Bleomycin-induced aggregation of presolubilized and nuclear chromatin from L1210 cells

These studies have identified a new activity of bleomycin (in addition to the well-known DNA cleavage): drug-induced chromatin aggregation. Bleomycin treatment of presolubilized chromatin from L1210 nuclei resulted in two types of effect as shown by sedimentation analysis of intact nucleoprotein. The first effect was a dose-dependent fragmentation of chromatin to mononucleosomes (12 S) and subnucleosomal fragments (under 5 S). The second effect was aggregation manifested by the generation of large chromatin particles (over 120 S) that sedimented faster than the original material (20-40 S). Bleomycin treatment of nuclei from L1210 cells resulted in a similar, almost bimodal, size distribution of drug-released chromatin fragments. Increasing levels of bleomycin produced a gradual enhancement of the amount of small fragments (under 5 S) accompanied by generation of large, aggregated particles. Aggregation caused by high drug concentrations significantly reduced the overall extent of chromatin solubilization and allowed only the release of the most degraded fragments from nuclei. The aggregation required intact nucleoprotein, since it was not detected after high-salt deproteinization of bleomycin-treated presolubilized or nuclear chromatin. The aggregation phenomenon reflects a novel activity of bleomycin which may contribute to the drug's antiproliferative properties.     

Topoisomerase-II-mediated lesions in nascent DNA: comparison of the effects of epipodophyllotoxin derivatives, VM-26 and VP-16, and 9-anilinoacridine derivatives, m-AMSA and o-AMSA

This study compares the effects of the epipodophyllotoxin derivatives, VM-26 and VP-16, and the 9-anilinoacridine derivatives, m-AMSA and o-AMSA, on nascent and mature DNA. Two types of lesion which are putatively mediated by topoisomerase II, DNA-protein crosslinks and DNA double-strand breaks, were analyzed in drug-treated nuclei from 3H/14C labelled L1210 cells. Potassium/dodecyl sulfate precipitation assay was used to assess DNA-protein crosslinks in mature and nascent (1 min old) DNA. Both epipodophyllotoxins and m-AMSA showed a strong preference for nascent DNA. DNA double-strand cleavage induced by VM-26 and m-AMSA also showed a preference for nascent DNA as indicated by neutral elution technique. Sedimentation on neutral sucrose gradients revealed that these drugs generated highly degraded fragments (under 30 S) in nascent DNA substantially faster than in mature DNA. Lesions in nascent DNA were diminished substantially by the omission of ATP or the addition of novobiocin. The ability to induce lesions in nascent DNA correlates with cytotoxic potency of the agents studied. The results suggest that replicating DNA may constitute a preferential target for antitopoisomerase II drugs.     

Changes in chromatin structure at the replication fork. II The DNPs containing nascent DNA and a transient chromatin modification detected by DNAase I.

Discrete deoxyribonucleoproteins (DNPs) containing nascent and/or bulk DNA, were obtained by fractionating micrococcal nuclease digests of nuclei form 3H-thymidine pulse (15-20 sec) and 14C-thymidine long (16 h) labeled sea urchin embryos in polyacrylamide gels. One of these DNPs was shown to contain the micrococcal nuclease resistant 300 bp "large nascent DNA" described in Cell 14, 259-267, 1978. The bulk and nascent mononucleosome fractions provided evidence for the preferential digestion by micrococcal nuclease of nascent over bulk linker regions to yield mononucleosome cores with nascent DNA. DNAase I was used to probe whether any nascent DNA is in nucleosomes. Nascent as well as bulk single-stranded DNA fragments occurred in multiples of 10.4 bases with higher than random frequencies of certain fragment sizes (for instance 83 bases) as expected from a nucleosome structure. However, a striking background of nascent DNA between nascent DNA peaks was observed. This was eliminated by a pulse-chase treatment or by digestion of pulse-labeled nuclei with micrococcal nuclease together with DNAase I. One of several possible interpretations of these results suggests that a transient change in nucleosome structure may have created additional sites for the nicking of nascent DNA by DNAase I; the micrococcal nuclease sensitivity of the interpeak radioactivity suggest its origin from the linker region. Endogenous nuclease of sea urchin embryos cleaves chromatin DNA in a manner similar to that of DNAase I.     

Influence of histone acetylation on the solubility, H1 content and DNase I sensitivity of newly assembled chromatin.

