Electrophoresis of Chromatin on Nondenaturing Agarose Gels Containing Mg2+

Abstract

We show that nondenaturing agarose gels can be used for the study of the structure and dynamic properties of native (uncross-linked) chromatin. In gels containing 1.7 mM Mg 2؉ , chicken erythrocyte chromatin fragments having from about 6 to 50 nucleosomes produce well defined bands. These bands have an electrophoretic mobility that decreases only slightly with molecular weight. This surprising behavior is not observed in low ionic strength gels. Fragments with less than 6 nucleosomes and low content of histones H1-H5 give rise to broad bands in gels with Mg 2؉. In contrast, fragments containing only 3-4 nucleosomes but with the normal H1-H5 content are able to form associated structures with a mobility similar to that observed for high molecular weight chromatin. Electron microscopy results indicate that the associated fragments and the fragments of higher molecular weight show similar electrophoretic properties because they become very compact in the presence of Mg 2؉ and form cylindrical structures with a diameter of ϳ33 nm. Our results suggest that the interactions involved in the self-assembly of small fragments are the same that direct the folding of larger fragments; in both cases, the resulting compact chromatin structure is formed from a basic element containing 5-7 nucleosomes.In chromatin an octamer of histones H2A, H2B, H3, and H4 associated with 146 bp 1 of DNA forms the core of the nucleosome (1, 2). The two turns of DNA (about 165 bp) around these core histones are sealed by histone H1 (H5 in avian erythrocytes) (3-5). Nucleosomes connected by linker DNA form a filament that can fold into a condensed chromatin fiber of about 30 nm in diameter (reviewed in Ref. 6).The mechanism of chromatin folding and the structure of the 30-nm fiber have been studied by sedimentation methods (7-9), enzymatic and chemical digestions (10 -12), electric, flow, and photochemical dichroism (13-15), x-ray diffraction (16, 17), small angle x-ray scattering (18 -20), neutron scattering (21, 22), transmission electron microscopy (23-32), and scanning force microscopy (33). The results obtained in these studies have suggested different models for the folding and structural organization of the 30-nm chromatin fiber. These models differ essentially in the location of the linker DNA. In the one-start helix models, the linker DNA is folded and connects laterally consecutive nucleosomes (8,13,17,23,32). In the twistedribbon models, a two-start helix is formed by pairs of nucleosomes with the linker DNA parallel to the fiber axis (24, 26). Finally, in several continuous (11,18,28) and discontinuous (27) crossed-linker models, and in the variable zigzag nucleosomal ribbon model (30, 31), the linker DNA is extended in the fiber interior.In this work we show that nondenaturing agarose gel electrophoresis can be used for the study of the folding of chromatin. We have found that, when electrophoresis is performed in the presence of Mg 2ϩ , chicken erythrocyte chromatin fragments of a relatively high molecular weight change dramaticall...

Interdigitated Solenoid Model for Compact Chromatin Fibers

“…The stacking of nucleosome cores in secondary helices is also in agreement with cross-linking (41) and electron microscopy (19,42,43) results demonstrating that histone octamers can associate, forming different oligomers of higher molecular mass and aggregated helical tubes. The stabilization given by these interactions could be responsible for the folding and selfassembly of chromatin fragments containing few nucleosomes found in electrophoretic studies (2). In our models the first turn is sealed when the fourth (Figure 1E), fifth (Figure 1G), sixth (Figures 1A,F and 2A), or seventh (Figure 1H) nucleosome interacts with the first nucleosome.…”

“…Early work performed with space-filling models built to scale showed us that, to construct a helix of nucleosomes that (i) must be very compact (1) and (ii) has to be initiated and stabilized with a basic element containing about 6 nucleosomes (2), it is necessary to interdigitate the nucleosomes of the successive helical turns. Compact helices, with a small pitch, can be constructed using a relatively large number of nucleosomes per turn, but we have excluded this possibility because these helices require more than 6 nucleosomes to be initiated.…”

In Vitro Binding of H1 Histone Subtypes to Nucleosomal Organized Mouse Mammary Tumor Virus Long Terminal Repeat Promotor

“…To check the properties of chromatin aggregation (condensation) of the various linker histones, we used an MMTV LTR polynucleosome core template of a defined length of 1.3 kbp, which is organized in six phased nucleosomes (24) after reconstitution with liver core histones, as described under "Experimental Procedures." Bartolomé et al (41) showed that about six nucleosomes are sufficient to produce compact structures, such as chromatin fragments, of higher molecular weight, and many other laboratories using different techniques reported results concerning the association of oligonucleosomes to form higher order structures (42,43). Therefore, our model consisting of chromatin fragments containing six nucleosomes is suitable for studying the influence of linker histones on the formation of the polynucleosome complex.…”

Electrostatic mechanism of nucleosomal array folding revealed by computer simulation

“…They differ significantly in how the linker DNA is arranged within the condensed chromatin fiber. In the solenoid model, the linker DNA is bent such that the consecutive nucleosomes interact with each other and form a helix (10)(11)(12)(13)(14); in the zig-zag model, the linker DNA remains straight and crosses the fiber axis so that the consecutive nucleosomes appear at the opposite side of the fiber axis (5,(15)(16)(17)(18).…”