Digestion Of Chromatin Research Articles

Ultrastructural studies on chromatin digestion by micrococcal nuclease in the presence of polyamines.

Nuclei purified from C57BL mouse submandibular salivary gland were treated with a range of micrococcal nuclease concentrations and times of treatment (from 0.5 unit for 2.5 min to 50 units for 30 min) in the presence of polyamines. About 50% of the chromatin was solubilized initially but with prolonged digestion this chromatin became insoluble again. Electron microscopy showed destruction of the finely dispersed chromatin with mild digestion, followed by aggregation of chromatin with more vigorous digestion. The early disappearance of finely dispersed chromatin filaments was not accompanied by preferential solubilization of chromatin associated with RNA polymerase II (euchromatin). These data suggest that the polyamines markedly reduce the susceptibility of euchromatin to micrococcal nuclease digestion.

Studies on the mechanism of aflatoxin B1 inhibition of rat liver nucleolar RNA synthesis.

In an earlier report, it was shown that aflatoxin B1 treatment strongly inhibits rat liver nucleolar RNA synthesis (Yu, F. L. (1977) J. Biol. Chem. 252, 3245-3251). The present paper is an attempt to elucidate the mechanism of this inhibition. Two h after aflatoxin B1 injection (0.3 mg/100 g body weight), rat liver nucleolar RNA synthesis, in vitro, was inhibited by an average of 90%. This inhibition could result from (a) inhibited RNA polymerase I activity per se, (b) impaired nucleolar DNA template, or (c) impaired nucleolar chromatin. Earlier studies found that the total RNA polymerase I activity was not affected by aflatoxin B1 treatment. In the present work the total nucleolar DNAs from control and from aflatoxin B1-treated groups were isolated and compared for template efficiencies in directing RNA synthesis with solubilized RNA polymerase I from the control group. No difference was found. However, when nucleolar chromatin function was analyzed, it was found that aflatoxin B1 treatment resulted in a dramatic reduction in the RNA chain elongation rate to only 13% of the control. The chain number, which is a measure of the number of engaged enzymes transcribing the nucleolar chromatin initiated in vivo, was only slightly reduced (33%). Furthermore since it was found that aflatoxin B1 treatment did not increase RNase activity in the treated nucleoli, the dramatic decrease in RNA chain elongation is therefore believed to be the major mechanism of aflatoxin B1 inhibition of rat liver nucleolar RNA synthesis. DNase I digestion of the nucleolar chromatin suggests that aflatoxin B1 treatment may have altered the conformation of the transcriptionally active regions of the nucleolar chromatin.

Micrococcal nuclease cleavage of chromatin displays nonrandom properties.

A statistical analysis of the products of digestion of chicken erythrocyte chromatin by micrococcal nuclease was used to test for randomness of the cutting process. DNA fragment size classes corresponding to mononucleosome, dinucleosome, trinucleosome, tetranucleosome, and all fragments larger than tetranucleosome were evaluated. In every case, fragments in the mononucleosome and greater-than-tetranucleosome classes were produced in excess of the level expected on the basis of random cleavage while those in the dinucleosome-tetranucleosome classes exhibited a shortage. The pattern of nonrandomness appears to depend on substrate size: the magnitude of deviations from randomness was large when substrates of genomic size are compared with polynucleosomal segments whereas the direction of deviation is identical. Nonrandomness was independent of ionic conditions known to affect the state of chromatin condensation and also appeared to be unaffected by depletion of histones H1 and H5. The possible universality of nonrandom cleavage was suggested when other data from the literature was analyzed. Some possible mechanisms to account for this property are discussed.

The protein composition of rat satellite chromatin

Although most attempts to fractionate chromatin have been designed to prepare transcriptiona~y active chromatin, the advent of restriction endonuclease has provided the technology to isolate chromatin fractions enriched in highly repeated satellite DNA [l-5]. Two approaches have been used: the first of which involves digestion of nuclei or chromatin with a restriction enzyme which cuts within the satellite sequence. Since this sequence is repeated very frequently within the genome, it follows that this region of the chromate will be cut into much smaller fragments than the bulk of the chromatin. By centrifugation of the digested chromatin through sucrose gradients, purification of the satellite chromatin to 70-100% has been reported for calf [I], rat [2] and African green monkey [3] tissues. Alternatively, a restriction endonuclease can be chosen such that main band DNA rather than satellite DNA is digested and satellite chromatin is found enriched in the insoluble pellet [4,5]. The function of satellite DNA is unknown (reviewed in [6]) but in situ hybridisation of radioactive satellite DNA probes shows that these sequences are largely associated with permanently condensed chromatin, which suggests that they are not involved in gene expression ([6] but see [7,8]). A detailed knowledge of the protein associated with satellite DNA will undoubtedly be a useful complement to the studies on the composition of transcriptionally active chromatin, and may afford further insight into the function of highly repeated DNA. The a-satellite DNA-containing cluomatin of African green monkey cells lacks histone Hl 231 and is enriched in a set of low molecular weight tightly bound non-h~tone proteins. We have prepared satel-

