Characterization and primary structures of DNA-binding HU-type proteins from Rhizobiaceae

The DNA-binding HU-type proteins from several species of Rhizobiaceae including Rhizobium meliloti, two strains of Rhizobium leguminosarum with highly different phenotypic characters and Agrobacterium tumefaciens, were characterized and their amino acid sequences were determined. HU-type proteins isolated from R. leguminosarum L18 and A. tumefaciens are identical and show slight differences with the R. meliloti HU-type protein. On the other hand the R. leguminosarum L53 HU-type protein is quite different from the proteins cited above; several amino acid substitutions encountered in this protein result in significant changes in the folding of the polypeptide chain. The biochemical characteristics of these proteins are in good agreement with the respective position of these bacteria in the phylogeny determined by numerical taxonomy.     

Comparative study of the DNA-binding HU-type proteins from slow growing and fast growing strains of Rhizobiaceae

The DNA-binding HU-type proteins have been isolated from two very different strains of Rhizobiaceae: Agrobacterium tumefaciens and Rhizobium japonicum. These proteins have been called HAt and HRj respectively. Their electrophoretic mobility on polyacrylamide gel, amino acid composition and crossed immunoreactivity have been compared to that of the homologous protein isolated from Rhizobium meliloti: the protein HRm. The proteins HAt and HRm show close similarities whereas the protein HRj differs markedly from the two others. The physico-chemical characteristics of the HU-type proteins from these Rhizobiaceae are in good agreement with the respective position of these bacteria in the taxonomy.     

Primary structure of the DNA-binding protein HRm from Rhizobium meliloti

The amino acid sequence of protein HRm, a DNA-binding HU-type protein of 90 residues (Mr 9303), isolated from Rhizobium meliloti, has been established from automated sequence analysis of the protein and from structural data provided by peptides derived from cleavage of the protein at arginine and aspartic acid residues. The comparison of the primary structure of protein HRm with that of other HU-type proteins shows that two short sequences, of 7 and 6 residues respectively, located in the median part of the molecule, appear highly conserved and may be important in the function of the protein.     

Nonenzymatic glycation of histones in vitro and in vivo

Purified histones in solution, purified nuclei, or whole endothelial cells in cell culture were used to study the reactivity of histones with various sugars. The sugar incubation of purified histones produced nonenzymatic glycation and formation of histone cross-links showing disappearance of individual histone molecules and appearance of dimers and polymers in SDS-PAGE. In solution, core histones react considerably faster with sugars as compared to H1 histones. In sugar-incubated nuclei where histones are nucleosomally organized, H1 histones, which are located at the periphery of the nucleosome, and H2A-H2B dimers, which are associated with the central H3(2)-H4(2) tetramer, are more reactive as compared to H3 and H4 histones, which are most protected from the glycation reaction. Our in vivo experiments using endothelial cells show that high concentrations of ribose are able to generate protein cross-links paralleled by apoptotic cell death. High concentrations of glucose or fructose do not increase histone glycation or cell death, even after 60 days of incubation of endothelial cells. In long-time glucose- or fructose-treated cells, under nondenaturing and nonreducing SDS-PAGE conditions part of the H3 histones shifted away from their normal location. Because it is known that the mitochondrial production of reactive oxygen species (ROS) increases after hyperglycaemia, we hypothesize that ROS could be responsible for the formation of a disulphide bridge between the side chain of the cysteine residues of H3 molecules.     

Digestion of chromosomal proteins in formaldehyde treated chromatin

Treatment of chromatin subunits (nucleosome monomers) with formaldehyde results in the formation of cross-links between DNA and histones and between histones and histones. Digestion of chromosomal proteins with proteinase K does not lower the protein/DNA weight ratio below 0.08 to 0.1 as determined by cesium chloride gradient centrifugation of the digestion product from formaldehyde-treated nucleosomes. In addition to proteinase K, formaldehyde-treated nucleosomes were tested for accessibility to trypsin and pronase. The CsCl gradient patterns show, that pronase digestion and proteinase K treatment yield similar results. Trypsin treatment of control and formaldehyde-treated nucleosomes shows, that the sites which are accessible for trypsin in native nucleosomes, are blocked after formaldehyde treatment. Analysis of the CsCl gradient peak fractions in polyacrylamide gels shows, that the reliability of DNA fragment size determinations depends on the completeness of deproteinization.     

Multiple forms of cytosol and membrane-bound protein kinase activity in human erythrocytes.

