Nucleosome-nucleosome interaction in chromatin.
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Animals↗
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Nucleosome structure I: all four histones, H2A, H2B, H3, and H4, are required to form a nucleosome, but an H3-H4 subnucleosomal particle is formed with H3-H4 alone.
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Animals↗
Nucleosome structure II: structure of the SV40 minichromosome and electron microscopic evidence for reversible transitions of the nucleosome structure.
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Chromatin↗
Chromatin reconstitution on small DNA rings. IV. DNA supercoiling and nucleosome sequence preference.
Nucleosome formation on inverted repeats or on some alternations of purines and pyrimidines can be inhibited in vitro by DNA supercoiling through their supercoiling-induced structural transitions to cruciforms or Z-form DNA, respectively. We report here, as a result of study of single nucleosome reconstitutions on a DNA minicircle, that a physiological level of DNA supercoiling can also enhance nucleosome sequence preference. The 357 base-pair minicircle was composed of a promoter of phage SP6 RNA polymerase joined to a 256 base-pair fragment containing a sea urchin 5 S RNA gene. Nucleosome formation on the promoter was found to be enhanced on a topoisomer with in vivo superhelix density when compared to topoisomers of lower or higher superhelical densities, to the nicked circle, or to the linear DNA. In contrast, nucleosomes at other positions appeared to be insensitive to supercoiling. This observation relied on a novel procedure for the investigation of nucleosome positioning. The reconstituted circular chromatin was first linearized using a restriction endonuclease, and the linear chromatin so obtained was electrophoresed as nucleoprotein in a polyacrylamide gel. The gel showed well-fractionated bands whose mobilities were a V-like function of nucleosome positions, with the nucleosome near the middle migrating less. This behavior is similar to that previously observed for complexes of sequence-specific DNA-bending proteins with circularly permuted DNA fragments, and presumably reflects the change in the direction of the DNA axis between the entrance and the exit of the particle. Possible mechanisms for such supercoiling-induced modulation of nucleosome formation are discussed in the light of the supercoiling-dependent susceptibility to cleavage of the naked minicircle with S1 and Bal31 nucleases; and a comparison between DNase I cleavage patterns of the modulated nucleosome and of another, non-modulated, overlapping nucleosome.
Animals↗
Involvement of histone H1 in the organization of the nucleosome and of the salt-dependent superstructures of chromatin.
We describe the results of a systematic study, using electron microscopy, of the effects of ionic strength on the morphology of chromatin and of H1-depleted chromatin. With increasing ionic strength, chromatin folds up progressively from a filament of nucleosomes at approximately 1 mM monovalent salt through some intermediate higher-order helical structures (Thoma, F., and T. Koller, 1977, Cell 12:101-107) with a fairly constant pitch but increasing numbers of nucleosomes per turn, until finally at 60 mM (or else in approximately 0.3 mM Mg++) a thick fiber of 250 A diameter is formed, corresponding to a structurally well-organized but not perfectly regular superhelix or solenoid of pitch approximately 110 A as described by Finch and Klug (1976, Proc. Natl. Acad. Sci. U.S.A. 73:1897-1901). The numbers of nucleosomes per turn of the helical structures agree well with those which can be calculated from the light-scattering data of Campbell et al. (1978, Nucleic Acids Res. 5:1571-1580). H1-depleted chromatin also condenses with increasing ionic strength but not so densely as chromatin and not into a definite structure with a well-defined fiber direction. At very low ionic strengths, nucleosomes are present in chromatin but not in H1-depleted chromatin which has the form of an unravelled filament. At somewhat higher ionic strengths (greater than 5 mM triethanolamine chloride), nucleosomes are visible in both types of specimen but the fine details are different. In chromatin containing H1, the DNA enters and leaves the nucleosome on the same side but in chromatin depleted of H1 the entrance and exit points are much more random and more or less on opposite sides of the nucleosome. We conclude that H1 stabilizes the nucleosome and is located in the region of the exit and entry points of the DNA. This result is correlated with biochemical and x-ray crystallographic results on the internal structure of the nucleosome core to give a picture of a nucleosome in which H1 is bound to the unique region on a complete two-turn, 166 base pair particle (Fig. 15). In the formation of higher-order structures, these regions on neighboring nucleosomes come closer together so that an H1 polymer may be formed in the center of the superhelical structures.