Human sperm chromatin has a nucleosomal structure.

Digestion of human sperm chromatin with micrococcal nuclease reveals an 160 base pair (b.p.) DNA fragment that is further degraded to a series of DNA fragments remarkably similar to the micrococcal nuclease digestion products of eukaryote somatic cellular chromatin. DNase I digestion of human sperm chromatin also yields an identical pattern of DNA fragments to that observed upon DNase I digestion of somatic chromatin. These data, together with earlier electron microscopic observations, suggest a nucleosomal structure for human sperm chromatin.

Influence of psychoactive and nonpsychoactive cannabinoids on chromatin structure and function in human cells

The influences of two naturally occurring psychoactive cannabinoids (Δ 9-tetrahydrocannabinol and Δ 8-tetrahydrocannabinol), of a psychoactive cannabinoid metabolite (11-OH-Δ 9-THC) and of a nonpsychoactive cannabinoid (cannabinol) on the composition, metabolism and structure of human (HeLa cell) chromatin were examined. The effects of all four cannabinoids on the composition and synthesis of chromosomal proteins were studied by pulse-labeling with [ 3H]leucine. The relative amounts of both histones and nonhistone chromosomal proteins remained unaffected, but a decrease in [ 3H]leucine incorporation into both histones and nonhistone proteins was observed. Post-translational acetylation and phosphorylation of chromosomal proteins were studied in cannabinoid-treated cells by pulse-labeling with [ 3H]acetate and [ 32P], Variations were observed in [ 3H]acetate incorporation into histones and [ 32P] incorporation into both histones and nonhistone chromosomal proteins. Pancreatic DNase I digestion of chromatin from cannabinoid-treated HeLa cells failed to demonstrate any drug-induced alteration in DNA-protein interactions.

Quantitation of DNase I sensitivity in Xenopus chromatin containing active and inactive globin, albumin and vitellogenin genes

The disappearance of defined restriction fragments of the beta 1-globin, an albumin and the A1 vitellogenin gene was quantitated after DNase I digestion and expressed by a sensitivity factor defined by a mathematical model. Analysis of naked DNA showed that the gene fragments have similar but not identical sensitivity factors. DNase I digestion of chromatin revealed for the same gene fragments sensitivity factors differing over a much wilder range. This is correlated to the activity of the genes analyzed: the beta 1-globin gene fragment is more sensitive to DNase I in chromatin of erythrocytes compared to hepatocytes whereas the albumin gene fragment is more sensitive to DNase I in chromatin of hepatocytes. The A1 vitellogenin gene has the same DNase I sensitivity in both cell types. Comparing the DNase I sensitivity of the three genes in their inactive state we suggest that different chromatin conformations may exist for inactive genes.

Nuclease sensitivity of chromatin containing active genes: kinetic analyses utilizing continuous elution of digestion products from an ultrafiltration cell.

Methods have been developed to analyze the kinetics of digestion of chromatin by nucleases. Radioactively labeled nuclei were incubated with enzyme in an ultrafiltration apparatus and digestion rates of different chromatin samples were computed employing a least-squares curve fitting technique to fit the data to zero-order and/or first-order kinetic models. These methods allow detailed kinetic analyses on small amounts of chromatin. Two biological systems were studied. 1) Tetrahymena thermophila macronuclei and micronuclei were compared; these nuclei differ in their transcriptional activities. 2) Ribosomal DNA (rDNA) of Tetrahymena pyriformis, approximately 60% of which codes for rRNA, can be preferentially labeled during starvation-refeeding; its digestion kinetics relative to bulk chromatin were studied. DNase I digested 20-40% of the macromolecular DNA about 3 times faster than bulk macronuclear or micronuclear DNA, and 60-80% of ribosomal gene-containing chromatin about 5 times faster than bulk chromatin. Filter hybridization studies of the DNAase I sensitivity of tRNA, 5S RNA, and ribosomal genes yielded similar results. These data are consistent with the observation that transcribed genes are especially sensitive to attach by DNase I and suggest that activated chromatin structure as probed by extensive DNase I digestion is the same in higher and lower eucaryotes for genes transcribed by all three RNA polymerases. Digestion kinetics of micrococcal nuclease were found to depend on the digestion conditions employed. These two biological systems and the methods we have developed should facilitate analyses of the factors responsible for maintaining an active chromatin structure.

