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.     

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.     

Binding of non-histone chromosomal protein HMG1 to histone H3 in nucleosomes detected by photochemical cross-linking

The interaction of non-histone chromosomal protein HMG1 with core histones in nucleosomes was studied via reconstitution and photochemical cross-linking. The results obtained indicated that photoaffinity-labeled HMG1 interacted in nucleosomes with histone H3. Similar experiments with peptides derived from HMG1 by V8 protease digestion allowed to identify N-terminal domain of HMG1 (peptide V3) as a binding region for histone H3 in nucleosomes.     

Studies on the oligomeric state of isolated cytochrome oxidase using cross-linking reagents

(1) Sucrose gradient centrifugation of cytochrome oxidase in the presence of Triton X-100 gave one slowly sedimenting green band. After cross-linking with dithiobis(succinimidylpropionate) (DSP), two green bands were observed, one sedimenting like the control and the other one more rapidly. Only the slowly sedimenting band was observed if the cross-linker was cleaved by dithiothreitol before centrifugation. (2) The rapidly sedimenting band in the Triton-containing sucrose gradient is probably the internally cross-linked dimer of cytochrome oxidase; the one sedimenting slowly is the monomeric enzyme. (3) Cross-linking with DSP after monomerization yields a small fraction of internally cross-linked dimers in addition to the internally cross-linked monomers. Under similar conditions, but using the shorter cross-linker disuccinimidyl tartarate (DST), no dimers are detected. (4) Both DSP and DST cross-link the dimeric enzyme so that it could no longer be monomerized by centrifugation in Triton, unless the cross-link is cleaved. (5) Polypeptide analysis using two-dimensional gel electrophoresis of cross-linked dimers and monomers suggest that subunit VIb is involved in intermonomeric cross-linking of dimeric enzyme by DSP.     

Photodynamic protein cross-linking

Exposure of spectrin to visible light in the presence of a photosensitizer results in photo-oxidation of sensitive amino acid residues and covalent cross-linking of the polypeptides. In a previous paper the cross-linking was ascribed to a secondary reaction between photo-oxidized histidine residues and amino groups. The following observations, described in this paper, are in accordance with this supposition. (1) During illumination of spectrin in the presence of a photosensitizer a pronounced photo-oxidation of histidine residues takes place. (2) Simultaneously a decrease of free amino groups is observed. (3) Semicarbazide protects against cross-linking and is bound to a histidine photo-oxidation product in spectrin. (4) The pH profile of histidine photo-oxidation and subsequent reaction with amino groups is similar to the pH profile of spectrin cross-linking. Amidination of NH₂ groups in spectrin does not inhibit cross-linking, as visualized by gel electrophoresis. On the other hand aminidation of denatured myoglobin causes a 50% inhibition of cross-linking. These observations support the notion of NH₂-involvement in cross-linking but also demonstrate, that other photodynamic cross-linking mechanisms exist.     

Structure of nucleosomes and organization of inter-nucleosomal DNA in chromatin

We have compared mononucleosomes that were obtained by hydrolysis of chromatin micrococcal nuclease from a number of sources with the length of a nucleosomal repeat 185--245 b. p. long. For hydrolysis of chromatin isolated from nuclei, a series of nucleosomes was formed: MN145 (core particle), MN165, MN175...MN205, MN215, the lengths of their DNAs differing (by approximately 10.n b.p. where n = 1, 2, 3...) by a factor of 10. A feature of hydrolysis of chromatin in nuclei was the appearance of an additional H1-depleted MN155 particle. It is suggested that upon isolation of chromatin from nuclei, its partial decompactization takes place. This decompactization changes the character of nuclease splitting and seems to be connected with rearrangement of histone H1. These observations demonstrate that besides core particles MN145 and chromatosomes MN165, the major particles of digest of nuclei appear to be MN155, and for isolated chromatin--MN175. Unlike this standard picture, mainly MN145, MN155, MN235 and MN245 are formed upon hydrolysis of sea urchin sperm nuclei.     