Both cytosol and membranes of human erythrocytes display protein kinase activity towards exogenous protein substrates such as casein, phosvitin and histones. The histone kinase activity, unlike casein kinase, of both cytosol and membranes is increased by cyclic AMP. The protein kinase forms removed from the membranes with 0.7 M NaCl, phosphorylate only serine residues of both casein and histones through a mechanism cyclic AMP-independent. The protein kinase activity located in the cytosol (hemolysate) is due also to enzyme forms phosphorylating both serine and threonine residues of casein, in addition to forms phosphorylating only serine residues of casein and histones. Also the cytosol kinase forms, once partially purified by Sepharose 6B filtration, appear to be cyclic AMP-independent.     

High mobility group protein 17 cross-links primarily to histone H2A in the reconstituted HMG 17-nucleosome core particle complex

The "neighbor relationship" of lamb thymus high mobility group (HMG) protein 17 to native HeLa nucleosome core particle histones in the reconstituted complex has been studied. 125I-Labeled HMG 17 was cross-linked to core histones using the protein-protein cross-linking reagent 2-iminothiolane. Specific cross-linked products were separated on a two-dimensional Triton-acid-urea/sodium dodecyl sulfate-gel system, located by autoradiography, excised, and quantified. Disulfide bonds in the cross-links were then cleaved, and the protein constituents were identified by sodium dodecyl sulfate-gel electrophoresis. HMG 17 cross-linked primarily to histone H2A while lower levels of cross-linking occurred between HMG 17 and the other histones. In contrast, cross-linking between 2 HMG 17 molecules bound on the same nucleosome core particle was relatively rare. We have concluded that H2A comprises part of the HMG 17 binding site. Less contact occurs between HMG 17 and the other core histones, and there is little contact possible between the 2 bound HMG 17 molecules. These results are in agreement with the current model for the structure of the nucleosome and the proposed binding sites for HMG 17.     

Studies on histone organization in the nucleosome using formaldehyde as a reversible cross-linking agent

A new procedure is described which allows selective reversal of formaldehyde cross-linking in both histone-histone and histone-DNA of nuclei isolated from calf thymus. All ten possible dimers of the four non-H1 histones, H3, H2B, H2A and H4, are observed, the major dimers being H3-H3, H3-H2A, H2B-H2A, H2a-H2A and two separate dimers of H2B-H4. Although oligomers of the non-H1 histones are formed by prolonged treatment with this reagent, 50% of the histones continue to remain resistant to cross-linking with each other. For those histones which cross-linking with each other. For those histones which cross-link, the site of cross-linking within the molecules is located in the "core" (trysin-resistant) regionand therfore indicates proximities for these molecules within the nucleosome. The core region also cross-links to DNA, indicating intimate interactions between this region in all the non-H1 histones with DNA.     

Analysis of the heterogeneity of chromatin proteins fractionated on hydroxyapatite

The protein composition of the liver chromatin has been studied by two techniques for fractionation of histones. The "lability" fraction of histones H2A-H2B is revealed. In these fractions histones H2B have many modified forms and they are not included into octamer (H3, H4, H2A, H2B)2. Young animals rather than old ones have much quantitative subfractions of histone H2B. The "lability" fraction of histones H2A-H2B is stated to be very significant in the activated and repressed chromatin structure.     

Multiple forms of cytosol and membrane-bound protein kinase activity in human erythrocytes

Both cytosol and membranes of human erythrocytes display protein kinase activity towards exogenous protein substrates such as casein, phosvitin andhistones. The histone kinase activity, unlike casein kinase, of both cytosol and membranes is increased by cyclic AMP. The protein kinase forms removed from the membranes with 0.7 M NaCl, phosphorylate only serine residues of both casein and histones through a mechanism cyclic AMP-independent.The protein kinase activity located in the cytosol (hemolysate) is due also to enzyme forms phosphorylating both serine and threonine residues of casein, in addition to forms phosphorylating only serine residues of casein and histones.Also the cytosol kinase forms, once partially purified by Sepharose 6B filtration, appear to be cyclic AMP-independent.     

Serratia marcescens contains a heterodimeric HU protein like Escherichia coli and Salmonella typhimurium.