The nuclear shell in hela cell nuclei: Whole-mount electron microscopy of the dissociated and isolated nuclear periphery

We have developed a method which allows rapid and reproducible isolation of the outermost layer of peripheral chromatin (cortical chromatin) from interphase HeLa cell nuclei, in association with the subjacent nuclear pore-lamina complex. This was achieved by chromatin lysis in a slightly alkaline Tris buffer, DNase digestion of the lysed chromatin, and subsequent low-speed centrifugation of the nuclease-resistant material through sucrose, under experimental conditions in which mechanical shearing was strictly avoided. Owing to the peripheral localization of the isolated fraction in the nucleus in situ , and to its particular rigidity, it was given the name of nuclear shell. The nuclear shell and its relationships with internal chromatin were visualized with the electron microscope, using very gentle Miller's spreading conditions. Cortical chromatin resembled a network of knobby fibers 22–28 nm in diameter, and an array of branched fibers and ring-like structures was identified with the nuclear pore-lamina layer. A model is presented for the organization of the nuclear periphery isolated under these conditions.

Deoxyribonuclease II as a probe for chromatin structure: II. Mode of cleavage

In the analysis of DNAase II digestion of chromatin, as described in the preceding paper, interactions between adjacent nucleosomes play an important part. In order to understand the mechanism of DNAase II cleavage we next investigated the role of histone H1 in these interactions and characterized the nucleoprotein particles arising in the course of DNAase II action. H1-free chromatin prepared by three different procedures, using either 0.6 m-NaCl, transfer RNA or an ion-exchange resin, can be cleaved by DNAase II only at the internucleosomal cleavage site leading to 200-bp † † Abbreviations used: bp, base-pairs; PMSF, phenylmethanesulfonyl fluoride; EGTA, ethyleneglycol bis(β-aminoethyl ether)- N,N′-tetraacetic acid. digestion patterns regardless of the ionic conditions. When H1 was added back to the three chromatin preparations the 100-bp cleavage pattern could be restored only with material prepared by the resin method at low concentrations of salt. Addition of polylysine instead of H1 has the same effect, but only with material prepared by that method. A direct correlation between extended and condensed states of chromatin as monitored by electron microscopy and DNAase II cleavage in the 200 and 100-bp modes, respectively, could be established. The continuity of the nucleosome chains in DNAase II-digested chromatin is maintained in spite of intranucleosomal cleavage in the terminal section of the core DNA, even in the absence of H1. Addition of 3 m-urea, however, disrupts the nucleosome chains at the intranucleosomal cleavage sites and leads to the formation of novel nucleoprotein particles as seen in sucrose gradient centrifugations. Those sedimenting between mononucleosomes and dinucleosomes contain, almost exclusively, DNA of 300 bp (mouse) or 315 bp (chicken erythrocyte). They can be formed from particles sedimenting in the absence of urea in the dinucleosome region by either a dissociation process or a massive conformational change. On the basis of the results presented here and in the preceding paper a mechanism for DNAase II cleavage of chromatin in the 200-bp and 100-bp modes is proposed and discussed in the context of structural features of chromatin recognized by DNAase II.

Aggregation of small oligonucleosomal chains into 300-A globular particles.

Chicken erythrocyte oligonucleosomes (trimers to about 20-mers) are able to interact with each other through the very lysine-rich histones (H1 and H5) and form heterogeneous globular particles with a mean diameter of about 300 A. These particles assemble spontaneously during micrococcal nuclease digestion of chromatin in the presence of 30 mM NaCl and contain approximately 25 nucleosomes. They are sensitive to ionic strength and unfold at lower salt concentrations but can be reconstituted by restoring the initial salt concentration. Even at 30 mM NaCl, the particles remain dynamic structures, being in equilibrium with their oligonucleosomal components as revealed by the fact that particle stability depends on the concentration of oligonucleosomes.