Structure of nucleosomes and organization of internucleosomal DNA in chromatin

We have compared the mononucleosomal pattern produced by micrococcal nuclease digestion of condensed and unfolded chromatin and chromatin in nuclei from various sources with the repeat length varying from 165 to 240 base-pairs (bp). Upon digestion of isolated H1-containing chromatin of every tested type in a low ionic strength solution (unfolded chromatin), a standard series of mononucleosomes (MN) was formed: the core particle, MN145, and H1-containing, MN165, MN175, MN185, MN195, MN205 and MN215 (the indexes give an approximate length of the nucleosomal DNA that differs in these particles by an integral number of 10 bp). In addition to the pattern of unfolded chromatin, digestion of whole nuclei or condensed chromatin (high ionic strength of Ca2+) gave rise to nuclei-specific, H1-lacking MN155. Digestion of H1-lacking chromatin produced only MN145, MN155 and MN165 particles, indicating that the histone octamer can organize up to 165 bp of nucleosomal DNA. Although digestion of isolated sea urchin sperm chromatin (repeat length of about 240 bp) at a low ionic strength gave a typical "unfolded chromatin pattern", digests of spermal nuclei contained primarily MN145, MN155, MN235 and MN245 particles. A linear arrangement of histones along DNA (primary organization) of the core particle was found to be preserved in the mononucleosomes, with the spacer DNA length from 10 to 90 bp on one (in MN155) or both sides of core DNA being a multiple of about 10 bp. In MN235, the core particle occupies preferentially a central position with the length of the spacer DNA on both sides of the core DNA being usually about 30 + 60 or 40 + 50 bp. Histone H1 is localized at the ends of these particles, i.e. close to the centre of the spacer DNA. The finding that globular part of histones H3 and sea urchin sperm H2B can covalently bind to spacer DNA suggests their involvement in the organization of chromatin superstructure. Our data indicate that decondensation of chromatin is accompanied by rearrangement of histone H1 on the spacer DNA sites adjacent to the core particle and thus support a solenoid model for the chromatin superstructure in nuclei in which the core DNA together with the spacer DNA form a continuous superhelix.     

The layered organization of nucleosomes in 30 nm chromatin fibers

We have used electron microscopy and established methods of three-dimensional reconstruction to obtain structural information on the 30 nm chromatin fibers from sea cucumber sperm and chicken erythrocytes. The fibers show a longitudinal periodicity of 10–11 nm. We have interpreted this periodicity as due to a grouping of nucleosomes into disks, each disk containing about 5–6 nucleosomes. These disks are closely stacked to form the chromatin fiber. We have built a detailed model for four fibers and we have determined the approximate coordinates of all the nucleosomes in them. The average distance found between neighboring nucleosomes has a value close to 11 nm. They may be connected either as a regularly distorted helix or as a layered zigzag. The second model appears more appropriate, since in the constrictions of the fibers the nucleosomes can only be connected as a zigzag.     

Microtubule organization by cross-linking and bundling proteins.

To understand microtubule function the factors regulating their spatial organization and their interaction with cellular organelles, including other microtubules, must be elucidated. Many proteins are implicated in these organizational events and the known consequences of their actions within the cell are increasing. For example, the function of microtubule bundles at the surfaces of polarized cells has recently received attention, as has the action in cortical rotation of a transient arrangement of microtubules found beneath the vegetal surface of fertilized frog eggs. The in vivo association of microtubules during early Xenopus oogenesis has added interest as microtubules bundled in cell-free extracts are protected against the action of a severing protein found in this animal. A 52 kDa F-actin bundling protein purified from Physarum polycephalum organizes microtubules and causes the cobundling of microtubules and microfilaments. These observations, in concert with others that are presented, emphasize the diversity within the family of microtubule cross-linking proteins. The challenge is to determine which proteins are relevant from a physiological perspective, to ascertain their molecular mechanisms of action and to describe how they affect cytoplasmic organization and cell function. To realize this objective, the proteins which cross-link and bundle microtubules must be investigated by techniques which reveal different but related aspects of their properties. Cloning and sequencing of genes for cross-linking proteins, their subcellular localization especially as microtubule-related changes in cell morphology are occurring and the application of genetic studies are necessary. Study of the neural MAP provides the best example of just how powerful current experimental approaches are and at the same time shows their limits. The neural MAP have long been noted for their enhancement of tubulin assembly and microtubule stability. Their spatial distribution has been studied during the morphogenesis of neural cells. Sequencing of cloned genes has revealed the functional domains of neural MAP including carboxy-terminal microtubule-binding sites. Similarities to microtubule binding proteins from other cell types stimulate interest in the neural MAP and further suggest their importance in microtubule organization. For example, MAP4 enjoys a wide cellular distribution and has microtubule-binding sequences very similar to those in the neural MAP. Moreover, the nontubulin proteins of marginal bands are immunologically related to neural MAP, indicating shared structural/functional domains. Even with these findings the mechanism by which neural MAP cross-link microtubules remains uncertain. Indeed, some researchers express doubt that microtubule cross-linking is actually a function of neural MAP in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

Investigation of the organization of rhodopsin in the sheep photoreceptor membrane by using cross-linking reagents.