In a previous report [Annunziato, A.T. and Seale, R.L. (1983) J. Biol. Chem. 258:12675] a novel intermediate in chromatin assembly was described (detected by labeling new DNA in the presence of the deacetylase inhibitor sodium butyrate), which retained approximately 50% of the heightened sensitivity of newly replicated chromatin to DNaseI. It is now reported that nucleosomes replicated in butyrate are considerably more soluble in the presence of magnesium, relative to chromatin replicated under control conditions, and that this heightened magnesium-solubility is reflected in a concomitant increase in the preferential solubility of nucleosomes containing newly synthesized core histones. This differential solubility was accompanied by a 5- to 6-fold depletion of histone H1, and was completely abolished by the selective removal of H1 from isolated nuclei. The removal of H1 also markedly reduced the preferential DNaseI sensitivity of chromatin replicated in butyrate. Further, when mononucleosomes of control and (acetylated) nascent chromatin were compared, no differences in DNaseI sensitivity were detected. These results provide evidence that the interactions between newly assembled nucleosomes and histone H1 are altered when histone deacetylation is inhibited during chromatin replication, and suggest a mechanism for the control of H1 deposition during nucleosome assembly in vivo.     

Histone acetylation reduces H1-mediated nucleosome interactions during chromatin assembly

During chromatin replication and nucleosome assembly, newly synthesized histone H4 is acetylated before it is deposited onto DNA, then deacetylated as assembly proceeds. In a previous study (Perry and Annunziato, Nucleic Acids Res. 17, 4275 [1989]) it was shown that when replication occurs in the presence of sodium butyrate (thereby inhibiting histone deacetylation), nascent chromatin fails to mature fully and instead remains preferentially sensitive to DNaseI, more soluble in magnesium, and depleted of histone H1 (relative to mature chromatin). In the following report the relationships between chromatin replication, histone acetylation, and H1-mediated nucleosome aggregation were further investigated. Chromatin was replicated in the presence or absence of sodium butyrate; isolated nucleosomes were stripped of linker histone, reconstituted with H1, and treated to produce Mg(2+)-soluble and Mg(2+)-insoluble chromatin fractions. Following the removal of H1, all solubility differences between chromatin replicated in sodium butyrate for 30 min (bu-chromatin) and control chromatin were lost. Reconstitution with H1 completely restored the preferential Mg(2+)-solubility of bu-chromatin, demonstrating that a reduced capacity for aggregation/condensation is an inherent feature of acetylated nascent nucleosomes; however, titration with excess H1 caused the solubility differences to be lost again. Moreover, when the core histone N-terminal "tails" (the sites of acetylation) were removed by trypsinization prior to reconstitution, H1 was unable to reestablish the altered solubility of chromatin replicated in butyrate. Thus, the core histone "tails," and the acetylation thereof, not only modulate H1-mediated nucleosome interactions in vitro, but also strongly influence the ability of H1 to differentiate between new and old nucleosomes. The data suggest a possible mechanism for the control of H1 deposition and/or chromatin folding during nucleosome assembly.     

Histone h3 lysine 4 methylation marks postreplicative human cytomegalovirus chromatin

In the nuclei of permissive cells, human cytomegalovirus genomes form nucleosomal structures initially resembling heterochromatin but gradually switching to a euchromatin-like state. This switch is characterized by a decrease in histone H3 K9 methylation and a marked increase in H3 tail acetylation and H3 K4 methylation across the viral genome. We used ganciclovir and a mutant virus encoding a reversibly destabilized DNA polymerase to examine the impact of DNA replication on histone modification dynamics at the viral chromatin. The changes in H3 tail acetylation and H3 K9 methylation proceeded in a DNA replication-independent fashion. In contrast, the increase in H3 K4 methylation proved to depend widely on viral DNA synthesis. Consistently, labeling of nascent DNA using "click chemistry" revealed preferential incorporation of methylated H3 K4 into viral (but not cellular) chromatin during or following DNA replication. This study demonstrates largely selective epigenetic tagging of postreplicative human cytomegalovirus chromatin.     

Assembly of newly replicated chromatin.

Mild staphylococcal nuclease digestions under isotonic conditions release fragments of a 200 Å diameter fiber from nuclei of Drosophila melanogaster tissue culture cells. These soluble fragments have high sedimentation coefficients (30–100S) and show tightly packed nucleosomes in the electron microscope. Under the same conditions, newly replicated chromatin is released as more slowly sedimenting fragments (14S). Within 20 min after DNA replication, the nascent chromatin gradually matures into compact supranucleosomal structures which are indistinguishable from bulk chromatin on the isokinetic sucrose gradients.We have used this fractionation technique to examine the question of the fate and assembly of the new histones. After short pulses with either ³⁵S-methionine or ³H-lysine, the radioactive histones do not co-sediment with the bulk chromatin but appear instead in the fractions where the newly replicated DNA is found. Furthermore, the various nascent histones appear in different fractions on the gradient: histones H3 and H4 in 10–15S structures, histones H2A and H2B in 15–50S structures and histone H1 in 30–100S structures. These results, together with the analysis of pulse and pulse-chase experiments of both nascent DNA and histones, strongly suggest that histones H3 and H4 are deposited first on the nascent DNA (during or slightly after the DNA is replicated), histones H2A and H2B are deposited next (2–10 min later) and histone H1 is deposited last (10–20 min after DNA replication). A high turnover 20,000 dalton protein is also associated with the newly replicated chromatin.     

