Undermethylation of DNA in mononucleosomes solubilized by micrococcal nuclease digestion of HeLa cell nuclei

To examine the distribution of 5-methylcytosine in chromatin DNA, DNA of HeLa cells was labeled with [3H-methyl]methionine and [14C] thymidine and analyzed after extensive digestion of the nuclei with micrococcal nuclease. When the chromatin solubilized with the nuclease was fractionated on a sucrose density gradient, DNA in mononucleosomes was considerably depleted in 5-methylcytosine, as compared with polynucleosomes. Electrophoretic separation of DNA from the chromatin also revealed the depletion of 5-methylcytosine in the mononucleosomal size of DNA. This was confirmed by the chromatographic analysis of 5-methyldeoxycytidine after enzymatic digestion of the DNA to nucleosides. Thus the DNA in mononucleosomes solubilized by extensive micrococcal nuclease digestion is depleted in 5-methylcytosine, suggesting that 5-methylcytosine is preferentially missing from the DNA in the nucleosome core particles.     

Isolation and characterisation of a 167 bp core particle isolated from stripped chicken erythrocyte chromatin

We have digested chicken erythrocyte soluble chromatin, both unstripped and stripped of histones H1 and H5 with either 0.6 M NaCl or DNA-cellulose, with micrococcal nuclease (MNase). Digestion of unstripped chromatin to monomeric particles initially paused at 188 bp DNA; continued digestion resulted in another pause at 177 before the 167 bp chromatosome and 146 bp core particle were obtained. Digestion of stripped chromatin to monomeric particles paused transiently at 177 bp; continued digestion resulted in marked pauses at 167 and 156 before the 146 bp core particle was obtained. These results suggested that 167 bp DNA representing two complete turns are bound to the histone octamer. Histone H1/H5 binds an additional two helical turns of DNA, thereby protecting up to 188 bp DNA against nuclease digestion. Monomeric particles containing 167 bp DNA were isolated from stripped chromatin and found by DNase I digestion to be a homogeneous population with a 10 bp DNA extension to either end relative to the 146 bp core particle. Thermal denaturation and circular dichroism spectroscopy showed stronger histone-DNA interactions and increased DNA winding as the length of DNA attached to the core histone octamer was decreased. Thermal denaturation also showed three classes of histone-DNA interaction: the core particle containing 167 bp DNA had tight binding of ten helical turns of DNA, intermediate binding of two helical turns and looser binding of four helical turns.     

A 21 KDa protein resides in the nucleosomes of micrococcal nuclease sensitive liver chromatin of Atlantic salmon treated with estradiol

Atlantic salmon was treated with 17-beta-estradiol to induce the process of vitellogenesis in liver. When the isolated liver nuclei were incubated with micrococcal nuclease at increasing enzyme/DNA ratios a 21 KDa protein appeared in the nuclease sensitive chromatin, the S-fraction. After HPLC gel filtration the 21 KDa protein resided with the oligo- and mononucleosomes. The S-fraction contained vitellogenin gene sequences at low nuclease/DNA ratios. The sequences were detectable also in the mononucleosomes derived from the S-fraction. After hormone treatment the vitellogenin gene exposed a higher sensitivity to micrococcal nuclease than the hormone-untreated controls. The results indicate that the 21 KDa protein took part in the hormone-mediated changes in gene expression by modulating the structure of estradiol responsive chromatin domains including the vitellogenin gene.     

Organizational changes in chromatin at different malignant stages of Friend erythroleukemia.

The chromatin structure of morphologically-similar, but increasingly-malignant erythroleukemia cells was investigated using milk micrococcal nuclease digestion of isolated nuclei. The maximum solubilization of chromatin was unique for each of the three cell types: the least malignant (our Stage II) released 61% of its chromatin DNA, the most malignant (Stage IV), 46%, and the intermediate (Stage III) released 36%. An analysis of the nucleosome oligomers liberated by digestion also demonstrated differences. After 15 minutes of digestion when release was reaching its maximum, a greater proportion of large nucleosomal oligomers (sizes > trinucleosome) was released from Stage II nuclei than from Stage III or IV nuclei. The cell types also differed in the relative amount of H1-depleted mononucleosomes released. Analysis of the size of the double-stranded DNA associated with mononucleosomal particles showed that Stage III mononucleosomes were smaller (148 bp) than Stage IV (167 bp) or Stage II (190 bp). In addition, while the DNA of mononucleosomes depleted in H1 was smaller than that in the H1-containing species, relative size differences among the different cell types were retained. These data suggested that the difference in the mononuocleosome particle size resistant to nuclease digestion was independent of histone H1. Differences in nucleosome repeat length were also noted among the cell types. These studies have demonstrated dramatic differences in chromatin structure associated with malignant potential of an otherwise morphologically identical cell type. These findings may reflect changes in the relative amounts of H2a variants which we have previously described among the different malignant cell types.     

