, including the eviction/deposition of nucleosomes or the covalent modification of histone subunits. This study examined the role of the histone H3/H4 chaperones, Asf1 and HIR, in histone mobility during transcription, with particular focus on the histone exchange pathway, using a dual histone expression system. The results showed that the exchange of H3/H4 normally occurs during transcription by the histone chaperones. Both Asf1 and HIR are important for histone deposition but have a different effect on histone exchange. While Asf1 mediated incorporation of external H3/H4 and renewal of pre-existing histones, HIR opposed it. The balance of two opposing activities might be an important mechanism for determining current chromatin states.

Impact of resuscitation strategies on the acetylation status of cardiac histones in a swine model of hemorrhage

Chromatin remodeling through histone acetylation is a key control mechanism in gene transcription. We have shown previously that fluid resuscitation in rodents is coupled with highly structured post-translational modifications of cardiac histones. The current experiment was performed to validate this concept in a clinically relevant large animal model of hemorrhage and resuscitation, and to correlate the changes in histone acetylation with altered expression of immediate-early response genes. Yorkshire swine (n=49, 7/group, weight=40-58kg) were subjected to combined uncontrolled and controlled hemorrhage (40% of estimated blood volume) and randomly assigned to the following resuscitation groups: (1) 0.9% saline (NS), (2) racemic lactated Ringer's (dl-LR), (3) l-isomer lactated Ringer's (l-LR), (4) Ketone Ringer's (KR), (5) 6% hetastarch in saline (Hespan). KR contained an equimolar substitution of lactate with beta-hydroxybutyrate. No hemorrhage (NH) and no resuscitation (NR) groups were included as controls. Cardiac protein was used in Western blotting to analyze total protein acetylation and histone acetylation specifically. Lysine residue-specific acetylation of histone subunits H3 and H4 was further evaluated. In addition, Chromatin Immunoprecipitation (ChIP) technique was used to separate the DNA bound to acetylated histones (H3 and H4 subunits), followed by measurement of genes that are altered by hemorrhage/resuscitation, including immediate-early response genes (c-fos and c-myc), and heat shock protein (HSP) 70. The type of fluid used for resuscitation influenced the patterns of cardiac histone acetylation. Resuscitation with dl-LR and KR induced hyperacetylation on H3K9. KR resuscitation was also associated with increased acetylation on H3K14 and H4K5, and hypoacetylation on H3K18. The expression of genes was also fluid specific, with the largest number of changes following KR resuscitation (increased c-fos and c-myc, HSP 70 linked with H3; and increased c-myc linked with H4). Among the histone subunits studied, altered H3 acetylations were associated with the majority of changes in immediate-early gene expression. Acetylation status of cardiac histones, affected by hemorrhage, is further modulated by resuscitation producing a fluid-specific code that is preserved in different species. Resuscitation with KR causes histone acetylation at the largest number of lysine sites (predominately H3 subunit), and has the most pronounced impact on the transcriptional regulation of selected (immediate-early response) genes.

Structural Ramification for Acetyl‐Lysine Recognition by the Bromodomain of Human BRG1 Protein, a Central ATPase of the SWI/SNF Remodeling Complex

Bromodomains represent an extensive family of evolutionarily conserved domains that are found in many chromatin-associated proteins such as histone acetyltransferases (HAT) and subunits of ATP-dependent chromatin-remodeling complexes. These domains are associated with acetylated lysine residues that bind both in vivo and in vitro; for example, they bind to the N-acetylated lysines of the histone tail of nucleosomes. In this report, we determined the structure of the bromodomain from human brahma-related gene 1 (BRG1) protein, a subunit of an ATP-dependent switching/sucrose nonfermenting (SWI/SNF) remodeling complex, and have also characterized its in vitro interaction with N-acetylated lysine peptides from histones. In addition to a typical all-alpha-helical fold that was observed in the bromodomains, we observed for the first time a small beta-sheet in the ZA loop region of the BRG1 protein. The BRG1 bromodomain exhibited binding, albeit weak, to acetylated peptides that were derived from histones H3 and H4. We have compared the acetyl-lysine binding sites of BRG1 bromodomain with the yGCN5 (general control of amino acid biosynthesis). By modeling the acetylated-lysine peptide into the BRG1 bromodomain structure, we were able to explain the weak binding of acetylated-lysine peptides to this bromodomain.