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.     

Immunoelectron microscopical distribution of poly(ADP-ribose)polymerase in the mammalian cell nucleus

The ultrastructural distribution of poly(ADP-ribose)polymerase has been studied using a specific antibody and immunocytochemistry with immunogold markers. In situ localization in synchronized Chinese hamster ovary cells reveals the antibody associated with mitotic chromosomes, and later with condensed chromatin and perichromatin regions in G1 phase. During S and G2, the label occurs mostly on perichromatin regions where perichromatin fibrils are also observed. In the nucleolus, the label appears especially on the dense fibrillar component and to a minor extent on the granular component. Immunolabeled spread active chromatin preparations from exponentially growing cultured mouse P815 cells indicate preferential association of the antibody with nascent nonribosomal RNP fibrils compared to inactive chromatin. The results, suggesting a relationship between the poly(ADP-ribose)polymerase occurrence and RNA (or RNP) formation, are discussed in view of the present knowledge about possible relations between poly(ADP-ribosylation) and synthesis of RNA and DNA.     

Reduced repeat length of nascent nucleosomal DNA is generated by replicating chromatin in vivo

Micrococcal nuclease digestion of nuclei from sea urchin embryos revealed transient changes in chromatin structure which resulted in a reduction in the repeat length of nascent chromatin DNA as compared with bulk DNA. This was considered to be entirely the consequence of in vivo events at the replication fork (Cell 14, 259, 1978). However, a micrococcal nuclease-generated sliding of nucleosome cores relative to nascent DNA, which might account for the smaller DNA fragments, was not excluded. In vivo [3H]thymidine pulse-labeled nuclei were fixed with a formaldehyde prior to micrococcal nuclease digestion. This linked chromatin proteins to DNA and thus prevented any in vitro sliding of histone cores. All the nascent DNAs exhibiting shorter repeat lengths after micrococcal nuclease digestion, were resolved at identical mobilities in polyacrylamide gels of DNA from fixed and unfixed nuclei. We conclude that these differences in repeat lengths between nascent and bulk DNA was generated in vivo by changes in chromatin structure during replication, rather than by micrococcal nuclease-induced sliding of histone cores in vitro.     

Effect of the antitumor antibiotic bleomycin on DNA polymerase activity

Treatment with bleomycin activates considerably a repair synthesis of DNA in rat liver chromatin in vitro and can cause loosening of the nucleoprotein complex, which facilitates the accessibility or repair enzymes for lesions in chromatin DNA. The bleomycin action on DNA-template increases severalfold the rate of synthesis catalyzed by DNA polymerase beta inhibits the activity of DNA polymerase I from Escherichia coli and suppresses severalfold the activity of DNA polymerase alpha and DNA polymerase of bacteriophage T4. The effect of bleomycin consists in a prevailing increase of nicks and minimal gaps in DNA as compared to the rise of moderate gaps, thus suggesting that bleomycin is a gamma-mimetic.     

Cycle specific association of nascent chromatin with nuclear envelope components in Physarum polycephalum.

The action of heparin on isolated nuclei derived from different phases of the mitotic cycle in plasmodia of Physarum polycephalum was studied. Heparin addition at two-fold excess over DNA concentration to nuclei in Mg-free low ionic strength buffer (10 mM TRIS-HC1, 10 mM Na2 HPO4, pH = 8) releases 60-80% of chromatin from S, G2, and mitotic phase nuclei. The RNA/protein ratio of herparin-solubilized cromatin is constant through S and G2 phases, but rises about two-fold at early prophase coincident with nucleolar breakdown. Purified nuclear envelopes were obtained from heparin-treated nuclei by sedimentation according to Bornens procedures (Nature 244, 28, 1973), and examined by transmission electron microscopy. Residual chromatin is seen at all stages with fine network of DNA fibrils in contact with the envelop. Regardless of time in S, 80% of 3H-labeled DNA was released into soluble chromatin with identical 3H/14C ratios. The residual chromatin in nuclear envelopes exhibited a preferential association of early S-DNA in nuclei engaged in early S replication, and late S preferential association in nuclei engaged in late S replication.     