Nick translation of HeLa cell nuclei as a probe for locating DNase I-sensitive nucleosomes

The technique of nick translation of nuclei (Levitt, A., Axel, R., and Cedar, H. (1979) Dev. Biol. 69, 496-505) has been used in HeLa cells to label DNase I-sensitive regions. Micrococcal nuclease digestion of the nick translated nuclei was followed by a low ionic strength gel electrophoresis system which separates different types of mononucleosomes. The major label was observed in the vicinity of high mobility group protein containing mononucleosomes. However, further analysis revealed that the particle does not sediment in the position of mononucleosomes on a sucrose gradient. Two alternative explanations are discussed as the possible source of this particle. It is either a high mobility group protein containing nucleosome in some unfolded conformation or the labeled particle originates from discrete DNA fragments, wrapped around some nonhistone proteins, located in a highly DNase I-sensitive region, which is resistant to micrococcal nuclease digestion.     

Digestion of insect chromatin with micrococcal nuclease, DNase I and DNase I combined with single-strand specific nuclease S1.

The chromatin of the lepidopteran Ephestia kuehniella was digested by micrococcal nuclease, DNase I and S1-nuclease combined with DNase I pretreatment. The resulting DNA fragments were analyzed by gel electrophoresis and compared with the DNA fragments of rat liver nuclei obtained by the same process. Extensive homology was revealed between insect and mammalian chromatin structure. The combined DNase I- S1-nuclease digestion yields double-stranded DNA fragments of lengths from 30 to 110 base-pairs. These DNA fragments are not obtained from nuclei predigested extensively with micrococcal nuclease. The results are discussed with respect to the internal structure of the chromatin subunit.     

Two-dimensional gel electrophoretic separation of the proteins present in chromatin of Escherichia coli

The polypeptides present in 35S-labelled chromatin prepared from Escherichia coli cells, and polypeptides present in the DNA and RNA complexes obtained by micrococcal nuclease digestion of the chromatin, were analysed by two-dimensional non-equilibrium polyacrylamide gel electrophoresis. Three hundred and thirty-five 35S-labelled polypeptides were detected in the chromatin whereas the DNA- and RNA-containing fractions of the micrococcal nuclease digest contained 126 and 183 polypeptides respectively. The major basic low-molecular-weight polypeptides were found in the DNA-containing fractions.     

Identification of nonhistone chromatin proteins in chromatin subunits (or mononucleosomes) devoid of histone H1.

Rat liver chromatin was digested by micrococcal nuclease. Chromatin subunits (or mononucleosomes) were isolated by sucrose density gradient and subsequently fractionated by 6% polyacrylamide gel electrophoresis into two major components. One component (MN1) of the mononucleosomes had a higher mobility, contained histones H2A, H2B, H3, H4, and shorter DNA fragments (140 base pairs) while the other (MN2) contained all five histones and longer DNA fragments (180 base pairs). Both submononucleosomes (MN1 and MN2) were found to contain nonhistone chromatin proteins (NHCP). By electrophoresis in 15% sodium dodecyl sulfate-polyacrylamide gel, 9 and 11 major fractions of NHCP were identified in the submononucleosomes MN1 and MN2, respectively. It was also observed that treatment of mononucleosomes with 0.6 M NaCl removes most of these NHCP and histone H1 except for two major NHCP which remain in the core particles.     

