Engeholm M et al. (2009), Nucleosomes can invade DNA territories occupied...

XB-ART-39251

Nat Struct Mol Biol 2009 Feb 01;162:151-8. doi: 10.1038/nsmb.1551.

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Nucleosomes can invade DNA territories occupied by their neighbors.

Engeholm M, de Jager M, Flaus A, Brenk R, van Noort J, Owen-Hughes T.

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Nucleosomes are the fundamental subunits of eukaryotic chromatin. They are not static entities, but can undergo a number of dynamic transitions, including spontaneous repositioning along DNA. As nucleosomes are spaced close together within genomes, it is likely that on occasion they approach each other and may even collide. Here we have used a dinucleosomal model system to show that the 147-base-pair (bp) DNA territories of two nucleosomes can overlap extensively. In the situation of an overlap by 44 bp or 54 bp, one histone dimer is lost and the resulting complex can condense to form a compact single particle. We propose a pathway in which adjacent nucleosomes promote DNA unraveling as they approach each other and that this permits their 147-bp territories to overlap, and we suggest that these events may represent early steps in a pathway for nucleosome removal via collision.

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Species referenced: Xenopus laevis
Genes referenced: prss1

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	Figure 2. Measuring the histone content of dimeric chromatin particlesa+b, Reconstitution and native-gel analysis. Reconstitutions were performed at the indicated [octamer]:[DNA] and [tetramer]:[dimer]:[DNA] ratios and analyzed by means of native-gel electrophoresis. Assembly was performed on a the (0bp) and b the(-44bp) construct. Each intermediate in assembly is labeled for a full description see text and Supplementary Figure 2. 1, mononucleosomes; 2, monotetrasomes; 3, monohexasomes; 4, fully assembled species; 5, ditetrasome; 6, tetrasome-hexasome; 7 dihexasome; 8 hexasome-nucleosome. c, The major fully assembled species (i.e. bands 4 in a+b) were purified by native-gel electrophoresis, digested with trypsin and analysed in triplicate by LC/ESI/MS/MS. Signal intensities of peptides in the (-44bp) samples were divided by the corresponding signal intensities in the (0bp) samples and the average over all replicates was calculated for each individual peptide (blue diamonds). Then these averaged normalized signal intensities were averaged separately for dimer-derived (green) and tetramer-derived (red) peptides yielding a [dimer]:[tetramer] ratio of 0.72. Standard deviations are shown as error bars.
	Figure 3. AFM imaging of dinucleosomesa-f, Dinucleosomes on the respective constructs were gel-purified, fixed in the absence (a-c) or presence (d-f) of 5 mM Mg2+ and imaged. In the presence of divalent cations the overlapping dinucleosomes on the (-44bp) construct appear as single particles of increased height. g, Maximal heights of the particles in the experiments in d-f. The average height of the nucleosomes on the (+48bp) and (0bp) constructs is indicated by the blue vertical line. h, Dinucleosomes on the (+48bp) (lanes 1-3) and (-44bp) (lanes 4-6) constructs were treated with MNase for the indicated amounts of time. An MNase resistant fragment of around 250 bp is observed for the dinucleosomes on the (-44bp) construct.
	Figure 4. Helical phasing is required for the condensation of overlapping dinucleosomesStructural models of dinucleosomes on the (-44bp) construct in the unfolded (left) and folded (right) states (a).. The exposed inner dimer on the complete nucleosome is highlighted red. Other histone proteins are shown in blue and DNA in yellow. b, A plot of the root mean square deviation (RMSD) of phosphorus atoms as a function of the overlap length for partial superimposition of two copies of the DNA superhelix in 1KX5. For a chosen overlap length n the last n base pairs in the first copy were superimposed with the first n base pairs in the second copy. The helical periodicity suggests that formation of compact structures is likely to require helical phasing. c-e, AFM imaging of dinucleosomes with overlaps lengths of -44, -49 and -54 bp following fixation in 5 mM Mg2+. Formation of compact particles is observed for the (-44bp) and (-54bp) constructs that coincide with minima in a, unfolded particles predominate for the -49bp construct.
	Figure 5. Formation of overlapping nucleosomes as a result of repositioninga, Samples of a dinucleosomal reconstitution on the MMTV were incubated at 0, 42, 47 or 52 °C for 60 min and analyzed by site-directed mapping. Mapping signals are labeled with the respective dyad positions. b, Dinucleosomes were gel-purified, temperature-treated and fixed in the presence of 5 mM Mg2+ followed by AFM imaging at room temperature. c-g, Volumetric analysis of particles on the MMTV fragment before (c) and following (d) temperature incubation and on the three 601 constructs in the presence of Mg2+. Where two particles could be resolved on a template molecule their volumes were determined and scored separately. Where only one large particle was present the total volume of the composite particle was used. h 2 pMoles of Dinucleosomes assembled on MMTV DNA were subject to remodeling with 0, .015, .03, 0.6, .012 fmoles RSC in the presence of 1mM ATP from 30 min at 30°C. Following remodeling the major new locations detected are consistent with nucleosomes moving into locations in which their DNA territories overlap. The loss of mapping signal in lanes 4 and 5 may be due to the increased heterogeneity in the remodeled chromatin and or additional alterations to chromatin structure.

References [+] :

Albert, Translational and rotational settings of H2A.Z nucleosomes across the Saccharomyces cerevisiae genome. 2007, Pubmed