Nuclear reassemblyin vitro is independent of nucleosome/chromatin assembly

It was show11 that nuclear reassembly was induced by small pieces of DNA fragments in cell-free extracts ofXenopus. In an attempt to learn the relationship between the nuclear reassembly and nucleosome/chromatin assembly, limited amounts of CM-Cellulose are used to eliminate the capacity of the egg extract S-150 to assemble chromatin. while the forming of nucleosomes is checked with DNA supercoiling by plasmid DNA pBR322 incubated in the extract, and further analysed by micrococcal nuclease digestion. This depleted extract is then used to induce nuclear reassembly around demembraned sperms with membrane vesicles. It is found that CM-Cellulose depletes histones H2A and H2B efficiently and blocks the assembly of nucleosomes, the demembraned sperms are yet reconstituted into nuclei in the treated S-150, although the chromatin in reassembled nuclei does not produce protected DNA fragments when digested with micrococcal nuclease. It suggests that in the cell-free system ofXenopus, DNA can be formed into nuclei without assembly of nucleosomes or chromatin.     

Nuclear reassembly in vitro is independent of nucleosome/chromatin assembly

It was show11 that nuclear reassembly was induced by small pieces of DNA fragments in cell-free extracts ofXenopus. In an attempt to learn the relationship between the nuclear reassembly and nucleosome/chromatin assembly, limited amounts of CM-Cellulose are used to eliminate the capacity of the egg extract S-150 to assemble chromatin. while the forming of nucleosomes is checked with DNA supercoiling by plasmid DNA pBR322 incubated in the extract, and further analysed by micrococcal nuclease digestion. This depleted extract is then used to induce nuclear reassembly around demembraned sperms with membrane vesicles. It is found that CM-Cellulose depletes histones H2A and H2B efficiently and blocks the assembly of nucleosomes, the demembraned sperms are yet reconstituted into nuclei in the treated S-150, although the chromatin in reassembled nuclei does not produce protected DNA fragments when digested with micrococcal nuclease. It suggests that in the cell-free system ofXenopus, DNA can be formed into nuclei without assembly of nucleosomes or chromatin. Projrrt supported by the National Natural Science Foundation of China (Grant No. 39730240)     

Assembly of correctly spaced chromatin in a nuclear extract from Xenopus laevis oocytes.

Assembly of nucleosomes on relaxed, covalently closed DNA has been studied in a nuclear extract of Xenopus laevis oocytes. Nucleosomes containing the four histones H3, H4, H2A and H2B but lacking histone H1 are readily assembled on the DNA. The pattern of micrococcal nuclease digestion shows that the nucleosomes assembled in the absence of ATP and Mg (II) are closely packed, with a periodicity of 150 base pairs (bp). In contrast, in the presence of ATP and Mg (II) the spacing of the nucleosomes is 180 bp, similar to that observed for nucleosomes assembled on DNA microinjected into oocyte nuclei. The ATP and Mg (II) requirements for the assembly of correctly spaced nucleosomes are unrelated to the activity of the ATP and Mg (II) dependent DNA topoisomerase II in the extract; addition of specific inhibitors of eukaryotic DNA topoisomerase II has no effect on the spacing of the reconstituted nucleosomes. The ATP requirement in the assembly of correctly spaced nucleosomes can be substituted by adenosine 5'-O-3'-thiotriphosphate (gamma-S-ATP) but not by adenyl-5'-yl imidodiphosphate (AMP-P-(NH)-P).     

Organization of 5S genes in chromatin of Xenopus laevis.

The chromatin organization of the genes coding for 5S RNA in Xenopus laevis has been investigated with restriction endonucleases and micrococcal nuclease. Digestion of nuclei from liver, kidney, blood and kidney cells maintained in culture with micrococcal nuclease reveals that these Xenopus cells and tissues have shorter nucleosome repeat lengths than the corresponding cells and tissues from other higher organisms. 5S genes are organized in nucleosomes with repeat lengths similar to those of the bulk chromatin in liver (178 bp) and cultured cells (165 bp); however, 5S gene chromatin in blood cells has a shorter nucleosome repeat (176 bp) than the bulk of the genome in these cells (184 bp). From an analysis of the 5S DNA fragments produced by extensive restriction endonuclease cleavage of chromatin in situ, no special arrangement of the nucleosomes with respect to the sequence of 5S DNA can be detected. The relative abundance of 5S gene multimers follows a Kuhn distribution, with about 57% of all HindIII sites cleaved. This suggests that HindIII sites can be cleaved both in the nucleosome core and linker regions.     

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.     

Chromatin assembly in isolated mammalian nuclei.

