The type of interaction of histone H5 wo ith DNA changes significantly at various stages of chromatin condensation

Histones were covalently bound to DNA by dimethylsulfate-induced crosslinking and DNA-contacting peptides of histone H5, thus modified, were mapped by a combination of peptide cleavage reactions and peptide gel electrophoresis. In the nucleosome, the only strong crosslinking point is His-25 which resides near the ends of nucleosomal DNA. This contact point persists throughout different steps of chromatin condensation--decondensation. In decondensed chromatin, it is supplemented by the contact with DNA of the N-terminus of the histone H5 molecule. The high level of chromatin condensation existing in the nuclei or induced by bivalent cations results in a new and considerably stronger crosslinking point His-62, which is also characteristic for cooperative H5-DNA complexes. This structural change is observed only on oligonucleosomal chains containing no less than 3 contiguous nucleosomes, and is absent in isolated mono- or dinucleosomes. We propose that the formation of the 30-nm chromatin fibre, typical for the nuclei, is accomplished in part by the histone H5-linker DNA cooperative interactions, manifested by strong His-62--linker DNA contact.     

Accessibility of some regions of DNA in chromatin (chicken erythrocytes) to single strand-specific nucleases.

The susceptibility of the DNA in chromatin to single strand-specific nucleases was examined using nuclease P1, mung bean nuclease, and venom phosphodiesterase. A stage in the reaction exists where the size range of the solubilized products is similar for each of the three nucleases and is nearly independent of incubation time. During this stage, the chromatin fragments sediment in the range of 30 to 100 S and contain duplex DNA ranging from 1 to 10 million daltons. Starting with chromatin depleted of histones H1 and H5 similar fragments are generated. In both cases these nucleoprotein fragments are reduced to nucleosomes and their multimers by micrococcal nuclease. Thus, chromatin contains a limited number of DNA sites which are susceptible to single strand-specific nucleases. These sites occur at intervals of 8 to 80 nucleosomes and are distributed throughout the chromatin. Nucleosome monomers, dimers, or trimers were not observed at any stage of single strand-specific nuclease digestion of nuclei, H1- and H5-depleted chromatin, or micrococcal nuclease-generated oligonucleosomes. Each of the three nucleases converted mononucleosomes (approximately 160 base pairs) to nucleosome cores (approximately 140 base pairs) probably by exonucleolytic action that was facilitated by the prior removal of H1 and H5. The minichromosome of SV40 is highly resistant to digestion by nuclease P1.     

SWI-SNF-mediated nucleosome remodeling: role of histone octamer mobility in the persistence of the remodeled state

SWI-SNF is an ATP-dependent chromatin remodeling complex that disrupts DNA-histone interactions. Several studies of SWI-SNF activity on mononucleosome substrates have suggested that remodeling leads to novel, accessible nucleosomes which persist in the absence of continuous ATP hydrolysis. In contrast, we have reported that SWI-SNF-dependent remodeling of nucleosomal arrays is rapidly reversed after removal of ATP. One possibility is that these contrasting results are due to the different assays used; alternatively, the lability of the SWI-SNF-remodeled state might be different on mononucleosomes versus nucleosomal arrays. To investigate these possibilities, we use a coupled SWI-SNF remodeling-restriction enzyme assay to directly compare the remodeling of mononucleosome and nucleosomal array substrates. We find that SWI-SNF action causes a mobilization of histone octamers for both the mononucleosome and nucleosomal array substrates, and these changes in nucleosome positioning persist in the absence of continued ATP hydrolysis or SWI-SNF binding. In the case of mononucleosomes, the histone octamers accumulate at the DNA ends even in the presence of continued ATP hydrolysis. On nucleosomal arrays, SWI-SNF and ATP lead to a more dynamic state where nucleosomes appear to be constantly redistributed and restriction enzyme sites throughout the array have increased accessibility. This random positioning of nucleosomes within the array persists after removal of ATP, but inactivation of SWI-SNF is accompanied by an increased occlusion of many restriction enzyme sites. Our results also indicate that remodeling of mononucleosomes or nucleosomal arrays does not lead to an accumulation of novel nucleosomes that maintain an accessible state in the absence of continuous ATP hydrolysis.     