Homologs of the dimeric HU protein of Escherichia coli can be found in every prokaryotic organism that has been analyzed. In this work, we demonstrate that Serratia marcescens synthesizes two distinct HU subunits, like E. coli and Salmonella typhimurium, suggesting that the heterodimeric HU protein could be a common feature of enteric bacteria. A phylogenetic analysis of the HU-type proteins (HU and IHF) is presented, and a scheme for the origin of the hup genes and the onset of HU heterodimericity is suggested.     

Linker histones do not interact with DNA containing a single interstrand cross-link created by cisplatin

During our earlier investigations we have observed a prominent preference of the linker histone H1 for binding to a cis-platinated DNA (a synthetic fragment with global type of platination in respect to targets for cisplatin) comparing with unmodified and trans-Pt-modified DNA. In the present work we report our recent experimental results on the binding of the linker histones H1 and H5 to a cisplatin-modified synthetic DNA fragment containing a single nucleotide target d(GC/CG) for inter-platination. Surprisingly, no preferential binding of linker histones to cis-inter-platinated DNA was observed by means of the electromobility-shift assay. The same negative results were obtained with a part of the linker histone molecule suggested to be responsible for DNA-binding--its globular domain. Contrary, the data with another nuclear protein with similar DNA-binding properties as linker histones--HMGB1--showed a strong afinity for interaction with DNA containing interstrand cross-links.     

A model two-component system for studying the architecture of elastin assembly in vitro

Tropoelastin is encoded by a single human gene that spans 36 exons and is oxidized in vivo by mammalian lysyl oxidase at the epsilon amino group of available lysines to give the adipic semialdehyde, which then facilitates covalent cross-link formation in an enzyme-free process involving tropoelastin association. We demonstrate here that this process is effectively modeled by a two protein component system using purified lysyl oxidase from the yeast Pichia pastoris to facilitate the oxidation and subsequent cross-linking of recombinant human tropoelastin. The oxidized human tropoelastin forms an elastin-like polymer (EL) that is elastic, shows hydrogel behavior and contains typical elastin cross-links including lysinonorleucine, allysine aldol, and desmosine. Protease digestion and subsequent mass-spectrometry analysis of multiple ELs allowed for the identification of specific intra- and inter-molecular cross-links, leading to a model of the molecular architecture of elastin assembly in vitro. Specific intra-molecular cross-links were confined to the region of tropoelastin encoded by exons 6-15. Inter-molecular cross-links were prevalent between the regions encoded by exons 19-25. We find that assembly of tropoelastin molecules in ELs are highly enriched for a defined subset of cross-links.     

EGFP-tagged core and linker histones diffuse via distinct mechanisms within living cells

The effect of chromatin organization on EGFP-tagged histone protein dynamics within the cell nucleus has been probed using fluorescence correlation and recovery measurements on single living HeLa cells. Our studies reveal that free fraction of core-particle histones exist as multimers within the cell nucleus whereas the linker histones exist in monomeric forms. The multimeric state of core histones is found to be invariant across mammalian and polytene chromosomes and this is ATP dependent. In contrast, the dynamics of the linker histones exhibits two distinct diffusion timescales corresponding to its transient binding and unbinding to chromatin governed by the tail domain residues. Under conditions of chromatin condensation induced by apoptosis, the free multimeric fraction of core histones is found to become immobile, while the monomeric linker histone mobility is partially reduced. In addition, we observe differences in nuclear colocalization of linker and core particle histones. These results are validated through Brownian dynamics simulation of core and linker histone mobility. Our findings provide a framework to understand the coupling between the state of chromatin assembly and histone protein dynamics that is central to accessing regulatory sites on the genome.     

Histones H3/H4 form a tight complex with the inner nuclear membrane protein LBR and heterochromatin protein 1

We have recently shown that heterochromatin protein 1 (HP1) interacts with the nuclear envelope in an acetylation-dependent manner. Using purified components and in vitro assays, we now demonstrate that HP1 forms a quaternary complex with the inner nuclear membrane protein LBR and a sub-set of core histones. This complex involves histone H3/H4 oligomers, which mediate binding of LBR to HP1 and cross-link these two proteins that do not interact directly with each other. Consistent with previous observations, HP1 and LBR binding to core histones is strongly inhibited when H3/H4 are modified by recombinant CREB-binding protein, revealing a new mechanism for anchoring domains of under-acetylated chromatin to the inner nuclear membrane.     

Identification of domains involved in nuclear uptake and histone binding of protein N1 of Xenopus laevis.

