Flexibility of nucleosomal DNA

In this study transient electric birefringence (TEB) has been used to investigate the molecular flexibility of short fragments of DNA. Nucleosomal DNA always exhibits negative birefringence and Kerr behavior was observed up to high field strengths (6 KV/cm). The value of the Kerr constant is 3.5 10^?2 e.s.u.. Birefringence decays were single exponentials and a field dependence of the molecular orientational relaxation time τ was found: it is explained by an inherent flexibility of the DNA molecule. A 20 % decrease in the calculated length was observed with fields applied as low as 2 KV/cm. The results obtained at very low fields establish TEB as a method well suited to calculate accurate values for the length of small fragments of DNA: the τ value of 4.3 μsec corresponds to a DNA length of 660 Å.     

DNA repair-deficient premature aging models display accelerated epigenetic age

Several premature aging mouse models have been developed to study aging and identify interventions that can delay age-related diseases. Yet, it is still unclear whether these models truly recapitulate natural aging. Here, we analyzed DNA methylation in multiple tissues of four previously reported mouse models of premature aging (Ercc1, LAKI, Polg, and Xpg). We estimated DNA methylation (DNAm) age of these samples using the Horvath clock. The most pronounced increase in DNAm age could be observed in Ercc1 mice, a strain which exhibits a deficit in DNA nucleotide excision repair. Similarly, we detected an increase in epigenetic age in fibroblasts isolated from patients with progeroid syndromes associated with mutations in DNA excision repair genes. These findings highlight that mouse models with deficiencies in DNA repair, unlike other premature aging models, display accelerated epigenetic age, suggesting a strong connection between DNA damage and epigenetic dysregulation during aging.     

Geometry of the nucleosomal DNA superhelix

Nucleosome stability is largely an indirect measure of DNA sequence based on the material properties of DNA and the ability of a sequence to assume the required left-handed superhelical conformation. Here we focus attention only on the geometry of the superhelix and present two distinct mathematical expressions that rely on the DNA helical parameters (Shift, Slide, Rise, Tilt, Roll, Twist). One representation requires torsion for superhelix formation; the other requires shear. To compare these mathematical expressions to experimental data we develop a strategy for Fourier-filtering the helical parameters that identifies necessary and sufficient conditions to achieve a high-resolution model of the nucleosome superhelix. We apply this filtering strategy to 24 high-resolution structures of the nucleosome and demonstrate that all structures have a highly conserved distribution of Roll, Slide and Twist that involves two length scales. One length scale spans the entire length of nucleosomal DNA. The other is associated with the helix repeat. Our strategy also enables us to identify ground state or simple nucleosomes and altered nucleosome structures. These results form a basis for characterizing structural variations in the emerging family of nucleosome structures and a method for further developing structure-based models of nucleosome stability.     

Rapid spontaneous accessibility of nucleosomal DNA

DNA wrapped in nucleosomes is sterically occluded, creating obstacles for proteins that must bind it. How proteins gain access to DNA buried inside nucleosomes is not known. Here we report measurements of the rates of spontaneous nucleosome conformational changes in which a stretch of DNA transiently unwraps off the histone surface, starting from one end of the nucleosome, and then rewraps. The rates are rapid. Nucleosomal DNA remains fully wrapped for only approximately 250 ms before spontaneously unwrapping; unwrapped DNA rewraps within approximately 10-50 ms. Spontaneous unwrapping of nucleosomal DNA allows any protein rapid access even to buried stretches of the DNA. Our results explain how remodeling factors can be recruited to particular nucleosomes on a biologically relevant timescale, and they imply that the major impediment to entry of RNA polymerase into a nucleosome is rewrapping of nucleosomal DNA, not unwrapping.     