Evidence for attachment of interphase chromatin to the nuclear matrix via matrix-bound nucleosomes

Chromatin structure has been studied in the sites of attachment to the nuclear matrix in interphase mouse liver and spleen nuclei. The patterns of fragmentation of the DNA belonging to these sites (0.3-2% of total DNA in spleen and liver, respectively) with staphylococcal nuclease and DNAase I were very close to those of usual nucleosomal chains. Moreover, the nuclear matrix preparations contained all five major histones, including H1, in almost stoichiometric amounts. The histone/DNA ratios for the matrix were also similar to those found in nuclei. These findings and the size of the matrix-protected DNA indicated that interphase chromatin was attached to the nuclear matrix via matrix-bound nucleosomes and, to a much lesser extent, oligonucleosomes up to 5-6 units long. Two-dimensional electrophoretic separation of the matrix-bound histones revealed that modifications of histone H1 and, probably, of other histones were distinguished from those in bulk chromatin. Study of binding of exogenously added labeled histone octamers or mononucleosomal size DNA to nuclear matrix excluded the possibility of their artifactual trapping during the isolation procedure.     

On the de novo formation of compact oligonucleosomes at high ionic strength. Evidence for nucleosomal sliding in high salt.

Histone H 1-depleted chromatin made from acid extracted, intact nuclei was exposed to various ionic strengths. NaCl concentrations above 0.3 M sufficed to generate novel oligonucleosomes formerly characterized as "compact oligomers" and "spacerless dinucleosomes". Such particles could not be identified within H 1-depleted nuclei or chromatin at low ionic strengths. Their formation, proceeding within days at 0 degrees C, was accelerated by increasing ionic strengths. The data was discussed in terms of a salt-induced motion of nucleosomal core particles along the DNA to form compact oligomers.     

Nucleosomes are assembled into discrete size structures by histone H1 in vitro

The involvement of histone H1 in the formation and maintenance of higher order chromatin structures in vitro was investigated biochemically. Addition of exogenous histone H1 to isolated calf thymus mononucleosomes in low ionic strength buffer resulted in the formation of electrophoretically distinct mononucleosome assemblies (supernucleosomes). The smaller supernucleosomes were composed of about 12, 18, 24, or 30 nucleosomes and one to two molecules of histone H1 per nucleosome. It was difficult to determine accurately the size of the larger supernucleosomes, but their bands from native gels contained probably between 60 and 300 nucleosomes or more. Similar supernucleosome size classes were also obtained when oligonucleosomes instead of mononucleosomes were employed. When the assembly of mono- and oligo-nucleosomes with histone H1 took place in 0.15 M NaCl, discrete supernucleosomes containing only mono- or di-nucleosomes, but not a mixture of both, were formed. It is proposed that the small supernucleosomes containing oligomers of 6 nucleosomes may represent integral multiples of the second-order chromatin structural subunit, whereas the larger supernucleosomes containing about 60 to 300 or more nucleosomes may correspond to chromatin domains or third-order chromatin structures observed by other techniques.     

Characterization of Dnmt1 Binding and DNA Methylation on Nucleosomes and Nucleosomal Arrays

The packaging of DNA into nucleosomes and the organisation into higher order structures of chromatin limits the access of sequence specific DNA binding factors to DNA. In cells, DNA methylation is preferentially occuring in the linker region of nucleosomes, suggesting a structural impact of chromatin on DNA methylation. These observations raise the question whether DNA methyltransferases are capable to recognize the nucleosomal substrates and to modify the packaged DNA. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the maintenance DNA methyltransferase Dnmt1. Our binding studies show that Dnmt1 has a DNA length sensing activity, binding cooperatively to DNA, and requiring a minimal DNA length of 20 bp. Dnmt1 needs linker DNA to bind to nucleosomes and most efficiently recognizes nucleosomes with symmetric DNA linkers. Footprinting experiments reveal that Dnmt1 binds to both DNA linkers exiting the nucleosome core. The binding pattern correlates with the efficient methylation of DNA linkers. However, the enzyme lacks the ability to methylate nucleosomal CpG sites on mononucleosomes and nucleosomal arrays, unless chromatin remodeling enzymes create a dynamic chromatin state. In addition, our results show that Dnmt1 functionally interacts with specific chromatin remodeling enzymes to enable complete methylation of hemi-methylated DNA in chromatin.     