The histone deacetylase inhibitor trichostatin A alters the pattern of DNA replication origin activity in human cells

Eukaryotic chromatin structure limits the initiation of DNA replication spatially to chromosomal origin zones and temporally to the ordered firing of origins during S phase. Here, we show that the level of histone H4 acetylation correlates with the frequency of replication initiation as measured by the abundance of short nascent DNA strands within the human c-myc and lamin B2 origins, but less well with the frequency of initiation across the β-globin locus. Treatment of HeLa cells with trichostatin A (TSA) reversibly increased the acetylation level of histone H4 globally and at these initiation sites. At all three origins, TSA treatment transiently promoted a more dispersive pattern of initiations, decreasing the abundance of nascent DNA at previously preferred initiation sites while increasing the nascent strand abundance at lower frequency genomic initiation sites. When cells arrested in late G₁ were released into TSA, they completed S phase more rapidly than untreated cells, possibly due to the earlier initiation from late-firing origins, as exemplified by the β-globin origin. Thus, TSA may modulate replication origin activity through its effects on chromatin structure, by changing the selection of initiation sites, and by advancing the time at which DNA synthesis can begin at some initiation sites.     

Interaction of histone H1 from sea urchin sperm with superhelical and relaxed DNA

Complexes of histone H1 from sea urchin sperm (H1S) and calf thymus (H1T) with superhelical DNA I and relaxed circular DNA II have been analyzed by analytical sedimentation. Similar to H1T, the highly basic and relatively arginine-rich histone H1S preferentially interacts with DNA I compared to DNA II under competition conditions. However, H1S induces a stronger aggregation of bothforms of DNA than H1T. Below 0.05 M NaCl, the soluble complexes formed by both histones have similar properties, but aggregation proceeds in a different manner: H1S induces a stronger aggregation of DNA II as compared to DNA I, whereas H1T fails to aggregate DNA I.The results are explained on the basis of differences in amino acid sequence and structure of the two histones and related to the special chromatin condensing ability of histone H1S.     

Daunomycin-induced unfolding and aggregation of chromatin.

Using equilibrium dialysis and sedimentation velocity analysis, we have characterized the binding of the anti-tumor drug daunomycin to chicken erythrocyte chromatin before and after depletion of linker histones and to its constitutive DNA under several ionic strengths (5, 25, and 75 mM NaCl). The equilibrium dialysis experiments reveal that the drug binds cooperatively to both the chromatin fractions and to the DNA counterpart within the range of ionic strength used in this study. A significant decrease in the binding affinity was observed at 75 mM NaCl. At any given salt concentration, daunomycin exhibits higher binding affinity for DNA than for linker histone-depleted chromatin or chromatin (in decreasing order). Binding of daunomycin to DNA does not significantly affect the sedimentation coefficient of the molecule. This is in contrast to binding to chromatin and to its linker histone-depleted counterpart. In these instances, preferential binding of the drug to the linker DNA regions induces an unfolding of the chromatin fiber that is followed by aggregation, presumably because of histone-DNA interfiber interactions.     

A general method for preparing chromatin containing intact DNA.

A simple and general method is described for preparing chromatin from eukaryotic cells using isotonic conditions. First, cells are encapsulated in agarose microbeads and then lysed using Triton X-100 in the presence of a chelating agent and a physiological concentration of salt. Most cytoplasmic proteins and RNA diffuse rapidly out through pores in the beads to leave encapsulated chromatin which is nevertheless completely accessible to enzymes and other probes. This chromatin can be manipulated freely without aggregation in a variety of different salt and detergent concentrations. It also contains intact DNA since removal of the histones releases superhelical DNA. Conditions are described for incubating this chromatin at 37 degrees C in the presence of Mg2+ ions without any nicking of the DNA. We illustrate the usefulness of this chromatin in investigations on the attachment of nascent RNA to the nucleoskeleton, the accessibility of the ribosomal locus to EcoRI and the properties of the endogenous RNA polymerase II. This type of chromatin preparation should prove useful for both structural and functional studies.     

Fine structural in situ analysis of nascent DNA movement following DNA replication

Nascent DNA (newly replicated DNA) was visualized in situ with regard to the position of the previously replicated DNA and to chromatin structure. Localization of nascent DNA at the replication sites can be achieved through pulse labeling of cells with labeled DNA precursors during very short periods of time. We were able to label V79 Chinese Hamster cells for as shortly as 2 min with BrdU; Br-DNA, detected by immunoelectron microscopy, occurs at the periphery of dense chromatin, at individual dispersed chromatin fibers, and within dispersed chromatin areas. In these regions DNA polymerase alpha was also visualized. After a 5-min BrdU pulse, condensed chromatin also became labeled. When the pulse was followed by a chase, a larger number of gold particles occurred on condensed chromatin. Double-labeling experiments, consisting in first incubating cells with IdU for 20 min, chased for 10 min and then labeled for 5 min with CldU, reveal CldU-labeled nascent DNA on the periphery of condensed chromatin, while previously replicated IdU-labeled DNA has been internalized into condensed chromatin. Altogether, these results show that the sites of DNA replication correspond essentially to perichromatin regions and that the newly replicated DNA moves rapidly from replication sites toward the interior of condensed chromatin areas.