A statistical thermodynamic model applied to experimental AFM population and location data is able to quantify DNA-histone binding strength and internucleosomal interaction differences between acetylated and unacetylated nucleosomal arrays

Imaging of nucleosomal arrays by atomic force microscopy allows a determination of the exact statistical distributions for the numbers of nucleosomes per array and the locations of nucleosomes on the arrays. This precision makes such data an excellent reference for testing models of nucleosome occupation on multisite DNA templates. The approach presented here uses a simple statistical thermodynamic model to calculate theoretical population and positional distributions and compares them to experimental distributions previously determined for 5S rDNA nucleosomal arrays (208-12,172-12). The model considers the possible locations of nucleosomes on the template, and takes as principal parameters an average free energy of interaction between histone octamers and DNA, and an average wrapping length of DNA around the octamers. Analysis of positional statistics shows that it is possible to consider interactions between nucleosomes and positioning effects as perturbations on a random positioning noninteracting model. Analysis of the population statistics is used to determine histone-DNA association constants and to test for differences in the free energies of nucleosome formation with different types of histone octamers, namely acetylated or unacetylated, and different DNA templates, namely 172-12 or 208-12 5S rDNA multisite templates. The results show that the two template DNAs bind histones with similar affinities but histone acetylation weakens the association of histones with both templates. Analysis of locational statistics is used to determine the strength of specific nucleosome positioning tendencies by the DNA templates, and the strength of the interactions between neighboring nucleosomes. The results show only weak positioning tendencies and that unacetylated nucleosomes interact much more strongly with one another than acetylated nucleosomes; in fact acetylation appears to induce a small anticooperative occupation effect between neighboring nucleosomes.     

DNA distortion as a factor in nucleosome positioning.

In a previous report we constructed a synthetic DNA sequence that directed the deposition of histone octamers to a single site, and it was proposed that DNA distortion was involved in the positioning effect. In the present study we utilized the chemical probe potassium permanganate to identify sites of DNA distortion in the synthetic positioning sequence. A permanganate hypersite was identified 15 bp from the nucleosome pseudo-dyad at a site known to display DNA distortion in the mature nucleosome. The sequence of the site contained a TA step flanked by an oligo-pyrimidine tract. A series of substitutions were made in the region of the permanganate hypersite and the resulting constructs tested for affinity for histone octamers and translational positioning in in vitro studies. The results revealed that either a single base substitution at the TA step or in the adjacent homopolymeric tract dramatically affected affinity and positioning activity. The rotational orientation of the permanganate-sensitive sequence was shown to be important for functions, since altering the orientation of the site in a positioning fragment reduced positioning activity and octamer affinity, while altering the rotational orientation of the sequence in a non-positioning fragment had the opposite effects. A reconstituted 5 S rDNA positioning sequence from Lytechinus variegatus was also shown to display a permanganate hypersite 16 bp from its pseudo-dyad.     

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.     

Attraction, phasing and neighbour effects of histone octamers on curved DNA

Nucleosome core particles were reconstituted on various DNA fragments containing a Crithidia fasciculata kinetoplast curved tract. The results show that, on curved DNA, nucleosome core particles form six- to sevenfold preferentially, relative to bulk sequences. The preferential deposition occurs at multiple periodic positions, whose distribution reveals a unique rotational setting of DNA with respect to the histone octamer surface and whose average periodicity is 10.26 +/- 0.04. Evidence is provided for a context effect in histone octamer deposition: octamers bound to a segment of curved DNA influence the positions of neighbour octamers. Taken together, the preferential formation of nucleosome core particles and the influence on the localization of neighbouring particles suggest for intrinsically bent sequences the biologically relevant role of organizers of nucleosomal arrays.     

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.     