On the origin of the histone fold

BackgroundHistones organize the genomic DNA of eukaryotes into chromatin. The four core histone subunits consist of two consecutive helix-strand-helix motifs and are interleaved into heterodimers with a unique fold. We have searched for the evolutionary origin of this fold using sequence and structure comparisons, based on the hypothesis that folded proteins evolved by combination of an ancestral set of peptides, the antecedent domain segments.ResultsOur results suggest that an antecedent domain segment, corresponding to one helix-strand-helix motif, gave rise divergently to the N-terminal substrate recognition domain of Clp/Hsp100 proteins and to the helical part of the extended ATPase domain found in AAA+ proteins. The histone fold arose subsequently from the latter through a 3D domain-swapping event. To our knowledge, this is the first example of a genetically fixed 3D domain swap that led to the emergence of a protein family with novel properties, establishing domain swapping as a mechanism for protein evolution.ConclusionThe helix-strand-helix motif common to these three folds provides support for our theory of an 'ancient peptide world' by demonstrating how an ancestral fragment can give rise to 3 different folds.

The Human Telomeric Protein TRF1 Specifically Recognizes Nucleosomal Binding Sites and Alters Nucleosome Structure

Telomeres are dynamic nucleoprotein structures that cap the ends of eukaryotic chromosomes. In humans, the long (TTAGGG) n double-stranded telomeric DNA repeats are bound specifically by the two related proteins TRF1 and TRF2, and are organized in nucleosomes. Whereas the role of TRF1 and TRF2 in telomeric function has been studied extensively, little is known about the involvement of telomeric nucleosomes in telomere structures or how chromatin formation may affect binding of the TRFs. Here, we address the question of whether TRF1 is able to bind to telomeric binding sites in a nucleosomal context. We show that TRF1 is able to specifically recognize telomeric binding sites located within nucleosomes, forming a ternary complex. The formation of this complex is strongly dependent on the orientation of binding sites on the nucleosome surface, rather than on the location of the binding sites with respect to the nucleosome dyad. Strikingly, TRF1 binding causes alterations in nucleosome structure without dissociation of histone subunits. These results indicate that nucleosomes contribute to the establishment of a telomeric capping complex, whose structure and dynamics can be modulated by the binding of telomeric factors.

Defined Structural Changes Occur in a Nucleosome upon Amt1 Transcription Factor Binding

Here, we study the binding of the transcription factor Amt1 to its recognition site near the dyad of a highly positioned nucleosome. We find that the DNA binding domain of Amt1 binds to nucleosomes with only threefold reduced affinity compared to free DNA. We show by fluorescence resonance energy transfer that factor binding at the nucleosomal dyad is accompanied by the partial dissociation of the DNA ends from the histone octamer surface; however, no dissociation or subtle rearrangements of histone subunits is observed. A poly(dA.dT) DNA sequence element adjacent to the transcription factor binding site appears to facilitate factor binding, but is not essential. The methods that we describe here characterize for the first time the subtle structural changes that occur upon transcription factor binding to nucleosomes, and demonstrate the ability of the nucleosome to structurally adapt in response to outside influences.

A protein complex containing the conserved Swi2/Snf2-related ATPase Swr1p deposits histone variant H2A.Z into euchromatin.

The conserved histone variant H2A.Z functions in euchromatin to antagonize the spread of heterochromatin. The mechanism by which histone H2A is replaced by H2A.Z in the nucleosome is unknown. We identified a complex containing 13 different polypeptides associated with a soluble pool of H2A.Z in Saccharomyces cerevisiae. This complex was designated SWR1-Com in reference to the Swr1p subunit, a Swi2/Snf2-paralog. Swr1p and six other subunits were found only in SWR1-Com, whereas six other subunits were also found in the NuA4 histone acetyltransferase and/or the Ino80 chromatin remodeling complex. H2A.Z and SWR1 were essential for viability of cells lacking the EAF1 component of NuA4, pointing to a close functional connection between these two complexes. Strikingly, chromatin immunoprecipitation analysis of cells lacking Swr1p, the presumed ATPase of the complex, revealed a profound defect in the deposition of H2A.Z at euchromatic regions that flank the silent mating type cassette HMR and at 12 other chromosomal sites tested. Consistent with a specialized role for Swr1p in H2A.Z deposition, the majority of the genome-wide transcriptional defects seen in swr1Δ cells were also found in htz1Δ cells. These studies revealed a novel role for a member of the ATP-dependent chromatin remodeling enzyme family in determining the region-specific histone subunit composition of chromatin in vivo and controlling the epigenetic state of chromatin. Metazoan orthologs of Swr1p (Drosophila Domino; human SRCAP and p400) may have analogous functions.

