Nucleosome positioning on yeast plasmids is determined only by the internal signal of DNA sequence occupied by the nucleosome

The possible role of border factors in determining the nucleosome positioning on a DNA sequence was investigated. To this end a family of recombinant plasmids based on Gal10Cyc1 promoter and neomycin phosphotransferase gene NPTII were created. A DNA sequence adjoining the GalCyc promoter was varied in these plasmids. Three nearly equally represented nucleosome positions on the GalCyc promoter were found. In the basal plasmid an FRT sequence adjoins the GalCyc promoter at the right. It contains an internal signal of multiple positioning. Its replacement with different DNA sequences does not affect nucleosome positioning on the GalCyc promoter. The nucleosome positioning on the GalCyc promoter does not depend on nucleosome positioning (or its absence) on adjoining sequences. The same is true for nucleosome positioning on FRT sequence. It was found also that nucleosomes' positioning on the NPTII gene and their mutual disposition, namely the spacing between neighboring nucleosomes (linker length) are determined by the location of positioning signals only. Generally the nucleosome positioning in our experimental model is determined solely by internal DNA sequence occupied by nucleosome. On the other hand, the action of this internal positioning signal does not extend to neighboring DNA sequences.     

Nucleosome positioning signals in the DNA sequence of the human and mouse H19 imprinting control regions

We have investigated the sequences of the mouse and human H19 imprinting control regions (ICRs) to see whether they contain nucleosome positioning information pertinent to their function as a methylation-regulated chromatin boundary. Positioning signals were identified by an in vitro approach that employs reconstituted chromatin to comprehensively describe the contribution of the DNA to the most basic, underlying level of chromatin structure. Signals in the DNA sequence of both ICRs directed nucleosomes to flank and encompass the short conserved sequences that constitute the binding sites for the zinc finger protein CTCF, an essential mediator of insulator activity. The repeat structure of the human ICR presented a conserved array of strong positioning signals that would preferentially flank these CTCF binding sites with positioned nucleosomes, a chromatin structure that would tend to maintain their accessibility. Conversely, all four CTCF binding sites in the mouse sequence were located close to the centre of positioning signals that were stronger than those in their flanks; these binding sites might therefore be expected to be more readily incorporated into positioned nucleosomes. We found that CpG methylation did not effect widespread repositioning of nucleosomes on either ICR, indicating that allelic methylation patterns were unlikely to establish allele-specific chromatin structures for H19 by operating directly upon the underlying DNA-histone interactions; instead, epigenetic modulation of ICR chromatin structure is likely to be mediated principally at higher levels of control. DNA methylation did, however, both promote and inhibit nucleosome positioning at several sites in both ICRs and substantially negated one of the strongest nucleosome positioning signals in the human sequence, observations that underline the fact that this epigenetic modification can, nevertheless, directly and decisively modulate core histone-DNA interactions within the nucleosome.     

Nucleosome positioning in relation to nucleosome spacing and DNA sequence-specific binding of a protein

Nucleosome positioning is an important mechanism for the regulation of eukaryotic gene expression. Folding of the chromatin fiber can influence nucleosome positioning, whereas similar electrostatic mechanisms govern the nucleosome repeat length and chromatin fiber folding in vitro. The position of the nucleosomes is directed either by the DNA sequence or by the boundaries created due to the binding of certain trans-acting factors to their target sites in the DNA. Increasing ionic strength results in an increase in nucleosome spacing on the chromatin assembled by the S-190 extract of Drosophila embryos. In this study, a mutant lac repressor protein R3 was used to find the mechanisms of nucleosome positioning on a plasmid with three R3-binding sites. With increasing ionic strength in the presence of R3, the number of positioned nucleosomes in the chromatin decreased, whereas the internucleosomal spacings of the positioned nucleosomes in a single register did not change. The number of the positioned nucleosomes in the chromatin assembled in vitro over different plasmid DNAs with 1-3 lac operators changed with the relative position and number of the R3-binding sites. We found that in the presence of R3, nucleosomes were positioned in the salt gradient method of the chromatin assembly, even in the absence of a nucleosome-positioning sequence. Our results show that nucleosome-positioning mechanisms are dominant, as the nucleosomes can be positioned even in the absence of regular spacing mechanisms. The protein-generated boundaries are more effective when more than one binding site is present with a minimum distance of approximately 165 bp, greater than the nucleosome core DNA length, between them.     

Nucleosome positioning signals and potential H-DNA within the DNA sequence of the imprinting control region of the mouse Igf2r gene

The imprinting control region within the second intron of the mouse Igf2r gene contains a CpG island comprising direct repeats, an imprinting box and the Air antisense promoter which is blocked by the methylation imprint on the active maternal allele. We have investigated the structural features of this DNA, including a mapping of all nucleosome positioning signals within the nucleotide sequence. A discrete series of strong positioning signals distinguished the direct repeat region from the much more diverse positioning capacity of the sequence encompassing the known regulatory elements. At only a few locations did CpG methylation modulate the use of this positioning information. Direct effects upon histone-DNA interactions are therefore unlikely to contribute significantly to the means by which the imprint may establish allele-specific chromatin architecture and determine Air expression. A strand-specific obstruction to DNA polymerase was observed between the repeat and regulatory regions. The same region adopts triple-stranded H-DNA structures in supercoiled DNA, according to pH and divalent cation exposure. Methylation did not modulate the occurrence or form of this structure under the conditions tested. This finding nevertheless adds to the repertoire of potential H-DNA structures found in the vicinity of regulatory sequences-here, in an imprinting context.     