Isw1a does not have strict limitations on the length of extranucleosomal DNAs for mobilization of nucleosomes assembled with HeLa cell histones

The Saccharomyces cerevisiae Isw1a and Isw2 ATP-dependent chromatin-remodeling complexes have important roles in vivo in the regulation of nucleosome positioning and modulation of gene activity. We studied the ability of the Isw1a- and Isw2-remodeling enzymes to reposition nucleosomes in mono- and dinucleosomes templates with variably positioned histone octamers (in the center or at the ends of the DNA fragment). To compare the Isw1a and Isw2 nucleosome-mobilizing activities, we utilized mono- and dinucleosome templates reconstituted with purified HeLa cell histones and DNA containing one or two copies of the “601” nucleosome high-affinity sequence used to specifically position nucleosomes on the DNA. The obtained data suggest that Isw1a is able to mobilize HeLa cell histone-assembled mononucleosomes with long (more than 30?bp) extranucleosomal DNAs protruding from both sides, which contrasts to the previously reported inability of Isw1 to mobilize similar nucleosomes assembled with recombinant yeast histones. The results also suggest that Isw1a and Isw2 can mobilize nucleosomes with unfavorably short linker DNA lengths, and the presence of internucleosomal interactions promotes mobilization of nucleosomes even when the linkers are short.     

Human centromere protein B induces translational positioning of nucleosomes on alpha-satellite sequences

The human centromere proteins A (CENP-A) and B (CENP-B) are the fundamental centromere components of chromosomes. CENP-A is the centromere-specific histone H3 variant, and CENP-B specifically binds a 17-base pair sequence (the CENP-B box), which appears within every other alpha-satellite DNA repeat. In the present study, we demonstrated centromere-specific nucleosome formation in vitro with recombinant proteins, including histones H2A, H2B, H4, CENP-A, and the DNA-binding domain of CENP-B. The CENP-A nucleosome wraps 147 base pairs of the alpha-satellite sequence within its nucleosome core particle, like the canonical H3 nucleosome. Surprisingly, CENP-B binds to nucleosomal DNA when the CENP-B box is wrapped within the nucleosome core particle and induces translational positioning of the nucleosome without affecting its rotational setting. This CENP-B-induced translational positioning only occurs when the CENP-B box sequence is settled in the proper rotational setting with respect to the histone octamer surface. Therefore, CENP-B may be a determinant for translational positioning of the centromere-specific nucleosomes through its binding to the nucleosomal CENP-B box.     

Nucleosome positioning as a critical determinant for the DNA cleavage sites of mammalian DNA topoisomerase II in reconstituted simian virus 40 chromatin.

We have assessed the ability of nucleosomes to influence the formation of mammalian topoisomerase II-DNA complexes by mapping the sites of cleavage induced by four unrelated topoisomerase II inhibitors in naked versus nucleosome-reconstituted SV40 DNA. DNA fragments were reconstituted with histone octamers from HeLa cells by the histone exchange method. Nucleosome positions were determined by comparing micrococcal nuclease cleavage patterns of nucleosome-reconstituted and naked DNA. Three types of DNA regions were defined: 1) regions with fixed nucleosome positioning; 2) regions lacking regular nucleosome phasing; and 3) a region around the replication origin (from position 5100 to 600) with no detectable nucleosomes. Topoisomerase II cleavage sites were suppressed in nucleosomes and persisted or were enhanced in linker DNA and in the nucleosome-free region around the replication origin. Incubation of reconstituted chromatin with topoisomerase II protected nucleosome-free regions from micrococcal nuclease cleavage without changing the overall micrococcal nuclease cleavage pattern. Thus, the present results indicate that topoisomerase II binds preferentially to nucleosome-free DNA and that the presence of nucleosomes at preferred DNA sequences influences drug-induced DNA breaks by topoisomerase II inhibitors.     

Structural features of a regulatory nucleosome

DNA sequences from the long terminal repeat of the mouse mammary tumor virus (MMTV-LTR) position nucleosomes both in vivo and in vitro. Here, were present chromatin reconstitution experiments showing that MMTV-LTR sequences from -236 to +204 accommodate two histone octamers in positions compatible with the in vivo data. This positioning is not influenced by the length of the DNA fragment and occurs in linear as well as in closed circular DNA molecules. MMTV-LTR DNA sequences show an intrinsic bendability that closely resembles its wrapping around the histone octamer. We propose that bendability is responsible for the observed rotational nucleosome positioning. Translational nucleosome positioning seems also to be determined by the DNA sequence. These data, along with the results from reconstitution experiments with insertion mutants, support a modular model of nucleosome phasing on MMTV-LTR, where the actual positioning of the histone octamer results from the additive effect of multiple features of the DNA sequence.     