Chromatin replication in a higher plant, Vicia faba

Embryo axes of Vicia faba seeds were collected and pulse-labeled with [ 3H]thymidine for 5–6 min at the first DNA synthesis period (34 h stage of germination) and chromatin was isolated. About 67% of the pulse-labeled DNA in the chromatin was converted to acid-soluble material after extensive digestion with micrococcal nuclease. On the other hand, bulk chromatin uniformly labeled with [ 14C]thymidine for a 12-h period showed acid-solubility of about 56% under identical conditions of digestion. Sedimentation analyses of the nuclease digests of chromatin and extracted DNA samples showed that the nucleosome structure was already organized in the chromatin containing 10–11-S single-strand DNA fragments of an intermediate molecule during DNA replication. These fragments were detected as a major component after labeling for 10 to 15 min with [ 3H]thymidine. Further-more, the result of the pulse-labeling for 5–6 min indicated the presence of the nucleosome structure in the chromatin containing 5–6-S and 8–9-S nascent DNA fragments as the main labeled components, thus suggesting the involvement of at least the 8–9-S intermediate fragment in the reorganization of the nucleosome structure. Electrophoresis of DNA fragments obtained after micrococcal nuclease digestion of chromatin showed that after a pulse of 5–6 min with [ 3H]thymidine, labeled dimer and trimer DNA fragments were significantly shorter than corresponding [ 14C]thymidine-labeled bulk dimer and trimer DNA fragments, though the peaks of pulse-labeled and uniformly labeled monomer DNA fragments were coincident with each other in 3.5% gels. The difference in size of dimer and trimer DNA fragments between the newly formed and non-replicating bulk chromatin disappeared after a prolonged labeling for 15 min, irrespective of the fact that the major part of labeled DNA was still the 10–11-S intermediate fragment. The mode of chromatin assembly in the replicating chromatin in higher plant cells is discussed.

Deoxyribonuclease I generates single-stranded gaps in chromatin deoxyribonucleic acid.

Production of 10-base multiple DNA ladder fragments during DNase I digestion of chromatin is explained by a model which does not involve site-specific nicking by the DNase I. This model was tested because it explains why 10-base (actually 10.4 base) multiple-related fragments are paradoxically generated by both endonucleolytic (DNase I) and exonucleolytic (exonuclease III) mechanisms. This new model also explains the phenomenon of substantial single-stranded DNA production during DNase I digestion of chromatin. The latter phenomenon has been widely observed but is not explained by previous models. The single-stranded gap model to be presented makes testable predictions. Primarily, these are that DNase I produces single-stranded gaps in chromatin DNA and that the termini of 10-base multiple ladder fragments are separated by single-stranded gaps. Single-stranded gap production by DNase I was confirmed by a number of methods. Sensitivity of ladder band components (from DNase I but not staphylococcal nuclease digests) to S1 nuclease suggested that the ladder fragments themselves may compose a significant portion of these gaps. Separation of ladder fragment termini by single-stranded gaps was verified by demonstrating both resistance to the nick-specific NAD+-dependent ligase and sensitivity to T4 ligase which can ligate across gaps. Many single-stranded gaps, occurring both individually and clusters, were observed by electron microscopy using either cytochrome c labeling (where the gaps) are thinner than duplex) or gene 32 protein labeling (gaps thicker than duplex). Gap sizes were estimated by protecting them with gene 32 protein and digesting away unprotected duplexes. By this method, gap sizes fall into a ladder distribution (from 10 or 20 bases up to 120 bases), which, at least in the region of the shorter sizes, clearly indicates the sizes of single-stranded gaps formed in chromatin by DNase I.

Nucleosomes structure and its dynamic transitions

The discovery of nucleosomes as a basic repeating unit of the chromatin structure organizing the major part of eukaryotic DNA greatly catalyzes the expansion of our knowledge on chromatin. Several lines of experimental evidence have led to the formulation of the nucleosome conception: the observation of chains of globular particles in electron micrographs of chromatin (Olins & Olins, 1974); the demonstration that DNA is released as a set of discrete sizes upon digestion of chromatin with endogenous nucleases (Hewish & Burgoyne, 1973); the isolation of discrete nucleoprotein particles upon digestion of chromatin with micrococcal nuclease (Rill & Van Holde, 1973).

Mitochondrial chromatin in Paramecium aurelia.