Formation of hybrid nucleosomes cantaining new and old histones.

5 mM hydroxyurea (HU) inhibits DNA synthesis in mouse P815 cells by 94-97% in less than 1 hr. Nevertheless, histone synthesis continues and newly-synthesised histones are incorporated into non-replicating chromatin at a rate of about 20% of that in control exponentially-growing cells. To study the organization of these histones in chromatin P815 cells were treated with 5 mM HU in medium containing dense (15N, 13C, 2H) - substituted amino acids. After inhibition of DNA synthesis, newly-synthesised histones were labelled with (3H)-arginine. The cells were harvested 90 min later, and mono- and oligonucleosomes were prepared and analysed on metrizamide-triethanolamine (MA-TEA density gradients. Analysis of the distribution of 3H-labelled histones in these gradients shows that they are incorporated into hybrid mononucleosomes containing both new and old histones. It is also shown that these hybrid nucleosomes are not randomly distributed, but show a certain tendency to be clustered in certain chromatin regions.     

Biochemical and cytological studies of bleomycin actions on chromatin and chromosomes

Interaction of bleomycin with nuclei isolated from a variety of mammalian cells resulted in the release of nucleosomes. When isolated mononucleosomes (core plus linker) were re-treated with bleomycin, no further degradation of DNA occurred. The results suggest that the bleomycin cleavage sites in chromatin are present only in the linker region and that there are probably only one or two cleavage sites per linker. The repeat lengths of nucleosomal DNA released by bleomycin from nuclei of different species are different; this variability is considered to reflect the length of the linker. Incorporation of BrdU into DNA did not alter the bleomycin action on nucleosomes. When mitotic cells were held at metaphase for a prolonged period, bleomycin caused a gradual disintegration of chromosomes, although the bleomycin cleavage sites in metaphase chromosomes were found to be the same as those in interphase nuclei.     

Selective arrangement of ubiquitinated and D1 protein-containing nucleosomes within the Drosophila genome

We have a new approach, two-dimensional hybridization mapping of nucleosomes, to compare the structures of mononucleosomes from different regions of the Drosophila melanogaster genome. Approximately one in two nucleosomes of the transcribed copia and heat-shock 70 (hsp 70) genes in nonshocked cultured cells contains ubiquitin-H2A (uH2A) semihistone, a covalent conjugate of histone H2A and a small protein, ubiquitin. In striking contrast, less than one in 25 nucleosomes of tandemly repeated, nontranscribed 1.688 satellite DNA contains uH2A, suggesting that most of the nucleosomal uH2A is located in transcribed genes. Approximately 25% of all nucleosomes are ubiquitinated in nonsynchronized cultured Drosophila cells. The hsp 70 genes in nonshocked cells occur in nucleosomes, are greatly enriched in uH2A and are not digested preferentially by staphylococcal nuclease. In contrast, the same genes in chromatin from heat-shocked cells are highly sensitive to staphylococcal nuclease and no longer possess nucleosomal organization recognizable with this probe. Histone ubiquitination in transcribed nucleosomes may prevent formation of higher order chromosomal structures by modifying nucleosome-nucleosome interactions. The observed loss of nucleosomal organization in very actively transcribed genes, such as the hsp 70 genes in shocked cells, may be related to the recent finding that ubiquitin conjugates are substrates for the cytoplasmic ATP-dependent proteolytic system. We have also found that 1.688 satellite mononucleotomes contain a specific approximately 50,000 dalton nonhistone protein, D1, in addition to being extremely under-ubiquitinated. D1 may be involved in formation of the highly compact structure of satellite heterochromatin.     