Cellular DNA replication was stimulated in confluent monolayers of CV-1 monkey kidney cells following infection with SV40. Nuclei were isolated from CV-1 cells labeled with [3H]thymidine and then incubated in the presence of [alpha-32P]deoxyribonucleoside triphosphates under conditions that support DNA replication. To determine whether or not the cellular DNA synthesized in vitro was assembled into nucleosomes the DNA was digested in situ with either micrococcal nuclease or pancreatic DNase I, and the products were examined by electrophoretic and sedimentation analysis. The distribution of DNA fragment lengths on agarose gels following micrococcal nuclease digestion was more heterogeneous for newly replicated than for the bulk of the DNA. Nonetheless, the state of cellular DNA synthesized in vitro (32P-labeled) was found to be identical with that of the DNA in the bulk of the chromatin (3H-labeled) by the following criteria: (i) The extent of protection against digestion by micrococcal nuclease of DNase I. (ii) The size of the nucleosomes (180 base pairs) and core particles (145 base pairs). (iii) The number and sizes of DNA fragments produced by micrococcal nuclease in a limit digest. (iv) The sedimentation behavior on neutral sucrose gradients of nucleoprotein particles released by micrococcal nuclease. (v) The number and sizes of DNA fragments produced by DNase I digestion. These results demonstrate that cellular DNA replicated in isolated nuclei is organized into typical nucleosomes. Consequently, subcellular systems can be used to study the relationship between DNA replication and the assembly of chromatin under physiological conditions.     

Mechanism of chromatin assembly in Xenopus oocytes

We have analyzed the chromatin assembly reaction catalyzed by the Xenopus oocyte extract (S-150). A 50 S complex is formed upon mixing the 17 S pUC DNA and the S-150. Mature histones are not detected in this complex, which contains relaxed DNA and protein, and generates subnucleosomal 7 S particles upon digestion with micrococcal nuclease. The relaxed nucleoprotein is gradually supercoiled into nucleosomal chromatin in the S-150, via a pathway that requires ATP and is blocked by novobiocin, and this process is accompanied by the appearance of mature histones H3 and H4. Isolated complexes also supercoil in vitro, which implies the complex is a kit that contains histone precursors, as well as topoisomerases and other enzymes required for assembly. We discuss the biological implications of these findings.     

Nuclear assembly of demembranated Xenopus sperm in plant cell-free extracts from Nicotiana ovules

The cell-free preparation derived from Nicotiana tabaccum ovules induced chromatin decondensation and pronuclear formation from demembranated Xenopus laevis sperm nuclei. Fluorescent microscope and phase-contrast microscope visualization of assembly intermediates indicated that 95.6% of X. leavis sperm changed their tadpole-like shape to circular shape or elliptical shape after over 1.5 h of incubation. Transmission electron microscope visualization showed that nuclear membrane was assembled around the periphery of the dispersed chromatin. Nuclear envelope of most reassembled nuclei was composed of a double membrane inlaid with a little single membrane. Nucleosome assembly was verified by means of micrococcal nuclease digestion. After 2 to 5 h of incubation, digestion of the product of nuclear assembly with micrococcal nuclease produced at least six nucleosome fragments of about 250 bp each.     

Fractionation of nucleosomes by salt elution from micrococcal nuclease- digested nuclei

The solubilization of nucleosomes and histone H1 with increasing concentrations of NaCl has been investigated in rat liver nuclei that had been digested with micrococcal nuclease under conditions that did not substantially alter morphological properties with respect to differences in the extent of chromatin condensation. The pattern of nucleosome and H1 solubilization was gradual and noncoordinate and at least three different types of nucleosome packing interactions could be distinguished from the pattern. A class of nucleosomes containing 13-- 17% of the DNA and comprising the chromatin structures most available for micrococcal nuclease attack was eluted by 0.2 M NaCl. This fraction was solubilized with an acid-soluble protein of apparent molecular weight of 20,000 daltons and no histone H1. It differed from the nucleosomes released at higher NaCl concentrations in content of nonhistone chromosomal proteins. 40--60% of the nucleosomes were released by 0.3 M NaCl with 30% of the total nuclear histone H1 bound. The remaining nucleosomes and H1 were solublized by 0.4 M or 0.6 M NaCl. H1 was not nucleosome bound at these ionic strengths, and these fractions contained, respectively, 1.5 and 1.8 times more H1 per nucleosome than the population released by 0.3 M NaCl. These fractions contained the DNA least available for micrococcal nuclease attach. The strikingly different macromolecular composition, availability for nuclease digestion, and strength of the packing interactions of the nucleosomes released by 0.2 M NaCl suggest that this population is involved in a special function.     

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.     

Disruption of the nucleosomes at the replication fork.

