Salt-induced structural changes in nucleosomes

Ehrlich ascites tumor (EAT) nucleosomes treated with increasing NaCl concentrations were analyzed by sucrose density gradient centrifugation. Two events were found to take place in the course of the salt treatment: a) increasing amounts of nucleosomes dissociated into free DNA and protein in the interval 0.6M–1.5M NaCl, and b) the sedimentation coefficient of the nucleosomes decreased from 11S to 8S in the interval 0.6M-1M NaCl. This decrease was not caused by loss of protein and was fully reversible upon slow and gradual lowering of the ionic strength. This shows that before dissociation of the protein core from DNA, nucleosomes undergo a structural transition. The electron microscopic observations revealed that it consisted in detachment of the ends of nucleosomal DNA from the protein core. It is suggested that an arginine-rich domain in the protein core exists, which holds more tightly the central part of the nucleosomal DNA, while its ends are relatively loosely bound to lysine-rich domains.     

Conserved Substitution Patterns around Nucleosome Footprints in Eukaryotes and Archaea Derive from Frequent Nucleosome Repositioning through Evolution

Nucleosomes, the basic repeat units of eukaryotic chromatin, have been suggested to influence the evolution of eukaryotic genomes, both by altering the propensity of DNA to mutate and by selection acting to maintain or exclude nucleosomes in particular locations. Contrary to the popular idea that nucleosomes are unique to eukaryotes, histone proteins have also been discovered in some archaeal genomes. Archaeal nucleosomes, however, are quite unlike their eukaryotic counterparts in many respects, including their assembly into tetramers (rather than octamers) from histone proteins that lack N- and C-terminal tails. Here, we show that despite these fundamental differences the association between nucleosome footprints and sequence evolution is strikingly conserved between humans and the model archaeon Haloferax volcanii. In light of this finding we examine whether selection or mutation can explain concordant substitution patterns in the two kingdoms. Unexpectedly, we find that neither the mutation nor the selection model are sufficient to explain the observed association between nucleosomes and sequence divergence. Instead, we demonstrate that nucleosome-associated substitution patterns are more consistent with a third model where sequence divergence results in frequent repositioning of nucleosomes during evolution. Indeed, we show that nucleosome repositioning is both necessary and largely sufficient to explain the association between current nucleosome positions and biased substitution patterns. This finding highlights the importance of considering the direction of causality between genetic and epigenetic change.     

Effects of sequence alterations in a DNA segment containing the 5 S RNA gene from Lytechinus variegatus on positioning of a nucleosome core particle in vitro

Reassociation of a 260-base pair cloned fragment of Lytechinus variegatus DNA with core histones has been shown to give rise to a uniquely positioned nucleosome (Simpson, R. T., and Stafford, D. W. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 51-55). In an attempt to define the features that dictate the unique positioning of the nucleosome, we have constructed a number of mutants of this DNA sequence. The ability of these mutants to form positioned nucleosomes was analyzed by DNase I digestion of the DNA after reassociation with chicken erythrocyte core histones. While all the mutants were efficiently incorporated into core particles, not all of these modified sequences were capable of forming a positioned nucleosome. Of the 13 mutants examined, 7 fell into a class that gave rise to nucleosomes in which no unique positioning could be demonstrated. While no specific feature of the DNA sequences has been identified as the critical factor in allowing, or dictating, the formation of positioned nucleosomes, our results do indicate that the region 20-30 bases either side of the center of the core particle appears to contain the major elements necessary for positioning. Additionally, these studies clearly show that differences in the digestion of naked and core particle DNA are related to specific interactions of the DNA and histones rather than to an altered specificity of the enzyme induced by the presence of the proteins.     

Reconstitution experiments show that sequence-specific histone-DNA interactions are the basis for nucleosome phasing on mouse satellite DNA

The molecular basis underlying the sequence-specific positioning of nucleosomes on DNA was investigated. We previously showed that histone octamers occupy multiple specific positions on mouse satellite DNA in vivo and have now reconstituted the 234 bp mouse satellite repeat unit with pure core histones into mononucleosomes. Histones from mouse liver or chicken erythrocytes bind to the DNA in multiple precisely defined frames in perfect phase with a diverged 9 bp subrepeat of the satellite DNA. This is the first time that nucleosome positions on a DNA in vivo have been compared to those found on the same DNA by in vitro reconstitution. Most of the nucleosomes occupy identical positions in vivo and in vitro. There are, however, some characteristic differences. We conclude that sequence-dependent histone-DNA interactions play a decisive role in the positioning of nucleosomes in vivo, but that the nucleosome locations in native chromatin are subject to additional constraints.     