ACF catalyses chromatosome movements in chromatin fibres

Nucleosome-remodelling factors containing the ATPase ISWI, such as ACF, render DNA in chromatin accessible by promoting the sliding of histone octamers. Although the ATP-dependent repositioning of mononucleosomes is readily observable in vitro, it is unclear to which extent nucleosomes can be moved in physiological chromatin, where neighbouring nucleosomes, linker histones and the folding of the nucleosomal array restrict mobility. We assembled arrays consisting of 12 nucleosomes or 12 chromatosomes (nucleosomes plus linker histone) from defined components and subjected them to remodelling by ACF or the ATPase CHD1. Both factors increased the access to DNA in nucleosome arrays. ACF, but not CHD1, catalysed profound movements of nucleosomes throughout the array, suggesting different remodelling mechanisms. Linker histones inhibited remodelling by CHD1. Surprisingly, ACF catalysed significant repositioning of entire chromatosomes in chromatin containing saturating levels of linker histone H1. H1 inhibited the ATP-dependent generation of DNA accessibility by only about 50%. This first demonstration of catalysed chromatosome movements suggests that the bulk of interphase euchromatin may be rendered dynamic by dedicated nucleosome-remodelling factors.     

Characteristics of the structural state of DNA in chromatin with short linker

Various fragments of pigeon brain neuron chromatin with very short linker DNA have been studied by circular dichroism (CD). The DNA structure in core particles of the brain and thymus chromatins is very similar. Linker DNA and a part of core DNA in the mononucleosomes of brain chromatin is extended. This conclusion follows from increasing CD amplitude of the brain mononucleosomes as compared with the corresponding value for thymus mononucleosomes. The internucleosome interactions stabilized the compactness of core DNA in the brain oligonucleosomes. The whole linker DNA of the brain chromatin unlike thymus chromatin is extended at low ionic strength. This fact can explain the brain chromatin ability to form a compact structure with increasing ionic strength.     

Stable remodeling of tailless nucleosomes by the human SWI-SNF complex

The histone N-terminal tails have been shown previously to be important for chromatin assembly, remodeling, and stability. We have tested the ability of human SWI-SNF (hSWI-SNF) to remodel nucleosomes whose tails have been cleaved through a limited trypsin digestion. We show that hSWI-SNF is able to remodel tailless mononucleosomes and nucleosomal arrays, although hSWI-SNF remodeling of tailless nucleosomes is less effective than remodeling of nucleosomes with tails. Analogous to previous observations with tailed nucleosomal templates, we show both (i) that hSWI-SNF-remodeled trypsinized mononucleosomes and arrays are stable for 30 min in the remodeled conformation after removal of ATP and (ii) that the remodeled tailless mononucleosome can be isolated on a nondenaturing acrylamide gel as a novel species. Thus, nucleosome remodeling by hSWI-SNF can occur via interactions with a tailless nucleosome core.     

The association of a magnesium-dependent endonuclease activity with a nucleosome fraction from rat-liver nuclei

The nucleosomes released by the incubation (autodigestion) of rat-liver nuclei were fractionated by sucrose-density gradient centrifugation, and subjected to nuclease assay with heat-denatured 3H-DNA from Escherichia coli as an exogenous substrate. With increasing incubation time, the nuclease activity was enhanced and localized in the mono/tetra-, hexa/hepta-, and long-chain oligonucleosome fractions. In contrast, independent of the nucleosome size, the activities of 0.35 M NaCl-soluble fractions from them were found to be almost equal in terms of specific activity (dpm/nucleosomal DNA). Such nuclease activity was not detected in the sucrose gradient (top region) lacking nucleosomes and/or chromatin. When the chromatin was dialyzed against a 0.35 M NaCl buffer and then fractionated in a sucrose gradient containing 0.35 M NaCl, most of the nuclease activity was solubilized into the above top region. On gel filtration of the mononucleosome fraction in the 0.35 M NaCl buffer, the nuclease activity was eluted at the position of 36,000 daltons. This nuclease cleaved heat-denatured DNA more rapidly than the native DNA in the presence of Mg2+, and had the ability to make both single-strand nicks and double-strand cuts in pBR322 DNA; in other words, it had an endonucleolytic activity. Moreover, four different classes of mononucleosomes were fractionated by electrophoresis of the nucleosomes released by autodigestion of the nuclei. These mononucleosomes also showed nuclease activity with the heat-denatured DNA. Thus, the present studies suggest that an Mg2+-dependent endonuclease of about 36,000 daltons is associated with the nucleosome particle(s) in rat-liver nuclei.