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.     

Biophysical characterization of the centromere-specific nucleosome from budding yeast

The centromeric DNA of all eukaryotes is assembled upon a specialized nucleosome containing a histone H3 variant known as CenH3. Despite the importance and conserved nature of this protein, the characteristics of the centromeric nucleosome are still poorly understood. In particular, the stoichiometry and DNA-binding properties of the CenH3 nucleosome have been the subject of some debate. We have characterized the budding yeast centromeric nucleosome by biochemical and biophysical methods and show that it forms a stable octamer containing two copies of the Cse4 protein and wraps DNA in a left-handed supercoil, similar to the canonical H3 nucleosome. The DNA-binding properties of the recombinant nucleosome are identical to those observed in vivo demonstrating that the octameric structure is physiologically relevant.     

DNA methylation, nucleosome formation and positioning.

Recent mapping of nucleosome positioning on several long gene regions subject to DNA methylation has identified instances of nucleosome repositioning by this base modification. The evidence for an effect of CpG methylation on nucleosome formation and positioning in chromatin is reviewed here in the context of the complex sequence-structure requirements of DNA wrapping around the histone octamer and the role of this epigenetic mark in gene repression.     

Generalized electrostatic model of the wrapping of DNA around oppositely charged proteins

Histonelike proteins in prokaryotes and histone octamers in eukaryotes carry large positive charges, which are responsible of strong electrostatic interactions with DNA. As a result, DNA wraps around proteins and genetic information is condensed. We describe a generalized model of these electrostatic interactions mediated by salt that explains the wrapping of DNA around the nucleosome octamer, around remodeling factors in eukaryotes and around histonelike proteins in prokaryotes. It comes out that small changes in protein dimension and charge produce large effects in the supramolecular DNA-protein architecture.     

Activity of FEN1 endonuclease on nucleosome substrates is dependent upon DNA sequence but not flap orientation

We demonstrated previously that human FEN1 endonuclease, an enzyme involved in excising single-stranded DNA flaps that arise during Okazaki fragment processing and base excision repair, cleaves model flap substrates assembled into nucleosomes. Here we explore the effect of flap orientation with respect to the surface of the histone octamer on nucleosome structure and FEN1 activity in vitro. We find that orienting the flap substrate toward the histone octamer does not significantly alter the rotational orientation of two different nucleosome positioning sequences on the surface of the histone octamer but does cause minor perturbation of nucleosome structure. Surprisingly, flaps oriented toward the nucleosome surface are accessible to FEN1 cleavage in nucleosomes containing the Xenopus 5S positioning sequence. In contrast, neither flaps oriented toward nor away from the nucleosome surface are cleaved by the enzyme in nucleosomes containing the high-affinity 601 nucleosome positioning sequence. The data are consistent with a model in which sequence-dependent motility of DNA on the nucleosome is a major determinant of FEN1 activity. The implications of these findings for the activity of FEN1 in vivo are discussed.     

Breaths,Twists, and Turns of Atomistic Nucleosomes

Gene regulation programs establish cellular identity and rely on dynamic changes in the structural packaging of genomic DNA. The DNA is packaged in chromatin, which is formed from arrays of nucleosomes displaying different degree of compaction and different lengths of inter-nucleosomal linker DNA. The nucleosome represents the repetitive unit of chromatin and is formed by wrapping 145–147 basepairs of DNA around an octamer of histone proteins. Each of the four histones is present twice and has a structured core and intrinsically disordered terminal tails. Chromatin dynamics are triggered by inter- and intra-nucleosome motions that are controlled by the DNA sequence, the interactions between the histone core and the DNA, and the conformations, positions, and DNA interactions of the histone tails. Understanding chromatin dynamics requires studying all these features at the highest possible resolution. For this, molecular dynamics simulations can be used as a powerful complement or alternative to experimental approaches, from which it is often very challenging to characterize the structural features and atomic interactions controlling nucleosome motions. Molecular dynamics simulations can be performed at different resolutions, by coarse graining the molecular system with varying levels of details. Here we review the successes and the remaining challenges of the application of atomic resolution simulations to study the structure and dynamics of nucleosomes and their complexes with interacting partners.     