Sequence and position-dependence of the equilibrium accessibility of nucleosomal DNA target sites

We have previously shown that nucleosomes are conformationally dynamic: DNA sequences that in the time-average are buried inside nucleosomes are nevertheless transiently accessible, even to large proteins (or any other macromolecule). We refer to this dynamic behavior as "site exposure". Here we show that: (i) the equilibrium constants describing this dynamic site exposure decrease progressively from either end of the nucleosomal DNA in toward the middle; and (ii) these position-dependent equilibrium constants are strongly dependent on the nucleosomal DNA sequence. The progressive decrease in equilibrium constant with distance inside the nucleosome supports the hypothesis that access to sites internal to a nucleosome is provided by progressive (transient) release of DNA from the octamer surface, starting from one end of the nucleosomal DNA. The dependence on genomic DNA sequence implies that a specific genomic DNA sequence could be a major determinant of target site occupancies achieved by regulatory proteins in vivo, by either governing the time-averaged accessibility for a given nucleosome position, or biasing the time-averaged positioning (of mobile nucleosomes), which in turn is a major determinant of site accessibility.     

Nucleosomal structure of two Drosophila melanogaster simple satellites

Nucleosomes have been fractionated on nondenaturing polyacrylamide gels, and nucleosome subtypes containing the Drosophila melanogaster specific protein D1 and ubiquitinated core histone H2A were identified by solubility in 0.1 M NaCl before nucleoprotein gel electrophoresis. Nucleosomes which contain DNA complementary to the 1.672 density simple satellite (sequence-AATAT-) bind protein D1, as demonstrated by two-dimensional hybridization mapping. This hybridization pattern allows the identification of D1 dinucleosomes, which, like D1 mononucleosomes, are reduced in mobility on the first dimension (nucleoprotein) gel by the addition of D1, an AT sequence-specific DNA-binding protein. The 1.705 density simple satellite (sequence-AAGAG-) is also found in nucleosomes, in a radically different subset from those of the -AATAT- DNA sequence. -AAGAG- nucleosomes do not contain D1 protein, but appear to be enriched in ubiquitinated core histone H2A. One-dimensional hybridization patterns suggest that -AAGAG- nucleosomal DNA is rapidly trimmed to a shorter DNA length than either bulk or -AATAT- nucleosomes.     

X-Ray Structure of the Nucleosome Core Particle

Abstract

Two monoclinic crystal forms (P2₁,C2) of chicken erythrocyte nucleosomes have been under study in this laboratory. The x-ray structure of the P2₁ crystal form has been solved to 15 Å resolution. The B-DNA superhelix has a relatively uniform curvature, with only several local distortions observed in the superhelix. The individual histone domains have been localized and specific contacts between each histone and the DNA can be observed. Histone contacts to the inner surface of the DNA superhelix occur predominantly at the minor groove sites. Most of the histone core is contained within the inner surface of the superhelical DNA, except for part of H2A which extends between the DNA gyres near the terminus of the DNA. No part of H2A blocks the DNA terminus or would prevent a smooth exit of the DNA into the linker region. A similar extension of a portion of histone H4 between the DNA gyres occurs close to the dyad axis. Both unique nucleosomes in the P2₁ asymmetric unit demonstrate good dyad symmetry and are similar to each other throughout the histone core and DNA regions.     

The core histone tail domains contribute to sequence-dependent nucleosome positioning

The precise positioning of nucleosomes plays a critical role in the regulation of gene expression by modulating the DNA binding activity of trans-acting factors. However, molecular determinants responsible for positioning are not well understood. We examined whether the removal of the core histone tail domains from nucleosomes reconstituted with specific DNA fragments led to alteration of translational positions. Remarkably, we find that removal of tail domains from a nucleosome assembled on a DNA fragment containing a Xenopus borealis somatic-type 5S RNA gene results in repositioning of nucleosomes along the DNA, including two related major translational positions that move about 20 bp further upstream with respect to the 5S gene. In a nucleosome reconstituted with a DNA fragment containing the promoter of a Drosophila alcohol dehydrogenase gene, several translational positions shifted by about 10 bp along the DNA upon tail removal. However, the positions of nucleosomes assembled with a DNA fragment known to have one of the highest binding affinities for core histone proteins in the mouse genome were not altered by removal of core histone tail domains. Our data support the notion that the basic tail domains bind to nucleosomal DNA and influence the selection of the translational position of nucleosomes and that once tails are removed movement between translational positions occurs in a facile manner on some sequences. However, the effect of the N-terminal tails on the positioning and movement of a nucleosome appears to be dependent on the DNA sequence such that the contribution of the tails can be masked by very high affinity DNA sequences. Our results suggest a mechanism whereby sequence-dependent nucleosome positioning can be specifically altered by regulated changes in histone tail-DNA interactions in chromatin.     