Tying C' Ends of H2A and H2B Using a Molecular Glue, Tissue-Type Transglutaminase

This chapter describes the procedure for Tissue-type transglutaminase (tTGase) purification and the in vitro cross-linking reaction by the tTGase using chromatin/nucleosome as a substrate, along with a discussion on the possible physiological implications of the histone cross-linking. The tTGase activity was determined by measurement of the incorporation of radiolabeled [1, 4- 14 C] putrescine (NEN-Du Pont) in the dimethyl casein. The radioactive dimethyl casein was freed of unbound putrescine by a filter paper procedure and quantified by liquid scintillation counting. The tTGase was most easily prepared from human erythrocyte following the procedure described. The fractions eluted from heparinsepharose column with NaCl gradient were analyzed for protein profiles on 8% denaturing SDS-PAGE and tTGase activity. An in vitro experiment is carried out to cross-link histone subunits by the purified tTGase. Cross-linking of a histone subunit is detected at the position roughly where a dimeric histone subunit should move to, around 25 kD. Formation of the cross-linked products was accompanied by a concomitant decrease of substrate histone subunits, notably H2A and H2B, two middle bands. Cross-linking of a histone subunit was detected at the position roughly where a dimeric histone subunit should move to, around 25 kD.

Histone cross-linking by transglutaminase

Transglutaminases irreversibly catalyze covalent cross-linking of proteins by forming isopeptide bonds between peptide-bound glutamine and lysine residues. Among several transglutaminases, tissue-type transglutaminase (tTGase) is most ubiquitously found in every type of cells and tissues in animals, but its natural substrate has yet to be identified. In an attempt to identify the natural substrate for tTGase, we examined in vitro if core histones were subject to cross-linking by tTGase. We found core histone subunits, H2A and H2B, were specifically cross-linked by tTGase. The cross-linking was between either one or both glutamines at C-terminal end of H2A (-VTIA Q 104 GGVLPNT Q 112 SVLLPKKTESSKSK—C ′ end) and the first and/or third lysine from C-terminal end of H2B (-AVESEG K 116 AVTKYTSS K 125 —C ′ end). The cross-linking occurred only when these subunits were released from nucleosome but not when these were organized in nucleosome. Most interestingly, in chicken erythrocyte the cross-linked H2A–H2B was present in a significant amount. From these results, it can be proposed that tTGase-mediated cross-linking is an another form of core histone modification and it may play a role of chromatin condensation during erythrocyte differentiation.

A gene, han1A, encoding an archaeal histone-like protein from the Thermococcus species AN1: homology with eukaryal histone consensus sequences and the implications for delineation of the histone fold

The han1A gene, encoding a subunit of the histone-like protein HAN1 from the Thermococcus species AN1, has been cloned and sequenced. Sequence analysis of the translation product of the gene demonstrates homology with other archaeal histone-like proteins of the ‘HMf family’ and eukaryal consensus sequences, particularly H4. The region of highest homology between the AN1 histone subunit, termed the HAN1A1 subunit, and the H4 consensus is suggested, by the 3-dimensional structure of the histone octaner, to interact with the minor groove of DNA. The results presented add further weight to the notion that the ‘archaeal histones’ and the eukaryal histones are indeed related and that the approximate 65 amino acid residue length of the archaeal histones represents the archaeal equivalent of the histone fold structural building block common to all eukaryal histones.