Sequence-specific positioning of core histones on an 860 base-pair DNA. Experiment and theory

Previous experiments have shown that the locations of the histone octamer on DNA molecules of 140 to 240 base-pairs (bp) are influenced strongly by the nucleotide sequence. Here we have studied the locations of the histone octamer on a relatively long DNA molecule of 860 bp, using two different nucleases, micrococcal and DNAase I. Data were obtained from both the protein--DNA complexes and from the naked DNA at single-bond resolution, and then were analyzed by densitometry to yield plots of differential cleavage, which show clearly the changes in cutting due to the addition of protein. Our results show that the placement of core histones on the 860 bp molecule is definitely non-random. The digestion data provide evidence for five nucleosome cores, the centers of which lie in defined locations. In all but one of these protein--DNA complexes, the DNA adopts a unique, highly preferred rotational setting with respect to the protein surface. Another protein--DNA complex is unusual in that it protects 200 bp from digestion, yet is cut in its very center as if it were split into two parts. The apparent average twist of the DNA within all of these protein--DNA complexes is 10.2(+/- 0.1) bp, as measured by the periodicity of DNAase I digestion. This value is in excellent agreement with the twist of 10.21(+/- 0.05) bp deduced from the periodicity of sequence content in chicken nucleosome core DNA. In addition, we observe a discontinuity in the periodic cutting by DNAase I of about -1 to -3 bonds in going from any nucleosome core to the next. The most plausible interpretation of this discontinuity is that it reflects the angle by which adjacent protein--DNA complexes are aligned. Thus, any nucleosome may be related to its neighbor by a left-handed rotation in space of -1/10.2 to -3/10.2 helix turns, or -35 degrees to -105 degrees. Repeated many times, this operation would build a long, left-handed helix of nucleosomes similar to that described by many workers for the packing of nucleosomes in chromatin. In order to look for any long-range influences on the positioning of the histone octamer in the 860 bp molecule (as would be expected if the nucleosomes have to fit into some higher-order structure), we have examined the locations of the histone octamer on five different isolated short fragments of the 860-mer, all of nucleosomal length.(ABSTRACT TRUNCATED AT 400 WORDS)     

Folding of single-stranded DNA on the histone octamer

A complex between the single-stranded DNA of the bacteriophage M13 and the histone octamer was analyzed by electron microscopy, low-angle X-ray diffraction and nuclease analysis. The morphology and the diffraction pattern of the complex strongly resemble those of the nucleosome. These results, as well as the finding of a protected DNA fragment about 100 nucleotides long following single-stranded DNA specific nuclease digestion, indicate that 'a nucleosome-like' complex can be formed between single-stranded DNA and the histone octamer. Competition experiments suggest that under physiological conditions the histone octamer is transferred from single- to double-stranded DNA.     

DNA rotational positioning in a regulatory nucleosome is determined by base sequence. An algorithm to model the preferred superhelix.

MMTV-LTR sequences -190/-45 position a histone octamer both in vivo and in vitro. Experimental evidence suggested that nucleosome rotational positioning is determined by the DNA sequence itself. We developed an algorithm that is able to predict the most favorable path of a given DNA sequence over a histone octamer, based on rotational preferences of different dinucleotides. Our analysis shows that these preferences are sufficient for explaining the observed rotational positioning of the MMTV-LTR nucleosome, at one base pair accuracy level. Computer-generated 3-D models of the experimentally calculated and predicted MMTV-LTR nucleosome show that the predicted orientation is fully compatible with the currently available data in terms of accessibility of relevant sequences to regulatory proteins.     

Influence of DNA topology and histone tails in nucleosome organization on pBR322 DNA.

Recently, we have found that the assembly of nucleosomes reconstituted on negatively supercoiled DNA is cooperative. In the present paper the role of DNA topology and of histone tails in nucleosome assembly was explored. Reconstituted minichromosomes on relaxed DNA at different histone/DNA ratios (R) were assayed by topological analysis and electron microscopy visualization. Both methods show a linear relationship between average nucleosome number (N) and R. This suggests that in the case of relaxed DNA, cooperative internucleosomal interactions are small or absent. The influence of histone tails in nucleosome assembly was studied on minichromosomes reconstituted with trypsinized histone octamer on negatively supercoiled DNA by topological analysis. The topoisomers distribution, after trypsinization, dramatically changes, indicating that nucleosome-nucleosome interactions are remarkably decreased. These results show that, in chromatin folding, in addition to the well known role of histone H1, the interactions between histone octamer tails and DNA are also of importance.     