X-ray structure of the nucleosome core particle

Two monoclinic crystal forms (P2(1),C2) of chicken erythrocyte nucleosomes have been under study in this laboratory. The x-ray structure of the P2(1) crystal form has been solved to 15 A resolution. The B-DNA superhelix has a relatively uniform curvature, with only several local distortions observed in the superhelix. The individual histone domains have been localized and specific contacts between each histone and the DNA can be observed. Histone contacts to the inner surface of the DNA superhelix occur predominantly at the minor groove sites. Most of the histone core is contained within the inner surface of the superhelical DNA, except for part of H2A which extends between the DNA gyres near the terminus of the DNA. No part of H2A blocks the DNA terminus or would prevent a smooth exit of the DNA into the linker region. A similar extension of a portion of histone H4 between the DNA gyres occurs close to the dyad axis. Both unique nucleosomes in the P2(1) asymmetric unit demonstrate good dyad symmetry and are similar to each other throughout the histone core and DNA regions.     

Nucleosomes facilitate their own invasion

DNA wrapped in nucleosomes is sterically occluded, creating obstacles for polymerase, regulatory, remodeling, repair and recombination complexes, which require access to the wrapped DNA. How such complexes recognize and gain access to their DNA target sites is not known. Here we report the direct detection of a dynamic equilibrium conformational transition in nucleosomes that greatly increases the distance between the end of the nucleosomal DNA and the histone core. We quantified the equilibrium constant for this transition under physiological conditions. As predicted by these findings, addition of LexA protein to nucleosomes containing the LexA target site drives this conformational equilibrium toward the unwrapped, accessible state, simultaneously allowing stable LexA binding. This inherent property of nucleosomes allows any protein, whether an energy-dependent machine or a passive binder, to gain access even to buried stretches of nucleosomal DNA.     

Synthesis and nucleosome structure of DNA containing a UV photoproduct at a specific site.

A strategy was developed to assemble nucleosomes specifically damaged at only one site and one structural orientation. The most prevalent UV photoproduct, a cis-syn cyclobutane thymine dimer (cs CTD), was chemically synthesized and incorporated into a 30 base oligonucleotide harboring the glucocorticoid hormone response element. This oligonucleotide was assembled into a 165 base pair double stranded DNA molecule with nucleosome positioning elements on each side of the cs CTD-containing insert. Proton NMR verified that the synthetic photoproduct is the cis-syn stereoisomer of the CTD. Moreover, two different pyrimidine dimer-specific endonucleases cut approximately 90% of the dsDNA molecules. This cleavage is completely reversed by photoreactivation with E. coli UV photolyase, further demonstrating the correct stereochemistry of the photoproduct. Nucleosomes were reconstituted by histone octamer exchange from chicken erythocyte core particles, and contained a unique translational and rotational setting of the insert on the histone surface. Hydroxyl radical footprinting demonstrates that the minor groove at the cs CTD is positioned away from the histone surface about 5 bases from the nucleosome dyad. Competitive gel-shift analysis indicates there is a small increase in histone binding energy required for the damaged fragment (DeltaDeltaG approximately 0.15 kcal/mol), which does not prevent complete nucleosome loading under our conditions. Finally, folding of the synthetic DNA into nucleosomes dramatically inhibits cleavage at the cs CTD by T4 endonuclease V and photoreversal by UV photolyase. Thus, specifically damaged nucleosomes can be experimentally designed for in vitro DNA repair studies.     

In vitro selection of DNAs with an increased propensity to form small circles

A protocol was devised to select for DNA molecules that efficiently form circles from a library of 126 base pair DNAs containing 90 randomized base pairs. After six rounds of selection, individual molecules from the library showed 20‐ to 100‐fold greater j‐factors compared with the starting library, validating the selection protocol. High‐throughput sequencing revealed a sinusoidal pattern of enrichment and de‐enrichment of A/T dinucleotides in the random region with a 10.4 base pair period associated with the helicity of DNA. A similar, but more moderate pattern of C/G dinucleotides was offset by precisely half a helical turn. While C/G dinucleotide enrichments were evenly distributed, A/T dinucleotide enrichments displayed a preference to cluster in individual DNA molecules. The most highly enriched 10 base pair sequences in the random region contained adjacent blocks of A/T and C/G trinucleotides present in some, but not all, rapidly cyclizing molecules. The phased dinucleotide enrichments closely match those present in accurately mapped yeast nucleosomes, confirming the importance of DNA bending in nucleosome formation. However, at certain sites the nucleosomal DNAs show dinucleotide enrichments that differ substantially from the cyclization data. These discrepancies can often be correlated with sequence specific contacts that form between histones and DNA. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 303–320, 2015.     