Chromatin↗
Evidence that nucleosomes on the mouse mammary tumor virus promoter adopt specific translational positions.
We have previously demonstrated that an array of six nucleosomes are phased on the mouse mammary tumor virus (MMTV) long terminal repeat (1,2). In this study, we devised a new assay to measure the translational positions of specific nucleosomes on the MMTV promoter. Nucleosome core particles were purified and shown to contain A and B nucleosomal DNA by Taq polymerase primer extension with nucleosome-specific primers. The 5' and 3' boundaries of A and B nucleosomes were measured by extending to the end of the core DNA with internal primers. This approach yielded results consistent with major translational positions of -23 to +123 and -221 to -75 for A and B nucleosomes, respectively. The micrococcal nuclease cleavage patterns of A and B nucleosome regions in isolated nuclei are conserved at base-pair resolution in multiple murine cell lines containing either stable MMTV-reporter chimeras or endogenous proviruses. As the refined nucleosome positions place important transcription factor binding sites at the 3' edge of the B nucleosome and in the nucleosome A/B linker, we propose that linker histone depletion and chromatin unfolding may be required to expose these cis-elements during steroid hormone-induced transcription initiation.
Animals↗
A phase relationship associates tRNA structural gene sequences with nucleosome cores.
DNA (760 bp) isolated from nucleosome tetramers of staphylococcal nuclease-digested chicken embryo chromatin was highly enriched for tRNA genes and subsequently cloned in E. coli chi 1776. The location of genes coding for chicken embryo tRNALys, tRNAPhe and tRNAiMet within the cloned nucleosome tetramer DNA was determined using restriction endonucleases for which single cleavage sites could be predicted from the respective tRNA base sequence. All our tRNA genes reside nonrandomly at four locations on nucleosome tetramer DNA. The spacing between the tRNA gene locations is approximately 190 bp, similar to the DNA repeat length of chicken embryo chromatin. The four tRNA gene locations were also defined in noncloned nucleosome tetramer DNA highly enriched for tRNA genes. The majority of genes coding for tRNALys, tRNAPhe and tRNAiMet, respectively, are located in equal proportion 40-45, 230, 420 and 610 bp distant from the 5' end of the tRNA-identical strand. Thus the tRNA structural gene sequences all appear to begin about 20 bp "inside" the nucleosome core. As observed with nucleosomal DNA not enriched for tRNA genes, the phase relationship between tRNA genes and nucleosome location is maintained over a distance of 4-6 subsequent nucleosomes. A cloned molecule of nucleosomal DNA containing both a tRNALys gene and a tRNAiMet gene in the same polarity reveals that a phase adjustment might be necessary for the nucleosomes between these two tRNA genes in chicken embryo chromatin.
Animals↗
Subunit structure of chromatin and the organization of eukaryotic highly repetitive DNA: nucleosomal proteins associated with a highly repetitive mammalian DNA.