Rabbit anti-chromatin antibodies recognize similar epitopes on a histone H1 molecule as lupus autoantibodies

Autoantibodies in systemic lupus erythematosus (SLE) are believed to be produced through an antigen-driven mechanism, i.e., autosensitization with autoantigens. If this is the case, it is conceivable that antigenic determinants reactive with SLE autoantibodies and induced antibodies raised by immunization are quite similar. To examine this issue, rabbits were immunized with purified histone H1 or chromatin. Although immunization with purified histone H1 did not produce detectable amounts of anti-histone H1 antibodies, immunization with chromatin mounted relatively good antibody responses against not only all histone subunits but also ssDNA. Epitopes on a histone H1 molecule reactive with SLE autoantibodies and rabbit antisera were determined using deletion mutants of a histone Hl molecule. At least two epitopes reactive with induced antibodies were found on a histone H1 and these were closely related to or the same as major autoepitopes in SLE. These data might indicate that B cells producing anti-histone Hl autoantibodies are quite similar to those induced by immunization and provide additional evidence that autosensitization with an autoantigen might be operative and a particulate antigen chromatin could be one of the immunogens for the production of anti-nuclear antibodies of various kinds in SLE.

Visualization of nucleosomal substructure in native chromatin by atomic force microscopy.

Intact rDNA minichromosomes from Tetrahymena thermophila were isolated as native chromatin and imaged by atomic force microscopy (AFM). AFM measurements of condensed rDNA chromatin were consistent with a 30 nm fiber that frequently (87% of molecules observed) contained stretches of nucleosome cores arranged in a zig-zag conformation. Examination of rDNA chromatin in a dispersed conformation by tapping mode AFM in low humidity resulted in high resolution images of partially dissociated nucleosome cores and associated linker DNA. A majority of these nucleosome cores contained six to eight smaller particles with dimensions consistent with those of individual histones. Many of the nucleosome cores showed a striking resemblance to the wedge (35%), axial (15%), and front (6%) views of the nucleosome histone octamer modeled by Arents et al. [Arents, G., Burlingame, R. W., Wang, B.-C., Love, W.E., & Moudrianakis, E. N. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 10148-10152]. This direct visualization of histone subunits and nucleosomal substructure in native chromatin illustrates the potential use of AFM to localize individual proteins in condensed cellular chromatin.

Purification of yeast histones competent for nucleosome assemblyin vitro

We have developed a procedure to purify nucleosomal assembly-competent histones as a mixture of H2A, H2B, H3 and H4 from isolated nuclei of the yeast Saccharomyces cerevisiae with a purity of 70-80%. The mixture contained each of the histone subunits approximately at the equi-molar ratio. Plasmid pBR322 DNA was assembled into nucleosomes with the purified yeast histones in the presence of nucleoplasmin from unfertilized eggs of the frog Xenopus laevis. The efficiency of assembly of yeast histones was comparable to that of core histones purified from HeLa cells. The length of DNA fragment wrapping around a core histone particle and the molar ratio of histone components in an assembled nucleosome particle were estimated to be 150 +/- 10 bp long and H2A:H2B:H3:H4 = 1.0:0.9:0:9:1.0, respectively.

Solution studies of the interactions between the histone core proteins and DNA using fluorescence spectroscope

The equilibrium interactions between histone H2A-H2B and H3/H4 subunits with 200 base pair chicken erythrocyte DNA have been studied by monitoring the fluorescence polarization of a long-lived fluorescence probe covalently bound to the histone subunits. These studies have brought to light the formation of highly asymmetric complexes exhibiting very high histone/DNA stoichiometries as well as very high apparent affinities. The stoichiometries observed for these non-nucleosome complexes depended both upon the concentration of the histones and the concentration of the DNA 200mer. The observed stoichiometries varied approximately between 4 and 16 histone octamers/DNA 200mer and the affinities were in the nanomolar range. These results are discussed in terms of their in vitro as well as their possible in vivo significance.

Salt effects on histone subunit interactions as studied by fluorescence spectroscopy.