The organization of rhodopsin in the photoreceptor membrane of sheep rod outer segments was investigated by using a variety of bifunctional reagents. Of the nine reagents used, seven gave oligomeric opsin species, whereas two, copper phenanthroline and dithiobisphenyl azide, failed to cross-link the protein. In general, the cross-linked species obtained showed diminishing yields from dimer to tetramer, together with some higher-molecular-weight aggregates. It is proposed that the patterns of cross-linking arise as a result of collision complexes and best describe a monomeric organization for native rhodopsin. No significant differences between the patterns obtained with dark-adapted bleached or regenerated protein states were observed. This interpretation is discussed in relation to the postulated mechanism of action of rhodopsin.     

Extracellular signal-regulated kinase/mitogen-activated protein kinase regulates actin organization and cell motility by phosphorylating the actin cross-linking protein EPLIN

Extracellular signal-regulated kinase (ERK) is important for various cellular processes, including cell migration. However, the detailed molecular mechanism by which ERK promotes cell motility remains elusive. Here we characterize epithelial protein lost in neoplasm (EPLIN), an F-actin cross-linking protein, as a novel substrate for ERK. ERK phosphorylates Ser360, Ser602, and Ser692 on EPLIN in vitro and in intact cells. Phosphorylation of the C-terminal region of EPLIN reduces its affinity for actin filaments. EPLIN colocalizes with actin stress fibers in quiescent cells, and stimulation with platelet-derived growth factor (PDGF) induces stress fiber disassembly and relocalization of EPLIN to peripheral and dorsal ruffles, wherein phosphorylation of Ser360 and Ser602 is observed. Phosphorylation of these two residues is also evident during wound healing at the leading edge of migrating cells. Moreover, expression of a non-ERK-phosphorylatable mutant, but not wild-type EPLIN, prevents PDGF-induced stress fiber disassembly and membrane ruffling and also inhibits wound healing and PDGF-induced cell migration. We propose that ERK-mediated phosphorylation of EPLIN contributes to actin filament reorganization and enhanced cell motility.     

Studies on crystallization and cross-linking of lipase for biocatalysis

The development of robust biocatalysts with increased stability and activity is a major challenge to industry. A major breakthrough in this field was the development of cross-linked enzyme crystals with high specificity and stability. A method is described to produce micro crystals of CLEC lipase, which is thermostable and solvent stable. Lipase from Burkholderia cepacia was crystallized using ammonium sulfate and cross-linked with glutaraldehyde to produce catalytically active enzyme. The maximum yield of CLEC was obtained with 70% ammonium sulfate and cross-linked with 5% (v/v) glutaraldehyde. SEM studies showed small hexagonal-shaped crystals of 2–5 μm size. CLEC lipase had improved thermal and reuse stability. It is versatile, having good activity in both polar and nonpolar organic solvents. CLEC lipase was coated using β cyclodextrin for improving the storage and reuse stability. CLEC was successfully used for esterification of Ibuprofen and synthesis of ethyl butyrate.     

Photo-assisted peptide enrichment in protein complex cross-linking analysis of a model homodimeric protein using mass spectrometry

MS analysis of cross-linked peptides can be used to probe protein contact sites in macromolecular complexes. We have developed a photo-cleavable cross-linker that enhances peptide enrichment, improving the signal-to-noise ratio of the cross-linked peptides in mass spectrometry analysis. This cross-linker utilizes nitro-benzyl alcohol group that can be cleaved by UV irradiation and is stable during the multiple washing steps used for peptide enrichment. The enrichment method utilizes a cross-linker that aids in eliminating contamination resulting from protein-based retrieval systems, and thus, facilitates the identification of cross-linked peptides. Homodimeric pilM protein from Pseudomonas aeruginosa 2192 (pilM) was investigated to test the specificity and experimental conditions. As predicted, the known pair of lysine side chains within 14?? was cross-linked. An unexpected cross-link involving the protein's amino terminus was also detected. This is consistent with the predicted mobility of the amino terminus that may bring the amino groups within 19?? of one another in solution. These technical improvements allow this method to be used for investigating protein-protein interactions in complex biological samples.     