Component alpha DNA is a homogeneous, highly repetitive fraction that comprises nearly a quarter of the African green monkey (Cercopithecus aethiops) genome. By restriction enzyme analysis, it has a repeat periodicity of 176 +/- 4 nucleotide base pairs, corresponding closely with the length of DNA contained within a nucleosome. The sequence is organized into large blocks of constitutive heterochromatin. A method is described here for the isolation of intact polynucleosomal arrays containing only component alpha sequences. Isolated monkey nuclei are treated with EcoRI, which releases only component alpha nucleosomal arrays; the arrays are then fractionated and purified by sedimentation in sucrose gradients. The method permits a compositional analysis of the proteins associated with a constitutively repressed, heterochromatic sequence. The major differences in the proteins associated with component alpha nucleosomes that distinguish them from the bulk DNA nucleosomes are a decrease in the content of the H1 histones in the component alpha nucleosomes and a concomitant increase in the amount of certain nonhistone proteins. The specific observations are: (i) In the component alpha nucleosomes, 65-70% of the proteins were nonhistone proteins; this contrasts with the value, 40%, for nonhistone proteins associated with nucleosomes containing bulk DNA. (ii) The amount of H1 histone in chromatin containing predominantly bulk DNA was about 13.7%. However, the H1 histone was depleted and possibly absent in component alpha oligonucleosomes. (iii) Coincident with the decrease in the H1 histones and in the same molecular weight range (24,000-43,000), there appeared five minor nonhistone proteins. The minor, low-molecular-weight, nonhistone proteins were not detected in chromatin containing bulk DNA but they represented nearly 12% of the protein in component alpha nucleosomes. The resistance to salt extraction (0.6-2.0 M NaCl) indicates that the low-molecular-weight nonhistone proteins are tenaciously bound to the component alpha nucleosomes. In addition, a class of high-molecular-weight (>100,000) nonhistone proteins was enriched 5- or 6-fold in component alpha oligonucleosomes. The relative amounts of the nucleosome core histones were not changed.
Cell Line↗
Assembly of new nucleosomal histones and new DNA into chromatin.
The assembly of chromatin from newly synthesized nucleosomal histones (labeled with [3H]arginine) and new DNA (density-labeled with [125I]iododeoxyuridine)was studied in growing cultured mouse cells. The nucleosomal histones were specifically examined by dissociating histone H1 and nonhistone proteins from unsheared chromatin either by incubation in 0.6 M NaCl or by digestion with micrococcal nuclease to release nucleosomes. In both cases, the four nucleosomal histones (H2A, H2B, H3, and H4) are essentially the only proteins that remain bound to DNA and that are labeled by [3H]arginine. After formaldehyde fixation, H1-depleted chromatin containing dense DNA can be completely resolved in CsCl buoyant density gradients from that containing unreplicated DNA; separation of nucleosomes is satisfactory although less complete. New DNA and new histones are already assembled into chromatin possessing characteristic nucleosomal structure after 3 min of synthesis (the shortest time studied), as shown by the kinetics of digestion of new DNA by micrococcal nuclease, by the distribution of new DNA and new histones in nucleosomes. However, after 3-30 min of synthesis most new nucleosomal histones are associated with unreplicated DNA rather than with new DNA. It is concluded that new nucleosomes are assembled on DNA at some distance from DNA replication sites, with concomitant migration of preexisting nucleosomes onto new DNA.
Cell Line↗
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.
Cell Nucleus↗
Reaction of nucleosome DNA with dimethyl sulfate.
We have measured the effect of the histones in the nucleosome core particle on methylation of purines in nucleosome DNA by dimethyl sulfate. By using 32P terminally labeled nucleosome cores, we have examined the pattern of strand cleavage at methylated sites in the nucleosome DNA and compared it to the pattern observed in histone-free DNA. We are unable to detect any significant difference between the reactivity of N7 of guanines in nucleosome DNA and of that in naked DNA, with the exception of a single site of enhanced reactivity at approximately nucleotide 62 from the 5' end of the nucleosome. Contrary to our expectation, there is no detectable periodic modulation of reactivity corresponding to the twist of the DNA on the nucleosome surface. We are able to place a low upper limit on the extent to which the histones of the nucleosome can protect N7 of guanine in the large groove. With somewhat less precision, we also conclude that the N3 of adenine in the small groove is largely unprotected. These results indicate that in nucleosome DNA the bases are nearly as accessible to solvent as they are in DNA free of protein.
Adenine↗
Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination.
DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of chromatin, show most DNA repair enzymes remove DNA damage at reduced rates as compared to free DNA. The molecular details on how base excision repair (BER) enzymes recognize and remove DNA damage in nucleosomes have not been elucidated. However, biochemical BER data of nucleosomal substrates suggest the nucleosome presents different structural barriers dependent on the location of the DNA lesion and the enzyme. This indicates the mechanisms employed by these enzymes to remove DNA damage in free DNA may be different than those employed in nucleosomes. Given that the majority of genomic DNA is assembled into nucleosomes, structural information of these complexes is needed. To date, the scientific community lacks detailed protocols to perform technically feasible structural studies of these complexes. Here, we provide two methods to prepare a complex of two genetically fused BER enzymes (Polymerase β and AP Endonuclease1) bound to a single-nucleotide gap near the entry-exit of the nucleosome for cryo-electron microscopy (cryo-EM) structural determination. Both methods of sample preparation are compatible for vitrifying quality grids via plunge freezing. This protocol can be used as a starting point to prepare other nucleosomal complexes with different BER factors, pioneer transcription factors, and chromatin-modifying enzymes.
Chromatin↗
Nucleosome mono, di, tri-, and tetramers from chicken embryo chromatin.
The fractionation of gram quantities of nuclease digested chromatin from chicken embryos into nucleosome mono-, di-, tri-, and tetramers is described in detail. Each of these nucleosomal species contains a fraction soluble in 0-1 M KC1 that decreases with increasing repeat number. Less histone H1 is associated with the nucleosome fractions soluble as compared to the respective fractions precipitated in 0.1 M KC1. Thermal denaturation profiles of the four nucleosomal species are monophasic. The same Tm of 78 degrees C has been determined for the KC1-soluble nucleosomes and for the KC1-insoluble monomer. The Tm of the KC1-insoluble oligomers is 79.8 degrees C. Multiphasic melting curves were recorded for nucleosomal material that was concentrated by lyophilisation or stored at 4 degrees C in 0.25 mM EDTA. Total nucleosome mono-, di-, tri-, and tetramers (consisting of both the fraction soluble and insoluble in 0.1 M KC1) have been analyzed concerning their sedimentation, diffusion, partial specific volume, and molecular weight and compared with the sedimentation and molecular weight data of KC1-soluble nucleosome mono- and tetramers.
Animals↗
Salt-induced structural changes in nucleosomes.
Nucleosomes and oligonucleosomes were prepared by digestion of human placental nuclei with staphlococcal nuclease and fractionated by gel filtration chromatography. The effect of increasing salt on the structure of nucleosomes was examined in the presence and absence of 10 mM MgCl2. Nucleosomes and oligonucleosomes are insoluble over a broad range of salt concentration. Nucleosomes are insoluble in larger than or equal to 120 mM (NH4)2SO4 containing 10 mM MgCl2 allowing analyses of changes in nucleosomal DNA by C.D. spectroscopy. Nucleosomes are insoluble in less than or equal to 120 mM (NH4)2SO4 containing 10 mM MgCl2 as demonstrated by turbidity measurements. We conclude that the insolubility of nucleosomes accompanies salt-induced structural changes possibly due to individual particle condensation. As the salt concentration is increased the nucleosomes condense and then relax at higher salt concentrations.
Ammonium Sulfate↗
Presence of non-histone proteins in nucleosomes.
It has been established that nucleosomes are made of histones and DNA fragments. The purpose of this work to establish whether some non-histone proteins are also present in these chromatin subunits. We have found that nucleosome preparations contain phosphorylated non-histone proteins and protein kinases by sucrose gradient analysis. In order to establish whether these proteins are actually bound to nucleosomes or if they represent unbound or aggregated proteins, the following experiments were performed. (a) Free non-histone proteins and proteins released from chromatin by DNase overdigestion were analyzed by sucrose gradient centrifugation. No phosphoproteins but some phosvitin kinase activity was found in the part of the gradient which contained the nucleosomes. It could be assumed that part of the phosphoproteins are bound to nucleosomes. (b) A digestion of nucleosomes with DNase I suppressed the phosvitin kinase activity in the 11-S region of the gradient. (c) High ionic strength, which extracted non-histone proteins, suppressed the phosvitin kinase activity in the nucleosome region. Part of phosvitin kinase and of nuclear phosphoproteins are therefore bound to nucleosomes and are released by nuclease digestion and by high ionic strength.
