An intranuclear frame for chromatin compartmentalization and higher-order folding

Recent ultrastructural, immunoelectron, and confocal microscopy observations done in our laboratory [Barboro et al. [2002] Exp. Cell. Res. 279:202-218] have confirmed that lamins and the nuclear mitotic apparatus protein (NuMA) are localized inside the interphase nucleus in a polymerized form. This provided evidence of the existence of a RNA stabilized lamin/NuMA frame, consisting of a web of thin ( approximately 3 and approximately 5 nm) lamin filaments to which NuMA is anchored mainly in the form of discrete islands, which might correspond to the minilattices described by Harborth et al. [1999] (EMBO. J. 18:1689-1700). In this article we propose that this scaffold is involved in the compartmentalization of both chromatin and functional domains and further determines the higher-order nuclear organization. This hypothesis is strongly supported by the scrutiny of different structural transitions which occur inside the nucleus, such as chromatin displacement and rearrangements, the collapse of the internal nuclear matrix after RNA digestion and the disruption of chromosome territories induced by RNase A and high salt treatment. All of these destructive events directly depend on the loss of the stabilizing effect exerted on the different levels of structural organization by the interaction of RNA with lamins and/or NuMA. Therefore, the integrity of nuclear RNA must be safeguarded as far as possible to isolate the matrix in the native form. This material will allow for the first time the unambiguous ultrastructural localization inside the INM of the components of the functional domains, so opening new avenues of investigation on the mechanisms of gene expression in eukaryotes.     

A critical role for linker DNA in higher-order folding of chromatin fibers

Nucleosome-nucleosome interactions drive the folding of nucleosomal arrays into dense chromatin fibers. A better physical account of the folding of chromatin fibers is necessary to understand the role of chromatin in regulating DNA transactions. Here, we studied the unfolding pathway of regular chromatin fibers as a function of single base pair increments in linker length, using both rigid base-pair Monte Carlo simulations and single-molecule force spectroscopy. Both computational and experimental results reveal a periodic variation of the folding energies due to the limited flexibility of the linker DNA. We show that twist is more restrictive for nucleosome stacking than bend, and find the most stable stacking interactions for linker lengths of multiples of 10 bp. We analyzed nucleosomes stacking in both 1- and 2-start topologies and show that stacking preferences are determined by the length of the linker DNA. Moreover, we present evidence that the sequence of the linker DNA also modulates nucleosome stacking and that the effect of the deletion of the H4 tail depends on the linker length. Importantly, these results imply that nucleosome positioning in vivo not only affects the phasing of nucleosomes relative to DNA but also directs the higher-order structure of chromatin.     

Changes in chromatin folding in solution

The formation of higher order structures by nucleosome oligomers of graded sizes with increasing ionic strength has been studied in solution, by measuring sedimentation coefficients. Nucleosome monomers and dimers show no effect of ionic strength at the concentrations used, while trimers to pentamers show a linear dependence of the logarithm of sedimentation coefficient upon the logarithm of ionic strength between 5 and 25 mm, but no dependence above 25 mm. Between pentamer and hexamer a change occurs and the linear relationship is observed up to ionic strength 125 mm with hexamer and above.The simple power-law dependence of the sedimentation coefficient upon the ionic strength (s ∝ Iⁿ) is observed up to nucleosome 30mers, but by 60mer a jump in the sedimentation coefficient occurs between ionic strengths 45 and 55 mm, with the power-law applying both above and below the jump. Removal of histone H1 and non-histone proteins lowers the overall sedimentation rate and abolishes the jump.Cross-linking large oligomers at ionic strength 65 mm stabilizes the structure in the conformation found above the jump, leading to a simple power-law dependence throughout the range of ionic strength for cross-linked material. Cleavage of the cross-links restores the jump, presumably by allowing the conformational transition that causes it. Large oligomers are indistinguishable in sedimentation behaviour whether extracted from nuclei at low ionic strength or at 65 mm and maintained in the presence of salt.We interpret these results, together with the detailed electron microscopic studies reported by Thoma et al. (1979) under similar salt conditions, as showing the histone H1-dependent formation of superstructures of nucleosomes in solution induced by increasing ionic strength. The unit of higher order structure probably contains five or six nucleosomes, leading to the change in stability with hexamer. Although this size corresponds to the lower limit of size suggested for “superbeads” (Renz et al., 1977), we see no evidence that multiples of six nucleosomes have any special significance as might be predicted if superbeads had any structural importance. Rather, our results are compatible with a continuous pattern of condensation, such as a helix of nucleosomes (see e.g. Finch & Klug, 1976). The jump in sedimentation observed between ionic strengths 45 and 55 mm, together with the effect of cross-linking, suggests the co-operative stabilization of this structure at higher ionic strengths. A plausible hypothesis is that the turns of the solenoid are not tightly bonded in the axial direction below 45 mm, but come apart due to the hydrodynamic shearing forces in the larger particles leading to less compact structures with slower sedimentation rates. Above 55 mm the axial bonding is strong enough to give a stable structure of dimensions compatible with the 30 nm structures observed in the cell nucleus.     