How objects are grasped: the interplay between affordances and end-goals

Background

Substantial literature has demonstrated that how the hand approaches an object depends on the manipulative action that will follow object contact. Little is known about how the placement of individual fingers on objects is affected by the end-goal of the action.

Methodology/Principal Findings

Hand movement kinematics were measured during reaching for and grasping movements towards two objects (stimuli): a bottle with an ordinary cylindrical shape and a bottle with a concave constriction. The effects of the stimuli''s weight (half full or completely full of water) and the end-goals (pouring, moving) of the action were also assessed. Analysis of key kinematic landmarks measured during reaching movements indicate that object affordance facilitates the end-goal of the action regardless of accuracy constraints. Furthermore, the placement of individual digits at contact is modulated by the shape of the object and the end-goal of the action.

Conclusions/Significance

These findings offer a substantial contribution to the current debate about the role played by affordances and end-goals in determining the structure of reach-to-grasp movements.     

Understanding the Connection between Epigenetic DNA Methylation and Nucleosome Positioning from Computer Simulations

Cytosine methylation is one of the most important epigenetic marks that regulate the process of gene expression. Here, we have examined the effect of epigenetic DNA methylation on nucleosomal stability using molecular dynamics simulations and elastic deformation models. We found that methylation of CpG steps destabilizes nucleosomes, especially when these are placed in sites where the DNA minor groove faces the histone core. The larger stiffness of methylated CpG steps is a crucial factor behind the decrease in nucleosome stability. Methylation changes the positioning and phasing of the nucleosomal DNA, altering the accessibility of DNA to regulatory proteins, and accordingly gene functionality. Our theoretical calculations highlight a simple physical-based explanation on the foundations of epigenetic signaling.     

The dynamic interplay in chromatin remodeling factors polycomb and trithorax proteins in response to DNA damage

The dynamic interplay in polycomb group (PcG) and trithorax group (TrxG) proteins in response to DNA damage directly involves in the DNA double strand breaks (DSBs) sites and potentially function in both homologous recombination (HR) and nonhomologous end joining (NHEJ) pathways. The process includes chromatin remodeling that is a major mechanism used by cells to relax chromatin in DNA damage response (DDR) and repair. PcGs show resistance ability to the process while, some tumor suppressor genes involves in the DDR and repair by interacting with TrxGs. Understanding how the dynamic interplay in PcGs and TrxGs impacts on DDR will shed light on the mechanisms of carcinogenesis and develop a new target from anti-DDR related drugs.     

Molecular mechanisms and kinetics between DNA and DNA binding ligands

Mechanical properties of single double-stranded DNA (dsDNA) in the presence of different binding ligands were analyzed in optical-tweezers experiments with subpiconewton force resolution. The binding of ligands to DNA changes the overall mechanic response of the dsDNA molecule. This fundamental property can be used for discrimination and identification of different binding modes and, furthermore, may be relevant for various processes like nucleosome packing or applications like cancer therapy. We compared the effects of the minor groove binder distamycin-A, a major groove binding alpha-helical peptide, the intercalators ethidium bromide, YO-1, and daunomycin as well as the bisintercalator YOYO-1 on lambda-DNA. Binding of molecules to the minor and major groove of dsDNA induces distinct changes in the molecular elasticity compared to the free dsDNA detectable as a shift of the overstretching transition to higher forces. Intercalating molecules affect the molecular mechanics by a complete disappearance of the B-S transition and an associated increase in molecular contour length. Significant force hysteresis effects occurring during stretching/relaxation cycles with velocities >10 nm/s for YOYO-1 and >1000 nm/s for daunomycin. These indicate structural changes in the timescale of minutes for the YOYO-DNA and of seconds for the daunomycin-DNA complexes, respectively.     

Single-molecule studies on the mechanical interplay between DNA supercoiling and H-NS DNA architectural properties

The Escherichia coli H-NS protein is a major nucleoid-associated protein that is involved in chromosomal DNA packaging and gene regulatory functions. These biological processes are intimately related to the DNA supercoiling state and thus suggest a direct relationship between H-NS binding and DNA supercoiling. Here, we show that H-NS, which has two distinct DNA-binding modes, is able to differentially regulate DNA supercoiling. H-NS DNA-stiffening mode caused by nucleoprotein filament formation is able to suppress DNA plectoneme formation during DNA supercoiling. In contrast, when H-NS is in its DNA-bridging mode, it is able to promote DNA plectoneme formation during DNA supercoiling. In addition, the DNA-bridging mode is able to block twists diffusion thus trapping DNA in supercoiled domains. Overall, this work reveals the mechanical interplay between H-NS and DNA supercoiling which provides insights to H-NS organization of chromosomal DNA based on its two distinct DNA architectural properties.     

Positioning of a Nucleosome on Mouse Satellite DNA Inserted into a Yeast Plasmid Is Determined by Its DNA Sequence and an Adjacent Nucleosome

It has earlier been shown that multiple positioning of nucleosomes on mouse satellite DNA is determined by its nucleotide sequence. To clarify whether other factors, such as boundary ones, can affect the positionings, we modified the environment of satellite DNA monomer by inserting it into a yeast plasmid between inducible GalCyc promoter and a structural region of the yeast FLP gene. We have revealed that the positions of nucleosomes on satellite DNA are identical to those detected upon reconstruction in vitro. The positioning signal (GAAAAA sequence) of satellite DNA governs nucleosome location at the adjacent nucleotide sequence as well. Upon promoter induction the nucleosome, translationally positioned on the GalCyc promoter, transfers to the satellite DNA and its location follows the positioning signal of the latter. Thus, the alternatives of positioning of a nucleosome on satellite DNA are controlled by its nucleotide sequence, though the choice of one of them is determined by the adjacent nucleosome.