The fate of parental nucleosomes during chromatin replication was studied in vitro using in vitro assembled chromatin containing the whole SV40 genome as well as salt-treated and native SV40 minichromosomes. In vitro assembled minichromosomes were able to replicate efficiently in vitro, when the DNA was preincubated with T-antigen, a cytosolic S100 extract and three deoxynucleoside triphosphates prior to chromatin assembly, indicating that the origin has to be free of nucleosomes for replication initiation. The chromatin structure of the newly synthesized daughter strands in replicating molecules was analysed by psoralen cross-linking of the DNA and by micrococcal nuclease digestion. A 5- and 10-fold excess of protein-free competitor DNA present during minichromosome replication traps the segregating histones. In opposition to published data this suggests that the parental histones remain only loosely or not attached to the DNA in the region of the replication fork. Replication in the putative absence of free histones shows that a subnucleosomal particle is randomly assembled on the daughter strands. The data are compatible with the formation of a H3/H4 tetramer complex under these conditions, supporting the notion that under physiological conditions nucleosome core assembly on the newly synthesized daughter strands occurs by the binding of H2A/H2B dimers to a H3/H4 tetramer complex.     

Assembly and properties of chromatin containing histone H1

The Xenopus oocyte supernatant (oocyte S-150) forms chromatin in a reaction that is affected by temperature and by the concentration of ATP and Mg. Under optimal conditions at 27 degrees C, relaxed DNA plasmids are efficiently assembled into supercoiled minichromosomes with the endogenous histones H3, H4, H2A and H2B. This assembly reaction is a gradual process that takes four to six hours for completion. Micrococcal nuclease digestions of the chromatin assembled under these conditions generate an extended series of DNA fragments that are, on average, multiples of 180 base-pairs. We have examined the effect of histone H1 in this system. Exogenous histone H1, when added at a molar ratio of H1 to nucleosome of 1:1 to 5:1, causes an increase in the micrococcal nuclease resistance of the chromatin without causing chromatin aggregation under these experimental conditions. Furthermore, the periodically arranged nucleosomes display longer internucleosome distances, and the average length of the nucleosome repeat is a function of the amount of histone H1 added, when this histone is present at the onset of the assembly process. In contrast, no major change in the length of the nucleosome repeat is observed when histone H1 is added at the end of the chromatin assembly process. Protein analyses of the purified minichromosomes show that histone H1 is incorporated in the chromatin that is assembled in the S-150 supplemented with histone H1. The amount of histone H1 bound to chromatin is a function of the total amount of histone H1 added. We define here the parameters that generate histone H1-containing chromatin with native nucleosome repeats from 160 to 220 base-pairs, and we discuss the implications of these studies.     

Internucleosome interactions: detecting the dinucleosome fragmentation of chromatin using micrococcal nuclease. Heterogeneity of nucleosomes and localization of histone H1

The content of histone H1 (H1/H4 ratio) in dinucleosomes with the DNA of various length liberated from L-cell nuclear chromatin by micrococcal nuclease was analyzed. It was found that the histone H1 content in the dichromatosome is two times as low as that in the largest dinucleosome and in the complete mononucleosome. The set of chromatin fragments liberated from the Triton X-100 pretreated nuclei differs considerably from that of chromatin sites devoid of histone H1 (the de novo replicating chromatin and the chromatin formed on the undermethylated DNA). A scheme for asymmetric distribution of histone H1 with molecules oriented along the nucleosomal fibril, which reflects the peculiarities of chromatin fragmentation by micrococcal nuclease with predominant liberation of the dichromatosome, is proposed.     

Variation in chromatin structure in two cell types from the same tissue: a short DNA repeat length in cerebral cortex neurons

We have used micrococcal nuclease as a probe of the repeating structure of chromatin in four nuclear populations from three tissues of the rabbit. Neuronal nuclei isolated from the cerebral cortex contain about 160 base pairs of DNA in the chromatin repeat unit, as compared with about 200 base pairs for nonastrocytic glial cell nuclei from the same tissue, neuronal nuclei from the cerebellum and liver nuclei. All four types of nuclei show the same features of nucleosomal organization as other eucaryotic nuclei so far studied: nucleosomes liberated by digestion with micrococcal nuclease give a “core particle” containing 140 base pairs as a metastable intermediate on further digestion and a series of single-strand DNA fragments which are mutiples of 10 bases after digestion with DNAase I. Nuclei from cerebral cortex neurons, which have a short repeat, are distinct from the others in being larger, in having a higher proportion of euchromatin (dispersed chromatin) as judged by microscopy and in being more active in RNA synthesis in vitro.     

