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↗
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↗
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↗
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↗
Nucleosomes associated with newly replicated DNA have an altered conformation.
In vitro DNA synthesis was studied in HeLa cell nuclei, with emphasis on the question of whether newly replicated DNA is associated with nucleosomes. The newly replicated DNA was twice as sensitive to digestion by micrococcal nuclease as mature chromatin DNA, reaching a limit digest at 20-25% acid-insoluble product. Examination of the intermediates of digestion by micrococcal nuclease showed the nuclease-resistant, new DNA to be complexed in nucleosomes. However, structural differences were evident at both the polynucleosomal and the core particle level. The nucleosomes on newly replicated DNA were arranged with a repeat size of 165-170 base pairs-i.e., smaller than the 185-base-pair repeat of mature chromatin. The heterogeneity of polynucleosomal multimers, evident in digests of whole chromatin, was reduced in newly replicated chromatin such that the multimers resolved as sharply defined bands. Nucleosomal core particles associated with newly replicated DNA had a different conformation from particles in mature chromatin based on the following lines of evidence: (i) during micrococcal nuclease digestion, the monomer nucleosomes did not accumulate but were rapidly degraded under certain conditions; (ii) micrococcal nuclease limit digest patterns and DNase I digestion patterns, both of which reflect internal nucleosomal protein DNA associations, differed significantly from control patterns. These findings bear directly on models postulated for nucleosome-DNA interactions during chromation replication. A possible mechanism to account for the conformational change and its role in replication are discussed.
Chromatin↗
Binding of cis- and trans-dichlorodiammineplatinum(II) to the nucleosome core.
The binding of the antitumor drug cis-dichlorodiammineplatinum(II) and its inactive trans isomer with the nucleosome core particle has been investigated. Kinetic studies show that platinum binding increases with incubation time, from a few bound platinum atoms per nucleosome core in the first 0.5 hr to 40-50 after 40 hr. There is no crosslinking or dissociation of nucleosome cores upon platinum binding, as revealed by sedimentation velocity measurements. Electrophoresis through 0.1% sodium dodecyl sulfate/18% polyacrylamide gels after platinum binding reveals striking differences in the DNA and protein band patterns for the two isomers. cis-Dichlorodiammineplatinum(II) binds first to the DNA, retarding and spreading its migration in the gel. A comparison study with the 146-base-pair nucleosome core DNA showed the binding to be little affected by the presence of the histone octamer. The trans complex, on the other hand, produces DNA-histone and histone-histone crosslinks that only appear for the cis isomer after long incubation times. The protein-protein crosslinks were reversed by soaking the gel in cyanide solution to form [Pt(CN)(4)](-2). Subsequent two-dimensional gel electrophoresis revealed that trans-dichlorodiammineplatinum(II) forms specific crosslinks between histone protein pairs H3 and H2a and H2b and H4 in the nucleosome core. The occurrence of DNA-protein crosslinks was demonstrated by treating the platinum/nucleosome core reaction mixtures with a protease or with nucleases prior to electrophoresis and observing changes in the gel patterns. Platinum was located in the gels through autoradiography using (195m)Pt-labeled complexes. This work clearly demonstrates the greater propensity of trans-dichlorodiammineplatinum(II) to form histone-histone and histone-DNA crosslinks compared with the antitumor active cis isomer, which binds first to the DNA and only forms crosslinks to the histones when the nucleosome core is heavily loaded with platinum.
Animals↗
The nature of the interaction of nucleosomes with a eukaryotic RNA polymerase II.
The integrity and stability of nucleosomes under transcription assay conditions has been found to depend on concentration and ionic environment. Rifamycin AF/013, a commonly used inhibitor of initiation, is particularly effective in destabilisation of nucleosomes. Intact nucleosomes are refractory to transcription by wheat RNA polymerase II, the histone core preventing initiation. Template titration suggests that the polymerase can, however, bind to nucleosomes, and a 15--16S complex has been observed on sucrose gradients. DNase I digestion of polymerase-nucleosome incubations indicates that whilst histone is still present in the complex, the nucleosome conformation is altered resulting in enhanced nucleolysis at sites near the DNA centre but reduced overall kinetics of digestion.
Animals↗
Histone H3 disulfide dimers and nucleosome structure.