Animals↗
A nucleosome core is transferred out of the path of a transcribing polymerase.
We have determined the fate of a nucleosome core on transcription. A nucleosome core was assembled on a short DNA fragment and ligated into a plasmid containing a promoter and terminators for SP6 RNA polymerase. The nucleosome core was stable in the absence of transcription. The distribution of nucleosome cores after transcription was examined. The histone octamer was displaced from its original site and reformed a nucleosome core at a new site within the same plasmid molecule, with some preference for the untranscribed region behind the promoter. These observations eliminate several models that have been proposed for transcription through a nucleosome core. Our results suggest that a nucleosome core in the path of a transcribing polymerase is displaced by transfer to the closest acceptor DNA.
Animals↗
The fate of parental nucleosomes during SV40 DNA replication.
The fate of parental nucleosomes during the replication of chromatin templates was studied using a modification of the cell-free SV40 DNA replication system. Plasmid DNA molecules containing the SV40 origin were assembled into chromatin with purified core histones and fractionated assembly factors derived from HeLa cells. When these templates were replicated in vitro, the resulting progeny retained a nucleosomal organization. To determine whether the nucleosomes associated with the progeny molecules resulted from displacement of parental histones during replication followed by reassembly, the replication reactions were performed in the presence of control templates. It was observed that the progeny genomes resulting from the replication of chromatin templates retained a nucleosomal structure, whereas the progeny of the control DNA molecules were not assembled into chromatin. Additional experiments, involving direct addition of histones to the replication reaction mixtures, confirmed that the control templates were not sequestered in some form which made them unavailable for nucleosome assembly. Thus, our data demonstrate that parental nucleosomes remain associated with the replicating molecules and are transferred to the progeny molecules without displacement into solution. We propose a simple model in which nucleosomes ahead of the fork are transferred intact to the newly synthesized daughter duplexes.
Cell-Free System↗
DNAase I, DNAase II and staphylococcal nuclease cut at different, yet symmetrically located, sites in the nucleosome core.
We have determined the relative location of pancreatic DNAase (DNAase I), spleen acid DNAase (DNAase II) and staphylococcal nuclease cleavage sites in the nucleosome core. Each of these three enzymes cleaves the DNA of chromatin at 10.n nucleotide intervals (n integer); this specificity presumably reflects the internal structure of the nucleosome. We have already reported that DNAase I cleaves nucleosomal DNA so that nearest adjacent cuts on opposite strands are staggered by 2 nucleotides, 3' end extending (Sollner-Webb and Felsenfeld, 1977). Here we show that the nearest cuts made by DNAase II in nucleosomal DNA are staggered by 4 nucleotides, 3' end extending, while cuts made by staphylococcal nuclease have a stagger of 2 nucleotides, 5' end extending. The cutting sites of the three enzymes thus do not coincide. Each pair of staggered cuts, however, is symmetrically located about a common axis-that is, the "dyad axes" that bisect nearest pairs of cutting sites coincide for all three enzymes. This result is consistent with the presence of a true dyad axis in the nucleosome core. Our results support the conclusion that a structural feature of the nucleosome, having a 10 nucleotide periodicity, is the common recognition site for all three nucleases. The position of the cut is determined, however, by the individual characteristics of each enzyme. Sites potentially available to nuclease cleavage span a region of 4 nucleotides out of this 10 nucleotide repeat, and a large fraction of these sites are actually cut. Thus much of the nucleosomal DNA must in some sense be accessible to the environment.
Animals↗