The karyophilic protein N1 (590 amino acids) is an abundant soluble protein of the nuclei of Xenopus laevis oocytes where it forms defined complexes with histones H3 and H4. The amino acid sequence of this protein, as deduced from the cDNA, reveals a putative nuclear targeting signal as well as two acidic domains which are candidates for the interaction with histones. Using two different histone binding assays in vitro we have found that the deletion of the larger acidic domain reduces histone binding drastically to a residual value of approximately 15% of the complete molecule, whereas removal of the smaller acidic domain only slightly reduces histone complex formation in solution, but infers more effectively with binding to immobilized histones. In the primary structure of the protein both histone-binding domains are distant from the conspicuous nuclear accumulation signal sequence (residues 531-537) close to the carboxy terminus which is very similar to the SV40 large T-antigen nuclear targeting sequence. Using a series of N1 mutants altered by deletions or point mutations we show that this signal is required but not sufficient for nuclear accumulation of protein N1. The presence of an additional, more distantly related signal sequence in position 544-554 is also needed to achieve a level of nuclear uptake equivalent to that of the wild-type protein. Results obtained with point mutations support the concept of two nuclear targeting sequences and emphasize the importance of specific lysine and arginine residues in these signal sequences.     

Photochemical cross-linking of histones to DNA nucleosomes.

Ultraviolet (UV)-induced cross-linking was utilized in order to identify histone-DNA interacting regions in the chromatin repeating unit. Fractionated mononucleosomes which contained 185 base pairs of DNA and a full complement of the histones, including histone H1, were irradiated with light of lambda greater than 290nm in the presence of a photosensitizer. Equimolar amounts of histones H2A and H2B were found, by two independent labeling experiments, to be cross-linked to the DNA. Based on previous finding that the UV irradiation specifically cross-links residues which are in close proximity, irrespective of the nature of the amino acid side chain or the nucleotide involved, our results indicate that the four core histones are not positioned equivalently with respect to the DNA. This arrangement allows histones H2A and H2B to preferentially cross-link to the DNA. A water soluble covalent complex of DNA and histones was isolated. This complex was partially resistant to mild nuclease digestion, it exhibited a CD spectrum similar to that of chromatin, and was found to contain histone H1. These results are compatible with a model which suggests that histone H1, though anchored to the linker, is bound to the DNA at additional sites. By doing so it spans the whole length of the nucleosome and clamps together the DNA fold around the histone core.     

Further characterization of the posttranslational modifications of core histones in response to heat and arsenite stress in Drosophila

The effects of a heat shock or arsenite treatment on the methylation and acetylation of core histones have been investigated in Drosophila cultured cells. The decrease in H3 methylation, which is observed during a heat shock, is not a demethylation process, but results from methylation arrest. Two-dimensional gel electrophoresis leaves no ambiguity concerning the identity of H2B as a methylated protein, since H2B and D2, a nuclear nonhistone protein, which comigrate on one-dimensional gels, are well separated on these gels. Two-dimensional gel electrophoresis in the presence of Triton X-100 resolves each of the core histones into multiple forms resulting from posttranslational modifications. There are apparently, however, no histone variants in cultured Drosophila cells. At 23 degrees C, the various forms of the core histones resolved on two-dimensional gels are methylated. Under heat-shock or arsenite treatment, the methylation of all forms of H3 is decreased, while that of the various forms of H2B increase. These stress conditions also induce a generalized diminution in the acetylation of all forms of core histones. In the course of a heat shock, the synthesis of H2B is increased and this newly synthesized histone remains unacetylated during the shock. These changes in the patterns of core histone methylation and acetylation may be correlated with the reorganization of gene activity brought about by the heat shock.     

Interstrand DNA-platinum-DNA/cross-links induced by cis- and trans-diamminedichloroplatinum(II) at elevated temperature

Trans-diamminedichloroplatinum(II) (trans-DDP) forms with DNA at 37 degrees C, more numerous interstrand cross-links than cis-DDP in the isolated DNA and DNA in the chromatin complex. An increase in the temperature to 42.5 degrees C had no effect on the interstrand cross-links of DND-Pt-DNA formed by the two isomers, both in DNA and in chromatin.     

Histones in crenarchaea

Archaeal histone-encoding genes have been identified in marine Crenarchaea. The protein encoded by a representative of these genes, synthesized in vitro and expressed in Escherichia coli, binds DNA and forms complexes with properties typical of an archaeal histone. The discovery of histones in Crenarchaea supports the argument that histones evolved before the divergence of Archaea and Eukarya.