The arginine-rich histone, H3, isolated from avian erythrocytes, can dimerize by forming a disulfide linkage between the single cysteine sulfhydryl residues at position 110 of the H3 polypeptide chain. The H3 dimer can be substituted for undimerized H3 in experiments in which the nucleosome is reconstituted from DNA and mixtures of the four "core" histones, H2A, H2B, H3, and H4. We report here that reconstituted nucleosomes containing H3 dimer are indistinguishable, by a number of criteria, either from native nucleosomes or from reconstitutes containing H3 monomer. The criteria include the pattern of susceptibility of the complex to nucleases, the amount of DNA supercoiling induced by histone binding, and the hydrodynamic properties of reconstituted nucleosome "core" preparations. The results suggest that the residues in the neighborhood of position 110 on each H3 molecule are in close contact in the nucleosome. If, as has been proposed, the nucleosome has a dyad axis, then the disulfide bridge between H3 molecules must lie on this axis.
Chromatin↗
Stability of nucleosomes in native and reconstituted chromatins.
The stability of nucleosomes of SV40 minichromosomes extracted from infected cells or reconstituted by association of SV40 DNA and the four histones H2A, H2B, H3 and H4 was studied as a function of the ionic strength. As a measure of the stability of the nucleosome, we followed the disappearance of the nucleosomes from the original chromatin and their appearance on a "competing" DNA. We show here that the DNA and the histone components of the nucleosomes do not apprecially dissociate below 800 mM NaCl. At 800 mM and above, the histone moiety of the nucleosomes can dissociate from the DNA and efficiently participate to the formation of nucleosomes on a "competing" DNA.
Animals↗
A correlation between nucleosome spacer region susceptibility to DNase I and histone acetylation.
Hepatoma tissue culture (HTC) cell nuclei were digested with either DNase I or micrococcal nuclease and the nucleohistone digestion products fractionated by gel electrophoresis or exclusion chromatography. Under appropriate conditions, gel electrophoresis demonstrates that for both nucleases, only cleavages within the nucleosome spacer regions and not within the nucleosome core lead to freely migrating nucleohistone particles. These particles consist of nucleosome cores, nucleosomes and nucleosome oligomers. Following DNase I digestion and fractionation by exclusion chromatography, analysis of the histones indicates a direct relationship between increased spacer region susceptibility to nuclease and increased nucleosomal histone acetylation. Evidently digestion sites outside the regions of DNA protected by core histones can reflect the degree of acetylation of core histones. Such a relationship is not found when micrococcal nuclease is used to digest the samples.
Acetylation↗
Nucleosome stability safeguards cell identity, stress resilience and healthy aging.
Nucleosomes are the minimal repeating units of chromatin. Their dynamic assembly and disassembly underpins chromatin organization and genome regulation. However, it remains unclear how intrinsic nucleosome stability contributes to higher-level yet fundamental cellular and organismal properties-such as preservation of cell identity, lineage specification, stress resilience and ultimately healthy aging. To address this, we tested the impact of decreased intrinsic nucleosome stability across multiple cell, tissue and organismal models by introducing histone mutants that weaken histone-histone interactions. While nucleosome instability did not broadly alter global chromatin accessibility, DNA damage, cell proliferation or viability, it impaired lineage-specific gene expression programs, altered lineage specification and activated intrinsic inflammatory and stress pathways in a manner reminiscent of aging in mouse tissues and human cells. Consistently, nucleosome instability accelerated the onset of age-associated transcriptional alterations and functional decline in Caenorhabditis elegans and Drosophila melanogaster, and reduced cellular resilience to exogenous perturbations-including environmental, epigenetic and mitotic stress-in human cells and Saccharomyces cerevisiae. These cross-species findings identify nucleosome stability as an evolutionarily conserved epigenetic safeguard that preserves cell identity and stress resilience and supports organismal function and healthy aging.
Journal Article↗
Oriented binding of transcription factors to nucleosomes remodels chromatin at human promoters.
Transcription factors (TFs) can access nucleosomes via five distinct modes: gyre-spanning, periodic-binding, dyad-binding, and end-binding modes as well as an oriented binding mode, where the TF binding motif shows orientational preference relative to the nucleosome. Here, we report the first structure of an oriented TF:nucleosome complex, where two ELF2 proteins bind to a double motif located at superhelical location +4, unwinding four helical turns of DNA from the nucleosome. We further show that unlike previously described pioneer factors, ELF2 is able to occupy all of its unmethylated, high-affinity double motifs in vivo. Motifs of ELF2 and another oriented nucleosome binder, YY1, are highly enriched downstream of transcription start sites (TSSs) of highly expressed genes, with the motifs oriented in such a way that the TSS becomes accessible upon TF binding. Our results suggest that oriented binding may be generally important for high transcriptional activity.
Nucleosomes↗