Structure and dynamics of nucleosomal DNA

The last five years have seen exciting advances in our understanding of the structure of the nucleosome core particle, the basic repeating unit in all eukaryotic chromatin. A picture emerges in which nucleosomal DNA, while distorted and compacted fivefold by tight interactions with the histone octamer core, is at the same time highly dynamic and adaptable. Here, we summarize the salient features from recent structural studies of nucleosome core particles (both published and unpublished) that concern the structure and dynamics of nucleosomal DNA, and the nature of protein-DNA interactions. Current mechanisms for chromatin remodeling and nucleosome sliding are discussed in light of new structural evidence. Finally, techniques to study nucleosome stability and ultimately dynamics are introduced.     

Chromatin remodeling through directional DNA translocation from an internal nucleosomal site

The RSC chromatin remodeler contains Sth1, an ATP-dependent DNA translocase. On DNA substrates, RSC/Sth1 tracks along one strand of the duplex with a 3' --> 5' polarity and a tracking requirement of one base, properties that may enable directional DNA translocation on nucleosomes. The binding of RSC or Sth1 elicits a DNase I-hypersensitive site approximately two DNA turns from the nucleosomal dyad, and the binding of Sth1 requires intact DNA at this location. Results with various nucleosome substrates suggest that RSC/Sth1 remains at a fixed position on the histone octamer and that Sth1 conducts directional DNA translocation from a location about two turns from the nucleosomal dyad, drawing in DNA from one side of the nucleosome and pumping it toward the other. These studies suggest that nucleosome mobilization involves directional DNA translocation initiating from a fixed internal site on the nucleosome.     

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.     

A tandem insertion vector for large-scale preparation of nucleosomal DNA

We developed a novel nucleosome DNA template vector, pWMD01, which is optimized for the large-scale preparation of nucleosomal DNA. By using restricted digestion by SapI or EarI within its multicloning site, multiple half-nucleosome DNA units can be introduced unidirectionally into the vector at each subcloning step. Through this method, we constructed a plasmid that has 18 tandem repeats of a half-nucleosome 90-bp DNA unit containing c-Myb-binding sites in two subcloning cycles. This method enables the rapid, large-scale preparation of nucleosomal DNA with crystallization-grade quality.     

Inner nuclear membrane protein LBR preferentially interacts with DNA secondary structures and nucleosomal linker

The lamin B receptor (LBR) is an integral protein of inner nuclear membrane whose nucleoplasmic amino-terminal domain contributes to the attachment of the membrane to chromatin. Here we analyzed the interactions of a recombinant GST protein containing the amino-terminal domain of the protein with in vitro reconstituted nucleosomes and short DNA fragments. Data show that the LBR amino-terminal domain (AT) binds linker DNA but does not interact with the nucleosome core. Titration and competition studies revealed that the interaction between LBR AT and DNA is saturable, of high affinity (K(D) approximately 4 nM), independent of DNA sequence, and enhanced by DNA curvature and supercoiling. In this respect, LBR amino-terminal domain binding to nucleosomes is similar to that of histone H1 and non histone proteins HMG1/2 which both bind preferentially to linker DNA and present a significant affinity for DNA secondary structures.     

A computational study of nucleosomal DNA flexibility

Molecular dynamics simulations of the nucleosome core particle and its isolated DNA free in solution are reported. The simulations are based on the implicit solvent methodology and provide insights into the nature of large-scale structural fluctuations and flexibility of the nucleosomal DNA. In addition to the kinked regions previously identified in the x-ray structure of the nucleosome, the simulations support the existence of a biochemically identified distorted region of the DNA. Comparison of computed relative free energies shows that formation of the kinks is associated with little, if any, energy cost relative to a smooth, ideal conformation of the DNA superhelix. Isolated nucleosomal DNA is found to be considerably more flexible than expected for a 147 bp stretch of DNA based on its canonical persistence length of 500 A. Notably, the significant bending of the DNA observed in our simulations occurs without breaking of Watson-Crick bonds. The computed relative stability of bent conformations is sensitive to the ionic strength of the solution in the physiological range; the sensitivity suggests possible experiments that might provide further insights into the structural origins of the unusual flexibility of the DNA.     