Eight different highly specific nucleosome phases on alpha-satellite DNA in the African green monkey.

The question of nucleosome phasing on African Green Monkey (AGM) alpha-satellite DNA has been addressed by employing a new approach. Nucleosome cores were prepared from AGM nuclei with micrococcal nuclease, exonuclease III and nuclease S1. The core DNA population derived from alpha-satellite DNA containing chromatin was purified from total core DNA by denaturation of the DNA, reassociation to a low Cot value, and hydroxyapatite chromatography to separate the renatured satellite fraction. After end-labeling the termini of the alpha-satellite containing core DNA fragments were mapped by high resolution gel electrophoresis relative to known restriction sites along the 172 bp repeat unit of the satellite DNA. The results show that nucleosomes occupy eight strictly defined positions on the alpha-satellite DNA which could be determined with an accuracy of +/- 1 base pair. Approximately 35% of all nucleosomes are organized in one of these frames while the other seven registers contribute about 10% each.     

Theoretical aspects of extinction by inbreeding depression

Populational extinction due to inbreeding depression is analyzed with simple population genetic and population ecological models. Two alternative genetic mechanisms of inbreeding depression, i.e. recessive deleterious genes and overdominant genes, are assumed in separate analyses in order to examine their relative importance. With both mechanisms the population size and the coefficient of inbreeding are maintained at stable equilibria if there is no non-genetic demographic disturbance or stress. With a certain amount of demographic disturbance the population declines rapidly due to interaction between the decrease of population size and the increase of inbreeding coefficient. Such rapid extinction occurs with both genetic mechanisms. However, in the case of overdominant genes extinction happens only if the equilibrium population size is small and the selection coefficient is large such that segregation load is large. In nature, extinction due to overdominant genes is considered to be much less likely than extinction due to recessive deleterious genes.     

The divalent cations Ca2+ and Mg2+ play specific roles in stabilizing histone-DNA interactions within nucleosomes that are partially redundant with the core histone tail domains

We previously reported that reconstituted nucleosomes undergo sequence-dependent translational repositioning upon removal of the core histone tail domains under physiological conditions, indicating that the tails influence the choice of position. We report here that removal of the core histone tail domains increases the exposure of the DNA backbone in nucleosomes to hydroxyl radicals, a nonbiased chemical cleavage reagent, indicative of an increase in the motility of the DNA on the histone surface. Moreover, we demonstrate that the divalent cations Mg(2+) and Ca(2+) can replace the role of the tail domains with regard to stabilization of histone-DNA interactions within the nucleosome core and restrict repositioning of nucleosomes upon tail removal. However, when nucleosomes were incubated with Mg(2+) after tail removal, the original distribution of translational positions was not re-established, indicating that divalent cations increase the energy barrier between translational positions rather than altering the free energy differences between positions. Interestingly, other divalent cations such as Zn(2+), Fe(2+), Co(2+), and Mn(2+) had little or no effect on the stability of histone-DNA interactions within tailless nucleosomes. These results support the idea that specific binding sites for Mg(2+) and Ca(2+) ions exist within the nucleosome and play a critical role in nucleosome stability that is partially redundant with the core histone tail domains.     

Interactions between core histones and chromatin at physiological ionic strength

Addition of core histones to chromatin or chromatin core particles at physiological ionic strength results in soluble nucleohistone complexes when polyglutamic acid is included in the sample. The interaction between nucleosomes and added core histones is strong enough to inhibit nucleosome formation on a closed circular DNA in the same solution. Complexes consisting of core particles and core histones run as discrete nucleoprotein particles on polyacrylamide gels. Consistent with the electrophoretic properties of these particles, protein cross-linking with dimethyl suberimidate indicates that added core histones are bound as excess octamers. Histones in the excess octamers do not exchange with nucleosomal core histones at an ionic strength of 0.1 M and can be selectively removed from core particles by incubating the complexes in a solution containing sufficient DNA. Under conditions where added histones are confined to the surface of chromatin, the excess histones are mobile and can migrate onto a contiguous extension of naked DNA and form nucleosomes.     

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.