Dissection of the unusual structural and functional properties of the variant H2A.Bbd nucleosome

The histone variant H2A.Bbd appeared to be associated with active chromatin, but how it functions is unknown. We have dissected the properties of nucleosome containing H2A.Bbd. Atomic force microscopy (AFM) and electron cryo-microscopy (cryo-EM) showed that the H2A.Bbd histone octamer organizes only approximately 130 bp of DNA, suggesting that 10 bp of each end of nucleosomal DNA are released from the octamer. In agreement with this, the entry/exit angle of the nucleosomal DNA ends formed an angle close to 180 degrees and the physico-chemical analysis pointed to a lower stability of the variant particle. Reconstitution of nucleosomes with swapped-tail mutants demonstrated that the N-terminus of H2A.Bbd has no impact on the nucleosome properties. AFM, cryo-EM and chromatin remodeling experiments showed that the overall structure and stability of the particle, but not its property to interfere with the SWI/SNF induced remodeling, were determined to a considerable extent by the H2A.Bbd docking domain. These data show that the whole H2A.Bbd histone fold domain is responsible for the unusual properties of the H2A.Bbd nucleosome.     

Relative affinities of DNA sequences for the histone octamer depend strongly upon both the temperature and octamer concentration

Using a novel competition assay to determine the relative strength of different histone octamer-binding sites, we have compared three natural and two synthetic sites. We show that the relative affinities of these sites for the histone octamer depend upon both the temperature and octamer concentration. In particular, under certain conditions, a natural octamer-binding site from a yeast promoter outcompetes a synthetic sequence of comparable affinity to the strongest previously described positioning sequence. Under other conditions, this synthetic sequence is the preferred octamer ligand. We infer that sequence selection by the histone octamer depends strongly upon both the sequence-dependent anisotropy of DNA bending and on DNA deformability and that these parameters may contribute differently to nucleosome formation. These findings indicate that previous studies designed to identify strong octamer-binding sites may fail to select some natural strong binding sites.     

Model of the packing of DNA and histone octamer in the structure of the nucleosome

A model is proposed which describes the packing of polypeptide chains of histone molecules in the octamer (H3--H4--H2A--H2B)2, and interlocation of DNA and octamer in the nucleosome. DNA packing in the nucleosome is provided for by electrostatic interactions between DNA phosphates and cationic groups located on the globular part surface of histones octamer. The cationic groups of N- and C-end regions of the histone molecules (histones H3 and H4 in particular) additionally stabilize the nucleosome structure.     

Sequence-specific recognition of DNA in the nucleosome by pyrrole-imidazole polyamides

The ability of DNA-binding proteins to recognize their cognate sites in chromatin is restricted by the structure and dynamics of nucleosomal DNA, and by the translational and rotational positioning of the histone octamer. Here, we use six different pyrrole-imidazole polyamides as sequence-specific molecular probes for DNA accessibility in nucleosomes. We show that sites on nucleosomal DNA facing away from the histone octamer, or even partially facing the histone octamer, are fully accessible and that nucleosomes remain fully folded upon ligand binding. Polyamides only failed to bind where sites are completely blocked by interactions with the histone octamer. Removal of the amino-terminal tails of either histone H3 or histone H4 allowed these polyamides to bind. These results demonstrate that much of the DNA in the nucleosome is freely accessible for molecular recognition in the minor groove, and also support a role for the amino-terminal tails of H3 and H4 in modulating accessibility of nucleosomal DNA.     

The mechanics behind DNA sequence-dependent properties of the nucleosome

Chromatin organization and composition impart sophisticated regulatory features critical to eukaryotic genomic function. Although DNA sequence-dependent histone octamer binding is important for nucleosome activity, many aspects of this phenomenon have remained elusive. We studied nucleosome structure and stability with diverse DNA sequences, including Widom 601 derivatives with the highest known octamer affinities, to establish a simple model behind the mechanics of sequence dependency. This uncovers the unique but unexpected role of TA dinucleotides and a propensity for G|C-rich sequence elements to conform energetically favourably at most locations around the histone octamer, which rationalizes G|C% as the most predictive factor for nucleosome occupancy in vivo. In addition, our findings reveal dominant constraints on double helix conformation by H3-H4 relative to H2A-H2B binding and DNA sequence context-dependency underlying nucleosome structure, positioning and stability. This provides a basis for improved prediction of nucleosomal properties and the design of tailored DNA constructs for chromatin investigations.     