Chromatin-associated DNA endonucleases from xeroderma pigmentosum cells are defective in interaction with damaged nucleosomal DNA

The influence of nucleosome structure on the activity of 2 chromatin-associated DNA endonucleases, pIs 4.6 and 7.6, from normal human and xeroderma pigmentosum, complementation group A (XPA), lymphoblastoid cells was examined on DNA containing either psoralen monoadducts or cross-links. As substrate a reconstituted nucleosomal system was utilized consisting of a plasmid DNA and either core (H2A, H2B, H3, H4), or total (core plus H1) histones from normal or XPA cells. Both non-nucleosomal and nucleosomal DNA were treated with 8-methoxypsoralen (8-MOP) plus long-wavelength ultraviolet radiation (UVA), which produces monoadducts and DNA interstrand cross-links, and angelicin plus UVA, which produces monoadducts. Both normal endonucleases were over 2-fold more active on both types of psoralen-plus-UVA-damaged core nucleosomal DNA than on damaged non-nucleosomal DNA. Addition of histone H1 to the system reduced but did not abolish this increase. By contrast, neither XPA endonuclease showed any increase on psoralen-treated nucleosomal DNA, with or without histone H1. Mixing the normal with the XPA endonucleases led to complementation of the XPA defect. These results indicate that interaction of these endonucleases with chromatin is of critical importance and that it is at this level that a defect exists in XPA endonucleases.     

Distinct strategies to make nucleosomal DNA accessible

One hallmark of ATP-dependent remodeling complexes is the ability to make nucleosomal DNA accessible to regulatory factors. We have compared two prominent human ATP-dependent remodelers, BRG1 from the SWI/SNF family and SNF2h from the ISWI family, for their abilities to make a spectrum of nucleosomal sites accessible. By measuring rates of remodeling at seven different sites on a mononucleosome and at six different sites on the central nucleosome of a trinucleosome, we have found that BRG1 opens centrally located sites more than an order of magnitude better than SNF2h. We provide evidence that this capability of BRG1 is caused by its ability to create DNA loops on the surface of a nucleosome, even when that nucleosome is constrained by adjacent nucleosomes. This specialized ability to make central sites accessible should allow SWI/SNF family complexes to facilitate binding of nuclear factors in chromatin environments where adjacent nucleosomes might otherwise constrain mobility.     

Experimental evidence for asymmetrical shielding of nucleosomal DNA by histones.

Electron spin resonance study of Mn (II) binding to chromatin and derivatives, including core particles, shows that Mn (II) is a good probe for testing the overall electrostatic balance of the nucleoproteic complex as well as DNA accessibility. Experimental results are in good agreement with a recent model proposed (Mirzabekov A. D. and Rich A. (1979) Proc. Natl. Acad. Sci. USA 76, 1118-1121), for the core particle, in which an asymmetrical shielding of DNA by the protein core is assumed. Furthermore, it was found that the histone H1 hinders a number of charges on the linker DNA in a proportion equal to the net positive charge of the histone itself. This result is interpreted as due to a tighter interaction between the linker DNA and the core histones in the presence of histone H1.     

Gammaretroviral integration into nucleosomal target DNA in vivo

Some of the earliest studies of retroviral integration targeting reported that sites of gammaretroviral DNA integration were positively correlated with DNase I-hypersensitive sites in chromatin. This led to the suggestion that open chromatin was favorable for integration. More recent deep sequencing experiments confirmed that gammaretroviral integration sites and DNase I cleavage sites are associated in genome-wide surveys. Paradoxically, in vitro studies of integration show that nucleosomal DNA is actually favored over naked DNA, raising the question of whether integration target DNA in chromosomes is wrapped in nucleosomes or nucleosome free. In this study we examined gammaretroviral integration by infecting primary human CD4(+) T lymphocytes with a murine leukemia virus (MLV)-based retroviral vector or xenotropic murine leukemia virus-related virus (XMRV), and isolated 32,585 unique integration sites using ligation-mediated PCR and 454 pyrosequencing. CD4(+) T lymphocytes were chosen for study because of the particularly dense genome-wide mapping of chromatin features available for comparison. Analysis relative to predicted nucleosome positions showed that gammaretroviruses direct integration into outward-facing major grooves on nucleosome-wrapped DNA, similar to the integration pattern of HIV. Also, a suite of histone modifications correlated with gene activity are positively associated with integration by both MLV and XMRV. Thus, we conclude that favored integration near DNase I-hypersensitive sites does not imply that integration takes place exclusively in nucleosome-free regions.     