The small chromatin fragments released by micrococcal nuclease from hepatoma tissue cultured cell nuclei are strongly enriched in coding DNA sequences and are related to an actively transcribed single-stranded DNA fraction

It was shown with the use of specific probes that mild micrococcal nuclease digestion releases from chromatin actively-transcribed genes as small nucleosome oligomers. In the present work we demonstrate that most if not all of the active genes are accessible to the nuclease. It was found that the short released fragments are greatly enriched in transcribed DNA sequences, the most enriched being the dimers of nucleosomes since 35% of their DNA could be hybridized to cytoplasmic RNA. The results of cDNA-DNA hybridizations indicate that the monomers and dimers of nucleosomes contain most of the DNA sequences which encode poly(A⁺) RNAs, however larger released fragments include some transcribed sequences, while the nuclease-resistant chromatin is considerably impoverished in coding sites. These evidences and the finding that about 25% of the DNA from the dimers of nucleosomes are exclusively located in this class of fragments, tend to prove that the active chromatin regions are attacked in a non-random way by micrococcal nuclease. We have previously isolated, without using exogenous nuclease, an actively transcribed genomic fraction amounting to 1.5–2% of the total nuclear DNA, formed of single-stranded DNA. In the present study we show that all or nearly all the single-stranded DNA sequences could be reassociated with the DNA fragments present in the released monomers and dimers of nucleosomes. Our observations confirmed our previous finding that the greatest part of single-stranded DNA selectively originates from the coding strand of genomic DNA.     

Chromatin organization in Paracentrotus lividus eggs

After purification by buoyant density centrifugation in ethidium bromide - CsCl gradient and electrophoretic fractionation, the DNA fragments isolated from P. lividus egg nuclei incubated with micrococcal nuclease exhibit a typical oligomeric pattern. Analysis of chromatin samples digested to an increasing extent by micrococcal nuclease reveals that the structural organization of egg chromatin is heterogeneous, both in terms of repeat size and degree of sensitivity to nuclease attack. The nucleosomal repeats of P. lividus sperms and embryos up to the mesenchyme blastula stage have also been determined, for comparison.     

The extraction by micrococcal nuclease of glucocorticoid receptors and mouse mammary tumor virus DNA sequences is dissociated.

Glucocorticoid receptors (RG) and mammary tumor virus (MM-TV) DNA sequences were extracted by micrococcal nuclease digestion from the nuclei of C3H mouse mammary tumor cells in order to specify their relative distribution in chromatin. RG was labelled and translocated into the nuclei by incubating cells with 3H Dexamethasone (3H Dex). The purified nuclei were then treated at 2 degrees C with micrococcal nuclease. Three chromatin fractions were successively obtained: an isotonic extract (ne3H1), ahypotonic extract (ne2) and the residual pellet (P). The Dex-RG complexes were measured by the hydroxyapatite technique. The MMTV DNA sequences were titrated by molecular hybridization with an excess of MMTV radioactive cDNA probe. Up to 75% of the nuclear 3H Dex and the MMTV radioactive cDNA probe. Up to 75% of the nuclear 3H Dex and MMTV DNA sequences were extracted in a concentration dependent manner while only 10-15% of nucleic acids became soluble in 10% perchloric acid. The extracted 3H Dex-RG complex was found to be partly bound to soluble chromatin and partly free. The free complex displayed similar sedimentation constants (4S, 7S) and DNA binding ability to the cytosol receptor. The 3H Dex-RG complexes were 2 to 8 fold more concentrated in ne1, which is known to be enriched in active chromatin, than in ne2. Conversely, the concentration of MMTV DNA sequences per microgram DNA was the same in the three nuclear fractions. These results suggest that the Dex-RG complexes are concentrated in an active fraction of chromatin. We propose that, among the 20-30 copies of MMTV genes per haploid genome, only a small proportion are transcribed or regulated.     

Nucleosome assembly in mammalian cell extracts before and after DNA replication.

Protein-free DNA in a cytosolic extract supplemented with SV40 large T-antigen (T-Ag), is assembled into chromatin structure when nuclear extract is added. This assembly was monitored by topoisomer formation, micrococcal nuclease digestion and psoralen crosslinking of the DNA. Plasmids containing SV40 sequences (ori- and ori+) were assembled into chromatin with similar efficiencies whether T-Ag was present or not. Approximately 50-80% of the number of nucleosomes in vivo could be assembled in vitro; however, the kinetics of assembly differed on replicated and unreplicated molecules. In replicative intermediates, nucleosomes were observed on both the pre-replicated and post-replicated portions. We conclude that the extent of nucleosome assembly in mammalian cell extracts is not dependent upon DNA replication, in contrast to previous suggestions. However, the highly sensitive psoralen assay revealed that DNA replication appears to facilitate precise folding of DNA in the nucleosome.