The salt concentration dependence of the aggregation properties of calf thymus and chicken erythrocyte histones has been investigated by using fluorescence spectroscopy. The isolated H2A/H2B and H3/H4 subunit preparations were labeled with 5-(dimethylamino)naphthalene-1- sulfonyl (dansyl). This long-lived fluorescence probe allows for the observation of rotations due to tumbling of the particle and thus is a probe for changes in the size of macromolecular assemblies. The fluorescence polarization and lifetime were measured as a function of salt concentration for these isolated preparations. Next, each labeled preparation was reconstituted with its unlabeled complement, and the salt concentration dependence of histone core octamer interactions was investigated in the same manner. Salt-induced core particle formation was observed by monitoring the dansyl-labeled dimers for both the calf thymus and chicken erythrocyte preparations. Evidence for subunit dissociation of the isolated H2A-H2B preparations was also found, as well as aggregation of the isolated H3/H4 subunits to at least dimers of tetramers. The calf thymus H3/H4 preparation was in aggregated form under all conditions studied, whereas the chicken erythrocyte H3/H4 only formed aggregates at high protein or salt concentrations. We have found evidence that the dimer can displace the tetramer from the higher order aggregate in order to form core particles. Such competition between the subunit interfaces in the histone system suggests that they may play a regulatory role in histone-DNA interactions.

Histone subunit interactions as investigated by high pressure.

High-pressure fluorescence polarization was used to investigate subunit interactions of the histone H2A-H2B dimer and the H3/H4 tetramer isolated from calf thymus (CT) and chicken erythrocyte (CE) chromatin. The proteins were individually labeled with the fluorescent probe 5-(dimethylamino)-naphthalene-1-sulfonate (dansyl or DNS), and the fluorescence polarization was measured as a function of pressure. The long fluorescence lifetime of the probe allows for the observation of global rotations of the protein, the rate of which is dependent upon the aggregation state. From the pressure dependence of the dansyl polarization, the Kd of H2A-H2B dissociation of the CE dimer was found to be approximately 1 X 10(-7) M at 2.0 M NaCl. Lowering the salt concentration to 200 mM slightly stabilized the protein to 6 X 10(-8) M. Our data indicate a small negative volume change for the dissociation of the core particle octamer. The (H3)2(H4)2 tetramer, as was shown in the previous paper (Royer et al., 1989), also formed predominantly dimers of tetramers at higher protein or salt concentrations. In the study presented here, we found the dissociation constant for the H3/H4 octamer to dimer transition to be 1 X 10(-21) M3 (C1/2 = 4 X 10(-8) M) at 2 M NaCl for the CT preparation. Decreasing the salt concentration to 200 mM reduced the stability of the CT H3/H4 octamer to 9 X 10(-21) M3 (C1/2 = 8 X 10(-8) M). The dimer of the CE tetramer also dissociated upon application of pressure in 2 M salt.(ABSTRACT TRUNCATED AT 250 WORDS)

Two interferon-induced proteins are involved in the protein kinase complex dependent on double-stranded RNA

The double-stranded (ds) RNA-dependent protein kinase is a 100,000-110,000 Mr complex of two interferon-induced subunits each having ATP binding sites: a 48,000 Mr protein (p48) which appears to be responsible for the phosphorylation of a 68,000 Mr protein (p68) in the presence of dsRNA. The p68 subunit once phosphorylated is converted to an active protein kinase capable of phosphorylating exogenous substrates such as the alpha subunit of protein synthesis initiation factor eIF2 or calf thymus histone. The phosphorylation of exogenous substrates is highly correlated with the degree of phosphate saturation of p68 and does not require the presence of dsRNA. Both the p68 and the p48 subunits of the protein kinase complex are purified by an immuno-affinity column containing monoclonal antibody specific for the p68 subunit.

Binding of epinephrine to chromatin components.

To clarify the mechanism of the cellular DNA-breaking reaction of epinephrine (Ep), we examined the interaction between Ep and chromatin components. The Ep-binding activity of histone increased after the dissociation of histone subunits. The Ep bound to DNA increased with the increase of Ep concentration and pH. Solubilized chromatin, v bodies, showed Ep-oxidizing activity in the absence of Cu2+. The binding of Ep to v bodies occurred immediately after mixing and was highly specific. These data suggest the presence of some Ep-specific binding protein(s) in chromatin which oxidizes Ep and induces DNA breakage.