Sequence periodicity in nucleosomal DNA and intrinsic curvature

Background

Most eukaryotic DNA contained in the nucleus is packaged by wrapping DNA around histone octamers. Histones are ubiquitous and bind most regions of chromosomal DNA. In order to achieve smooth wrapping of the DNA around the histone octamer, the DNA duplex should be able to deform and should possess intrinsic curvature. The deformability of DNA is a result of the non-parallelness of base pair stacks. The stacking interaction between base pairs is sequence dependent. The higher the stacking energy the more rigid the DNA helix, thus it is natural to expect that sequences that are involved in wrapping around the histone octamer should be unstacked and possess intrinsic curvature. Intrinsic curvature has been shown to be dictated by the periodic recurrence of certain dinucleotides. Several genome-wide studies directed towards mapping of nucleosome positions have revealed periodicity associated with certain stretches of sequences. In the current study, these sequences have been analyzed with a view to understand their sequence-dependent structures.

Results

Higher order DNA structures and the distribution of molecular bend loci associated with 146 base nucleosome core DNA sequence from C. elegans and chicken have been analyzed using the theoretical model for DNA curvature. The curvature dispersion calculated by cyclically permuting the sequences revealed that the molecular bend loci were delocalized throughout the nucleosome core region and had varying degrees of intrinsic curvature.

Conclusions

The higher order structures associated with nucleosomes of C.elegans and chicken calculated from the sequences revealed heterogeneity with respect to the deviation of the DNA axis. The results points to the possibility of context dependent curvature of varying degrees to be associated with nucleosomal DNA.

     

Methylation of nucleosomal and nuclease sensitive DNA.

The proportion of cytosines methylated in the DNA of nucleosome oligomers and of core particles appears indistinguishable from that of total nuclear DNA from CHO cells. However the DNA in nucleoprotein which is initially released from nuclei by treatment with very low levels of micrococcal nuclease and the first 10% of material rendered acid soluble by treatment of nuclei with DNase I are enriched 2 fold in their content of 5 methylcytosine. (Cessation of hydrolysis by nuclease occurs concomitantly with precipitation of nucleosomal core particles).     

Superhelicity of nucleosomal DNA changes its double-helical repeat

Winding DNA in a superhelix can be considered a process consisting of two smooth deformations: bending and twisting. The extra twist angle introduced by winding DNA into the nucleosomal superhelix is calculated by means of the Crick formula to be -0.5 degrees per base pair (bp). This is equivalent to a change of -0.15 +/- 0.015 bp in the DNA double-helical repeat. Free DNA in solution is known to have a helical repeat of 10.55 +/- 0.1 bp. On the other hand, a weighted average of various estimates of the DNA repeat in the nucleosome is 10.38 +/- 0.02. The difference happens to be perfectly accounted for by the superhelicity of the nucleosomal DNA. This implies that the latter is essentially nonconstrained .     

Superhelicity of nucleosomal DNA changes its double-helical repeat

Winding DNA in a superhelix can be considered a process consisting of two smooth deformations: bending and twisting. The extra twist angle introduced by winding DNA into the nucleosomal superhelix is calculated by means of the Crick formula to be −0.5° per base pair (bp). This is equivalent to a change of −0.15±0.015 bp in the DNA double-helical repeat. Free DNA in solution is known to have a helical repeat of 10.55±0.1 bp. On the other hand, a weighted average of various estimates of the DNA repeat in the nucleosome is 10.38±0.02. The difference happens to be perfectly accounted for by the superhelicity of the nucleosomal DNA. This implies that the latter is essentially nonconstrained.