Revisiting higher-order and large-scale chromatin organization

The past several years has seen increasing appreciation for plasticity of higher-level chromatin folding. Four distinct '30nm' chromatin fiber structures have been identified, while new in situ imaging approaches have questioned the universality of 30nm chromatin fibers as building blocks for chromosome folding in vivo. 3C-based approaches have provided a non-microscopic, genomic approach to investigating chromosome folding while uncovering a plethora of long-distance cis interactions difficult to accommodate in traditional hierarchical chromatin folding models. Recent microscopy based studies have suggested complex topologies co-existing within linear interphase chromosome structures. These results call for a reappraisal of traditional models of higher-level chromatin folding.     

Effects of histone acetylation on the solubility and folding of the chromatin fiber

The folding ability of chromatin fractions containing approximately identical nucleosome numbers and the same linker histone composition, but with different extents of core histone acetylation, were analyzed by analytical ultracentrifugation. It was found that the acetylated fractions consistently exhibited a relatively small but significantly lower extent of compaction than that of their native nonacetylated counterparts. This was regardless of the extent of the size distribution heterogeneity of the fractions analyzed. Furthermore the acetylated chromatin fibers exhibited an enhanced solubility in both NaCl and MgCl(2), which is neither the result of a differential binding affinity of the linker histones to chromatin nor of an alteration in the relative amounts of the histone H1 variants.     

Towards microfluorometric quantitation of polyamines in situ

Summary Two different fluorescence cytochemical methods, the formaldehyde-fluorescamine (FF) method and the orthophthalaldehyde (OPT) method as well as an immunocytochemical method have been developed for the localization of spermidine and spermine. Of these three methods, the FF-method is the most easy to perform. We have studied the relationship between fluorescence intensity induced by the FF-method and cellular polyamine levels measured by HPLC in MCF-7 cells and HeLa cells. The experiments were designed to obtain different cell concentrations of polyamines. Cells grown on microscope slides in Petri-dishes were partly depleted of spermidine by two days inhibition of their ornithine decarboxylase activity using -difluoromethylornithine. One hr before harvest the cells were exposed to different concentrations (0–30 M) of spermidine. Microfluorometric results and chemical determinations of spermidine and spermine were obtained from each separate slide. The cellular total polyamine (spermidine + spermine) concentration on the slides varied between 4 and 15 nmol per mg protein (MCF-7 cells) and 5 and 26 nmol per mg protein (HeLa cells) and the corresponding microfluorometric results between 60 and 115 arbitrary units (MCF-7 cells) and 80 and 160 arbitrary units (HeLa cells). Simple regression analysis showed a good linear relationship between cellular polyamine concentration and FF-fluorescence yield. The correlation coefficient for MCF-7 cells was 0.86 and for HeLa cells 0.82, significance of the correlations was p0.0001. Our results add further credence to the specificity of the FF-method and indicate that the method may be useful for microfluorometric quantitation of polyamines in situ.     