Effects of DNA sequence and histone-histone interactions on nucleosome placement

Using competitive reconstitution, we have refined the parameters for the binding of histone octamers to artificial nucleosome-positioning sequences of the form: (A/T3nn(G/C)3nn. We find that the optimal period between flexible segments is approximately 10.1 base-pairs, supporting the view that the DNA on the nucleosome surface is overwound. The strongest requirement for flexible DNA is near the protein dyad. However, we see no indication of changes in DNA helical repeat in this region. Using a series of repetitive sequences, we confirm that neither all A/T-rich nor all G/C-rich regions are identical in promoting nucleosome formation. Surprisingly, A/T-rich segments containing the TpA step, subject to purine-purine clash in the minor groove, favor nucleosome formation over sequences lacking this step. Short tracts of adenine residues are found to position on the histone surface like other A/T-rich regions, in the manner predicted by the direction of their sequence-directed bends as determined by electrophoretic methods. Tracts containing five adenine residues are extremely aniostropic in their flexibility and are strongly detrimental to nucleosome formation when positioned for major groove compression. Longer adenine tracts are found to position near the ends of the nucleosomal DNA. However, other positions may be occupied by an A12 tract, with only a minor penalty in the free energy of nucleosome formation. Overall, reconstituted nucleosome positions are translationally degenerate, suggesting a weak dependence on DNA flexibility for nucleosome positioning. Dinucleosomal reconstitutions on tandem dimers of the 5 S RNA gene of Lytechinus variegatus demonstrate a weak phasing dependence for the interaction between nucleosomes. This interaction is maximal for the 202 base-pair repeat and suggests a co-operative mechanism for the formation of ordered nucleosomal arrays based on a combination of DNA flexibility and nucleosome-nucleosome interactions.     

A histone octamer blocks branch migration of a Holliday junction.

The Holliday junction is a key intermediate in genetic recombination. Here, we examine the effect of a nucleosome core on movement of the Holliday junction in vitro by spontaneous branch migration. Histone octamers consisting of H2A, H2B, H3, and H4 are reconstituted onto DNA duplexes containing an artificial nucleosome-positioning sequence consisting of a tandem array of an alternating AT-GC sequence motif. Characterization of the reconstituted branch migration substrates by micrococcal nuclease mapping and exonuclease III and hydroxyl radical footprinting reveal that 70% of the reconstituted octamers are positioned near the center of the substrate and the remaining 30% are located at the distal end, although in both cases some translational degeneracy is observed. Branch migration assays with the octamer-containing substrates reveal that the Holliday junction cannot migrate spontaneously through DNA organized into a nucleosomal core unless DNA-histone interactions are completely disrupted. Similar results are obtained with branch migration substrates containing an octamer positioned on a naturally occurring sequence derived from the yeast GLN3 locus. Digestion of Holliday junctions with T7 endonuclease I establishes that the junction is not trapped by the octamer but can branch migrate in regions free of histone octamers. Our findings suggest that migration of Holliday junctions during recombination and the recombinational repair of DNA damage requires proteins not only to accelerate the intrinsic rate of branch migration but also to facilitate the passage of the Holliday junction through a nucleosome.     

Unique positioning of reconstituted nucleosomes occurs in one region of simian virus 40 DNA

A direct end label method was used to study the positioning of nucleosome arrays on several long (greater than 2200 base pairs) SV40 DNA fragments reconstituted in vitro with core histones. Comparison of micrococcal nuclease cutting sites in reconstituted and naked DNA fragments revealed substantial differences in one DNA region. When sufficient core histones were annealed with the DNA to form closely spaced nucleosomes over most of the molecule, a uniquely positioned array of four nucleosomes could be assigned, by strict criteria, to a 610-base pair portion of the SV40 "late region," with a precision of about +/- 20 base pairs. In some other DNA regions, a number of alternative nucleosome positions were indicated. The uniquely positioned four-nucleosome array spanned the same 610 nucleotides on two different DNA fragments that possessed different ends. Removal of a DNA region that had contained a terminal nucleosome of the array, by truncation of the fragment before reconstitution, did not affect the positioning of the other three nucleosomes. As the core histone to DNA ratio was lowered, evidence for specific positioning of nucleosomes diminished, except within the region where the four uniquely positioned nucleosomes formed. This region, however, does not appear to have a higher affinity for core histones than other regions of the DNA.     