Chromatin-like structures have been observed in material extracted from the mitochondria of Paramecium aurelia and evidence is presented which establishes that these structures do not originate from nuclear contamination of mitochondrial preparations but are exclusively of mitochondrial origin. Extraction of these chromatin-like structures with dilute acid followed by gel electrophoresis of the extract shows the presence of five major basic proteins which are of similar electrophoretic mobility to histones. Digestion of mitochondrial extracts with endogenous nuclease, followed by electrophoresis of the extracted DNA, demonstrated that the mitochondrial genome is protected from nuclease digestion by regular repeating units very similar to those observed in the nuclease digestion of chromatin.

Rapid electrophoretic analysis of DNA in limited nuclease digests of chromatin and nuclei

Addition of Sarkosyl to complex protein-nucleic acid mixtures allows direct analysis of the component nucleic acids by either polyacrylamide or agarose gel electrophoresis. Since no other deproteinization, concentration, or purification steps are necessary for analysis of DNA fragments generated by nuclease digestion of chromatin in intact nuclei or RNA components in ribosomes, these nucleic acids can be analyzed within 1–2 h of preparing digests or cell lysates. It is demonstrated that with this modification of standard gel techniques, nucleic acids can be separated by electrophoresis in the presence of high concentrations of proteins with a resolution comparable to that obtained with fully purified nucleic acids.

Analysis of DNA associated with nucleosomes in pea chromatin

Micrococcal nuclease digestion of chromatin from ungerminated and 48 h-germinated pea embryos yields DNA fragments which are multiples of basic units of 194-195 base pairs. Extensive digestion produces a core particle of 145 base pairs. Deoxyribonuclease I gives rise to fragments which are multiples of 10 bases upon analysis on denaturing gels. These values are comparable with those found for other plant materials. These results indicate that gross changes in nucleosomal organization do not accompany the onset of germination.

Hydrodynamic shearing by VirTis blending conserves nucleosome structure of rat liver chromatin

The effects of VirTis shearing on chromatin subunit structure were investigated by enzymatic digestion, thermal denaturation, and electron microscopy. While initial rates of micrococcal nuclease and DNase I digestion were greater postshearing, limit digest values were similar to those for unsheared chromatin. Fractionated chromatin digestion kinetics varied with sedimentation. Digestion of all chromatins produced monomer and dimer DNA fragment lengths, but only unsheared chromatins exhibited higher order nucleosome oligomer lengths. Mononucleosomes and core particles were resolved in digests of sheared and gradient fractions analyzed by electrophoresis. All chromatins exposed to DNase I showed discrete 10-base pair nicking patterns. The presence of nucleosomes was confirmed by electron microscopy. Electron microscopy and histone content of gradient fractions showed that nucleosome density along the chromatin axis increased in rapidly sedimenting fractions. Thermal denaturation detected no appreciable generation of protein-free DNA fragments as a result of shearing. The results indicate that VirTis blending conserves subunit structure with loss of less than 12–15% of nucleosome structure.

Selective release of HMG nonhistone proteins during DNase digestion of Tetrahymena chromatin at different stages of the cell cycle.

The possible role of LG-1, a Tetrahymena specific HMG protein found in the macronuclear chromatin (Hamana, K. and Iwai, K. (1979) J. Biochem. 86, 789-794), was examined in relation to the chromatin structure. The chromatin isolated from cells synchronized at different stages of the cell cycle contained about one molecule of LG-1 per nucleosome. Limited digestion of the chromatin with DNase I or micrococcal nuclease selectively released LG-1 with the nucleosomal core histones and H1 remained insoluble, bound to the resistant DNA. Depending on the cell stages several types of chromatin structure were distinguished by their nuclease sensitivity. However, the chromatin at different stages exhibited the similar behavior of the LG-1 release with the nucleases as a function of the degree of chromatin solubilization. The results suggest that LG-1 proteins play a role in the chromatin organization which is rather independent of the cell stages.

Nuclease digestion promotes structural rearrangements in H1-depleted chromatin.

Digestion of H1-depleted chromatin with micrococcal nuclease at an ionic strength of 0.35M gives rise to structural rearrangements indicating nucleosomal sliding. The ionic strength necessary to reveal this effect is significantly lower than that required in the absence of an accompanying digestion. As an explanation, a model is presented in which the progressing terminal degradation of oligomeric nucleosomes is made responsible for promoting structural rearrangements.