The organization of oligonucleosomes in yeast

We have developed a method of preparing yeast chromatin that facilitates the analysis of nucleoprotein organization. Yeast chromatin, isolated as an insoluble complex, is digested with micrococcal nuclease and fractionated into major insoluble and soluble fractions. No nucleosomal repeat is seen early in digestion for the insoluble fraction. Nucleosomal complexes of the soluble fraction are excised by nuclease in a distinctive non-random pattern; they are markedly depleted in mononucleosomes. When we analyze the soluble material by high resolution native electrophoresis, we find that the nucleoproteins resolve into two bands for each DNA multimer of the nucleosomal repeat. Our results suggest that there are structural similarities between bulk yeast chromatin and chromatin configurations found in transcribing genes of complex eukaryotes.     

The structure of sub-nucleosomal particles. The octameric (H3/H4)4--125-base-pair-DNA complex

Chicken erythrocyte chromatin was depleted of histones H1, H5, H2A and H2B. The resulting (H3/H4)-containing chromatin was digested with micrococcal nuclease to yield monomer, dimer, trimer etc. units, irregularly spaced on the DNA, with even-number multimers being more prominent. Sucrose density gradient centrifugation separated monomers and dimers (7.7 S and 10.5 S). Sodium dodecyl sulphate gel electrophoresis and cross-linking indicated: the monomer contains 50-base-pair (bp), 60-bp and 70-bp DNA and the dimer 125-bp DNA; the monomer contains a tetramer and the dimer an octamer of H3 and H4. Partial association of monomer units to dimers inhibits structural studies of monomers. The internal structure of the dimer, i.e. and (H3/H4)4-125-bp-DNA particle, was studied using circular dichroism, thermal denaturation and nuclease digestion. Both micrococcal nuclease and DNase I digestion indicate that, unlike core particles, accessible sites occur in the centre of the particle and it is concluded that the (H3/H4)4-125-bp-DNA particle is not a 'pseudo-core particle' in which the 'extra' H3 and H4 replace H2A and H2B. It is proposed that the octamer particle is formed by the sliding together of two 'monomer' units, each containing the (H3/H4)2 tetramer and 70 bp of DNA. Excision of this dimer unit with micrococcal nuclease results in the loss of 10 readily digestible base pairs at each end, leaving 125 bp.     

The structural organization of sperm chromatin

The structure of rabbit, fowl, and Xenopus laevis sperm chromatin was explored by study of the reaction of their decondensed nuclei with DNase 1 and micrococcal nuclease. Those of rabbit and fowl were readily digested by DNase 1, and the polyacrylamide gel electrophoresis profiles of DNAs extracted from the digests were similar, each being polydisperse with a single discrete band of DNA smaller than 72 base pairs. There were differences, however, between the sperm chromatins in the course of their digestion by micrococcal nuclease. A limit digest at about 45% acid solubility was obtained with Xenopus sperm chromatin, while 90% of fowl sperm DNA was rendered acidsoluble by the enzyme. The gel profiles of the limit digests were polydisperse, but only those of rabbit and fowl sperm chromatins possessed a discrete band of DNA smaller than 72 base pairs. Bleomycin did not react with DNA of rabbit, fowl, or Xenopus spermatozoa. Since bleomycin reacts with somatic cell chromatin, and the course of DNase 1 or micrococcal nuclease digestion of sperm chromatin was different from that found for somatic cell chromatin, it would appear that sperm chromatin does not have the repeating nucleosometype structure of somatic cell chromatin. The nuclease digestion studies further suggest that the organization of rabbit and fowl sperm chromatins is similar, and is different from that of Xenopus sperm chromatin. The dependence of the structure of sperm chromatin on the composition of its basic proteins, and a possible structure for a protamine-type sperm chromatin, are discussed.     