Nucleosome movement by CHRAC and ISWI without disruption or trans-displacement of the histone octamer.

The chromatin accessibility complex (CHRAC) belongs to the class of nucleosome remodeling factors that increase the accessibility of nucleosomal DNA in an ATP-dependent manner. We found that CHRAC induces movements of intact histone octamers to neighboring DNA segments without facilitating their displacement to competing DNA or histone chaperones in trans. CHRAC-induced energy-dependent nucleosome sliding may, in principle, explain nucleosome remodeling, nucleosome positioning, and nucleosome spacing reactions known to be catalyzed by CHRAC. The catalytic core of CHRAC, the ATPase ISWI, also mobilized nucleosomes at the expense of energy. However, the directionality of the CHRAC- and ISWI-induced nucleosome movements differed drastically, indicating that the geometry of the native complex modulates the activity of its catalytic core.     

Archaeal nucleosome positioning sequence from Methanothermus fervidus.

DNA in Methanothermus fervidus, a hyperthermophilic archaeon, is constrained into archaeal nucleosomes in vivo by the archaeal histones HMfA and HMfB. Here, we document the translational and rotational positioning of archaeal nucleosome assembly in vitro by a sequence from the 7S RNA encoding region of the M. fervidus genome. The minor groove of the DNA at the center of the DNA sequence, protected from micrococcal nuclease digestion by incorporation into a positioned archaeal nucleosome, faces away from the archaeal histone core.     

Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. Here we describe the protocols necessary to reconstitute milligram quantities of length and compositionally defined nucleosomal arrays suitable for biochemical and biophysical studies of chromatin structure and function.     

Structural analysis of a reconstituted DNA containing three histone octamers and histone H5

Previous work has shown that DNA and the histone proteins will combine to form structures of a complex, yet definite nature. Here, we describe three experiments aimed at a better understanding of the interactions of DNA with the histone octamer and with histone H5. First, there has been some question as to whether the methylation of DNA could influence its folding about the histone octamer. To address this point, we reconstituted the histone octamer onto a 440 base-pair DNA of defined sequence at various levels of cytosine methylation, and also onto the unmethylated DNA. The reconstituted structures were probed by digestion with two different enzymes, micrococcal nuclease and DNase I. All samples were found to contain what appear to be three histone octamers, bound in close proximity on the 440 base-pair DNA. The cutting patterns of micrococcal nuclease and DNase I remain the same in all cases, even if the DNA has been extensively methylated. The results show, therefore, that methylation has little, or no, influence on the folding of this particular DNA about the histone octamer. Second, there has been concern as to whether the base sequence of DNA could determine its folding in a long molecule containing several nucleosomes, just as it does within any single, isolated nucleosome core. In order to deal with this problem, we cut the 440 base-pair DNA into three short fragments, each of nucleosomal length; we reconstituted each separately with the histone octamer; and then we digested the reconstituted complexes with DNase I for comparison with similar data from the intact 440 base-pair molecule. The results show that the folding of this DNA is influenced strongly by its base sequence, both in the three short fragments and in the long molecule. The rotational setting of the DNA within each of the three short fragments is as predicted from a computer algorithm, which measures its homology to 177 known examples of nucleosome core DNA. The rotational setting of the DNA in the 440 base-pair molecule remains the same as in two of the three short fragments, but changes slightly in a third case, apparently because of steric requirements when the nucleosomes pack closely against one another. Finally, there has been little direct evidence of where histone H5 binds within a DNA-octamer complex.(ABSTRACT TRUNCATED AT 400 WORDS)