The nature of forces stabilizing nucleosome structure. Dissociation of histone octamers from DNA

We have used the measurements of the histone fluorescence parameters to study the influence of the ionic strength on histone-DNA and histone-histone interactions in reconstructed nucleosomes. The ionic strength increase lead to the two-stage nucleosome dissociation. The dimer H2A-H2B dissociates at the first stage and the tetramer (H3-H4)2 at the second one. The dimer H2A-H2B dissociation from nucleosome is a two-stage process also. The ionic bonds between (H2A-H2B) histone dimer and DNA break at first and then the dissociation of dimer from histone tetramer (H3-H4)2 occurs. According to the proposed model the dissociation accompanying a nucleosome "swelling" and an increase of DNA curvature radius. It was shown that the energy of electrostatic interactions between histone dimer and DNA is sufficiently less than the energy of dimer-tetramer interaction. We propose that the nucleosome DNA ends interact with the dimer and tetramer simultaneously. The calculated number (approximately 30 divided by 40) of ionic bonds between DNA and histone octamer globular part practically coincides with the number of exposed cationic groups on the surface of octamer globular head. On this basis we have assumed that the spatial distribution of these groups is precisely determined, which explains the high evolutionary conservatism of the histone primary structure.     

Uracil DNA Glycosylase Activity on Nucleosomal DNA Depends on Rotational Orientation of Targets

The activity of uracil DNA glycosylases (UDGs), which recognize and excise uracil bases from DNA, has been well characterized on naked DNA substrates but less is known about activity in chromatin. We therefore prepared a set of model nucleosome substrates in which single thymidine residues were replaced with uracil at specific locations and a second set of nucleosomes in which uracils were randomly substituted for all thymidines. We found that UDG efficiently removes uracil from internal locations in the nucleosome where the DNA backbone is oriented away from the surface of the histone octamer, without significant disruption of histone-DNA interactions. However, uracils at sites oriented toward the histone octamer surface were excised at much slower rates, consistent with a mechanism requiring spontaneous DNA unwrapping from the nucleosome. In contrast to the nucleosome core, UDG activity on DNA outside the core DNA region was similar to that of naked DNA. Association of linker histone reduced activity of UDG at selected sites near where the globular domain of H1 is proposed to bind to the nucleosome as well as within the extra-core DNA. Our results indicate that some sites within the nucleosome core and the extra-core (linker) DNA regions represent hot spots for repair that could influence critical biological processes.     

The translational placement of nucleosome cores in vitro determines the access of the transacting factor suGF1 to DNA.

The sea urchin G-string binding factor (suGF1) is one of several proteins that bind sequence-specifically to oligo(dGxdC) motifs, frequently present upstream of eukaryotic genes. In this study we investigate the interaction of suGF1, purified to near homogeneity, with its oligo(dGxdC) binding site in a reconstituted nucleosome core in vitro. We show that the in vitro reconstitution of a 214 bp fragment containing a suGF1 binding site results in the appearance of five distinct nucleosome core species. These species contain the histone octamer in an identical rotational setting but in different translational frames. The resulting different nucleosomal locations of the suGF1 binding site in the five core species are shown to modulate the ability of suGF1 to bind to nucleosomal DNA, even though the rotational setting of the DNA in the nucleosome cores maximally exposes the suGF1 binding site. We propose that a direct protein-protein steric clash between suGF1 and the histone octamer is the most likely determinant in modulating the binding of suGF1 to its nucleosomally wrapped binding site. This result suggests that in vivo suGF1, like TBP, NF1 and heat shock factor, may require a complementary nucleosome disrupting activity or that suGF1 binds to free nascent replicated DNA prior to nucleosome deposition.     

Working the kinks out of nucleosomal DNA

Condensation of DNA in the nucleosome takes advantage of its double-helical architecture. The DNA deforms at sites where the base pairs face the histone octamer. The largest so-called kink-and-slide deformations occur in the vicinity of arginines that penetrate the minor groove. Nucleosome structures formed from the 601 positioning sequence differ subtly from those incorporating an AT-rich human α-satellite DNA. Restraints imposed by the histone arginines on the displacement of base pairs can modulate the sequence-dependent deformability of DNA and potentially contribute to the unique features of the different nucleosomes. Steric barriers mimicking constraints found in the nucleosome induce the simulated large-scale rearrangement of canonical B DNA to kink-and-slide states. The pathway to these states shows nonharmonic behavior consistent with bending profiles inferred from AFM measurements.