Nascent DNA in nucleosome like structures from chromatin

We have used chromatin sensitivity to cleavage by micrococcal nuclease as a probe for differences between chromatin containing nascent DNA and that containing bulk DNA. Micrococcal nuclease digested the nascent DNA in chromatin of swimming blastulae of sea urchins more rapidly to acid-soluble nucleotides than the DNA of bulk chromatin. A part of the nascent DNA occurred in micrococcal nuclease-resistant structures which were either different from, or temporary modifications of, the bulk nucleosomes. This was inferred from the size differences between bulk and nascent DNA fragments in 10% polyacrylamide gels after micrococcal nuclease digestion of nuclei from a mixture of ¹⁴C-thymidine long- and ³H-thymidine pulse-labeled embryos. Bulk monomer and dimer DNA fragments contained about 170 and 410 base pairs (bp), respectively, when 18% of the bulk DNA had been rendered acid-soluble. At this level of digestion, “nascent monomer DNA” fragments of about 150 bp as well as 305 bp “large nascent DNA fragments” were observed. Increasing levels of digestion indicated that the large nascent DNA fragment was derived from a chromatin structure which was more resistant to micrococcal nuclease cleavage than bulk dimer chromatin subunits. Peaks of ³H-thymidine-labeled DNA fragments from embryos which had been pulse-labeled and then chased or labeled for several minutes overlapped those of ¹⁴C-thymidine long-labeled monomer, dimer and trimer fragments. This indicated that the chromatin organization at or near the replication fork which had temporarily changed during replication had returned to the organization of its nonreplicating state.     

Sequence specific cleavage of African green monkey alpha-satellite DNA by micrococcal nuclease

The sequence specificity of micrococcal nuclease complicates its use in experiments addressed to the still controversial issue of nucleosome phasing. In the case of alpha-satellite DNA containing chromatin from African green monkey (AGM) cells cleavage by micrococcal nuclease in the nucleus was reported to occur predominantly at only one location around position 126 of the satellite repeat unit (Musich et al. (1982) Proc. Natl. Acad. Sci. USA 79, 118-122). DNA control experiments conducted in the same study indicated the presence of many preferential cleavage sites for micrococcal nuclease on the 172 bp long alpha-satellite repeat unit. This difference was taken as evidence for a direct and simple phase relationship between the alpha-satellite DNA sequence and the position of the nucleosomes on the DNA. We have quantitatively analyzed the digestion products of the protein-free satellite monomer with micrococcal nuclease and found that 50% of all cuts occur at positions 123 and 132, 5% at position 79, and to a level of 1-3% at about 20 other positions. We also digested high molecular weight alpha-satellite DNA from AGM nuclei with micrococcal nuclease. Again cleavage occurred mostly at positions 123 and 132 of the satellite repeat unit. Thus digestion of free DNA yields results very similar to those reported by Musich et al. for the digestion of chromatin. Therefore no conclusions on a possible phase relationship can be drawn from the chromatin digestion experiments.     

DNA repeat size in chick embryonic lens epithelium, lens fiber, brain and liver cell nuclei

The DNA repeat size is determined by micrococcal nuclease digestion kinetics and subsequent electrophoresis of the products among various chick embryonic tissues. The repeat size is found to be not significantly different from 193 to 197 bp, for brain and liver at 11 days and for lens epithelium and fiber at different embryonic stages. However, the pattern of micrococcal digestion seems to reveal an overall chromatin modification as a function of development in the lens fibers.     

Release of nucleosomes from nuclei by bleomycin-induced DNA strand scission

Mononucleosomes were released from both isolated mammalian (hog thyroid) and protozoan (Tetrahymena) nuclei by the bleomycin-induced DNA-strand breaking reaction. Trout sperm nuclei, on the other hand, were protected from the bleomycin-mediated DNA degradation. The mononucleosomes released from the bleomycin-treated nuclei contained the core histones H2A, H2B, H3, and H4; while HMG1 and HMG2 proteins, in addition to the core histones, were detected in the mononucleosomes obtained by micrococcal nuclease digestion of nuclei. HMGs, but not H1 histone, were dissociated into the supernatant by cleavage of chromatin DNA with bleomycin, whereas both HMGs and H1 were found in that fraction by digestion of nuclei with micrococcal nuclease. HMG1 and HMG2 were exclusively dissociated from chromatin with 1 mM bleomycin under the solvent condition where the DNA strand-breaking activity of the drug is repressed. These observations suggest the possibility that bleomycin preferentially binds to linker DNA regions not occupied by H1 histone in chromatin and exclusively dissociates HMG proteins and breaks the DNA strand. The results of the effects on bleomycin-induced DNA cleavage of nuclei of various drugs including polyamines, chelating agents, intercalating antibiotics such as mitomycin C or adriamycin, and radical scavengers are also presented.     

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.