Alignment of nucleosomes along DNA and organization of spacer DNA in Drosophila chromatin

A series of mono- and dinucleosomal DNAs characterized by an about ten-base periodicity in the size were revealed in the micrococcal nuclease digests of Drosophila chromatin which have 180 +/- 5 base pair (bp) nucleosomal repeat. 20, 30, and 40 bp spacers were found to be predominant in chromatin by trimming DNA in dinucleosomes to the core position. Among several identified mononucleosomes (MN), MN170, MN180 and MN190 were isolated from different sources (the figures indicate the DNA length in bp). The presence of the 10, 20, and 30 bp long spacers was shown in these mononucleosomes by crosslinking experiments. The interaction of histone H3 with the spacer in the Drosophila MN180 particle was also shown by the crosslinking /5/. We conclude from these results that the 10 n bp long intercore DNA (n = 2, 3 and 4) is organized by histone H3, in particular, and together with the core DNA forms a continuous superhelix. Taken together, these data suggest that Drosophila chromatin consists of the regularly aligned and tightly packed MN180, as a repeating unit, containing 10 and 20 bp spacers at the ends of 180 bp DNA. Within the asymmetric and randomly oriented in chromatin MN180, the cores occupy two alternative positions spaced by 10 bp.     

Germ cell nucleosomes contain remodeled core protein complex

Electrophoretic analysis of the nucleosomal histones from MN1 and MN2 subpopulations of the seminiferous tubules in gels containing either 6.25 or 2.5 M urea revealed the presence of testis specific histone H2S, H1 and protein ‘A’ in addition to the somatic histones in the core protein complex. Size analysis indicated the presence of a 150–160 bp DNA segment in the MNI subpopulation, whereas, an approx 180 bp DNA fragment was present in the MN2 subpopulation of both liver and tubule nucleosomes. These data suggest an extensive remodeling of the nucleosomal core protein complex during mammalian spermatogenesis.     

Structural organization and dynamics of the endoplasmic reticulum during spermatogenesis of Drosophila melanogaster: Studies using PDI-GFP chimera protein

An important property of the endoplasmic reticulum (ER) is its ability to change morphology and intracellular localization during the cell cycle and differentiation. Visualization of the ER membranes using the protein disulphide isomerase (PDI) GFP chimeric protein makes it possible to trace the dynamics of all these processes and expose transitional forms and intermediate configurations. In this article the results of the study of the ER morphology during spermatogenesis of D. melanogaster are presented. It was shown that ER membranes retain high level of GFP-fluorescence through all stages of spermatogenesis, so that revealing of the stage-specific features of the ER organization was possible. The ER network has distinctive reticular morphology during the interphase and early prophase. Right before the cell division, this morphology changes and ER forms a system of branchless filamentous membranes. In prometaphase, these membranes form concentric circles adjacent to the nuclear membrane; from metaphase to telophase, they lengthen along the axis of cell division and resemble a spindle. Later, in the next interphase, this configuration transforms to a reticular structure seen previously. At the beginning of spermatid differentiation, the ER encompasses nebenkern and nucleus and elongates adjacent to them. During the latest stages of spermatogenesis, the ER network dissociates into separate membranous granules that are eliminated from the cyst with the individualization complex. Possible mechanisms of the ER dynamics and reorganization are discussed.     

Human centromere protein B induces translational positioning of nucleosomes on alpha-satellite sequences

The human centromere proteins A (CENP-A) and B (CENP-B) are the fundamental centromere components of chromosomes. CENP-A is the centromere-specific histone H3 variant, and CENP-B specifically binds a 17-base pair sequence (the CENP-B box), which appears within every other alpha-satellite DNA repeat. In the present study, we demonstrated centromere-specific nucleosome formation in vitro with recombinant proteins, including histones H2A, H2B, H4, CENP-A, and the DNA-binding domain of CENP-B. The CENP-A nucleosome wraps 147 base pairs of the alpha-satellite sequence within its nucleosome core particle, like the canonical H3 nucleosome. Surprisingly, CENP-B binds to nucleosomal DNA when the CENP-B box is wrapped within the nucleosome core particle and induces translational positioning of the nucleosome without affecting its rotational setting. This CENP-B-induced translational positioning only occurs when the CENP-B box sequence is settled in the proper rotational setting with respect to the histone octamer surface. Therefore, CENP-B may be a determinant for translational positioning of the centromere-specific nucleosomes through its binding to the nucleosomal CENP-B box.