Possible nucleosome arrangements in the higher-order structure of chromatin

Consideration has been given to possible sequences of nucleosomes which can produce a ‘thick fibre’-like structure. Only a few basic requirements were imposed: (i) the thick fibre is a regular single helix with about 7 nucleosomes per turn; (ii) the nucleosomes are equidistant along the polynuclesome chain; (iii) the helix is flexible having variable pitch. It was found that in addition to the straightforward sequential arrangement there is only one other nonsequential arrangement which satisfies these requirements. This is a helix with around 8 nucleosomes per turn in which all nucleosomes are identically placed. It is possible in the region of 200 to 218 ± 10 base pairs (b.p.) DNA repeats lengths. The linker DNA is straight or almost straight and crosses the internal ‘hollow’ cylinder which is not occupied by nucleosomes. This structure satisfies the experimental data for the distance distribution function, and the observed mass per unit length and changes noted in the mass per unit length. Further, if it is assumed that the core particle axis of symmetry is in the plane of the two linkers and bisects them then this makes the core particles oblique to the thick fibre radii with alternate angles of ± 20 to 30°. This orientation of the nucleosomes can explain the DNA digestion patterns obtained with DNase II and with DNase I.     

Reconstitution of chromatin higher-order structure from histone H5 and depleted chromatin

Reconstitution of the 30 nm filament of chromatin from pure histone H5 and chromatin depleted of H1 and H5 has been studied using small-angle neutron-scattering. We find that depleted, or stripped, chromatin is saturated by H5 at the same stoichiometry as that of linker histone in native chromatin. The structure and condensation behavior of fully reconstituted chromatin is indistinguishable from that of native chromatin. Both native and reconstituted chromatin condense continuously as a function of salt concentration, to reach a limiting structure that has a mass per unit length of 6.4 nucleosomes per 11 nm. Stripped chromatin at all ionic strengths appears to be a 10 nm filament, or a random coil of nucleosomes. In contrast, both native and reconstituted chromatin have a quite different structure, showing that H5 imposes a spatial correlation between neighboring nucleosomes even at low ionic strength. Our data also suggest that five to seven contiguous nucleosomes must have H5 bound in order to be able to form a higher-order structure.     

Size-dependence of a stable higher-order structure of chromatin

We have recently reported a study of the formation of higher-order structures of chromatin with increasing ionic strength, in which we measured sedimentation coefficients of long nucleosome oligomers. We found that somewhere in the range of 30 to 60mer the sedimentation coefficient developed a jump of about 10% between ionic strengths of 45 mm and 55 mm, which persisted for larger oligomers (Butler & Thomas, 1980).This posed the question of whether the jump observed represented a greater relative compaction at high ionic strength on the part of long polymers, or a relatively lesser resistance to hydrodynamic shear forces at low ionic strength.We now define the dependence of the jump upon oligomer size in detail, the critical size being 50 nucleosomes. We also show that it occurs because the sedimentation of a large oligomer appears “slow” for its size at lower ionic strength, but “normal” at higher ionic strengths. We interpret this as the consequence of insufficient axial interaction to stabilize the helical coiling of long nucleosome filaments at low ionic strength, leading to a more open and slowly sedimenting structure.     

Regulation of higher-order chromatin structures by nucleosome-remodelling factors

Nucleosome-remodelling factors are key facilitators of chromatin dynamics. At the level of single nucleosomes, they are involved in nucleosome-repositioning, altering histone-DNA interactions, disassembly of nucleosomes, and the exchange of histones with variants of different properties. The fundamental nature of chromatin dictates that nucleosome-remodelling affects all aspects of eukaryotic DNA metabolism, but much less is known about the functional interactions of nucleosome-remodelling factors with folded chromatin fibres. Because remodelling machines are abundant constituents of eukaryotic nuclei and, therefore, have ample potential to interact with chromatin, they might also affect higher-order chromatin architecture. Recent observations support roles for nucleosome-remodelling factors at the supra-nucleosomal level.     

Visualization of chromatin higher-order structures and dynamics in live cells

Chromatin has highly organized structures in the nucleus, and these higher-order structures are proposed to regulate gene activities and cellular processes. Sequencing-based techniques, such as Hi-C, and fluorescent in situ hybridization (FISH) have revealed a spatial segregation of active and inactive compartments of chromatin, as well as the non-random positioning of chromosomes in the nucleus, respectively. However, regardless of their efficiency in capturing target genomic sites, these techniques are limited to fixed cells. Since chromatin has dynamic structures, live cell imaging techniques are highlighted for their ability to detect conformational changes in chromatin at a specific time point, or to track various arrangements of chromatin through long-term imaging. Given that the imaging approaches to study live cells are dramatically advanced, we recapitulate methods that are widely used to visualize the dynamics of higher-order chromatin structures.