Stepwise unfolding of chromatin by urea

Abstract

The unfolding of chromatin by urea (0 -7 M) was studied by means of flow linear dichroism, photoaffinity labeling and nuclease digestion. The linear dichroism results indicate that the unfolding of the DNA is accomplished through two distinct transitions at 1 -2 M urea and 6 -8 M urea, respectively. The photoaffinity labeling studies indicate that an opening of the nucleosome histone core occurs above 2 M urea, accompanied by general loosening of the structure. Based on the results a model for the unfolding of chromatin fibers by urea is proposed, which includes a stretching of the linker DNA (0-2 M urea) followed by a "loosening" of the nucleosome core, possibly to a one-loop DNA conformation (2 -6 M urea), and finally resulting in an almost total stretching of the DNA (> 6 M urea).The nucleosomal chromatin structure (for reviews [I, 21) is heavily perturbed by urea as revealed by several studies employing a variety of techniques. These include hydrodynamic [3,4], spectroscopic (in particular circular dichroism) [5, 61 and electron microscopic [7, 81 as well as enzymatic (nuclease digestion) [9, 101 analyses. Increasing concentrations of urea result in a gradual increase of the intrinsic viscosity of the DNA-histone complex [7], a decrease of the sedimentation coefficient [4] as well as a loss of the beaded appearance on electron micrographs [7], which is otherwise characteristic for native nucleosomal chromatin. Furthermore, even high concentrations of urea (8 M) do not result in histone-DNA dissociation [6,7, 111, but rather causes a denaturation of individual histones and a weakening and eventually disruption of intra-nucleosomal histone-histone interactions [7]. However, regardless of the urea concentration limit-digestion with micrococcal nuclease still results in distinct DNA fragments of about 70-200 base pairs [6], and the characteristic ten-base-pair repeats upon digestion with DNase I are analogously retained in 5 M urea [S].Employing two new techniques, i.e. flow linear dichroism, by which information concerning the conformation and orientation of the DNA helix may be obtained [12, 131, and photoaffinity labeling, reflecting changes in protein-DNA interactions [14, 151, we now report results on the influence of urea on chromatin structure. Our data indicate that the unfolding as a function of the urea concentration is a stepwise process in terms of the DNA conformation, showing distinct transitions at 1-2 M urea and 6-8 M urea. Furthermore, on the basis of accumulated results we propose a model for the unfolding process which involves: a stretching of the linker DNA (0 -2 M urea) followed by a "loosening" of the nucleosome histone-core structure, probably resulting in a one-loop Abbreviation. LD,, reduced linear dichroism DNA conformation (2-6 M urea), which is finally also unfolded, resulting in a structure where the DNA is almost totally extended (8 M urea) but still covered with histones. MATERIALS AND METHODSWhole chromatin containing all histones (HI, H2A, H2B, H3 and H4) was isolated from mo...

Photoaffinity labeling of chromatin using a tritiated (azidoaryl)bis(acridinyl)spermidine photoprobe

“…In order to evaluate the usefulness of the histone-labeling pattern for probing of the chromatin structure/conformation, the effect of urea, which causes unfolding of the nucleosomal structure [see, e.g., Olins et al (1977)], was investigated. These results are discussed in full in a separate paper (Nielsen et al, 1985), but as seen from Figure 3c, the histone-labeling pattern changes drastically in the presence of urea. A significant increase in the relative, as well as the absolute, labeling intensity of the core histones is observed, and furthermore, histone H2B is being labeled above 3 M urea.…”

Chemical and photochemical probing of DNA complexes

“…The diacridine ensures high affinity for DNA by bis-intercalation while the azidobenzoyl ligand photoreacts with nearby proteins (or the DNA). The technique is probably most correctly termed affinity mediated photolabeling and has been used for studies of histone DNA interactions in chromatin, and can also be applied to in vivo situations (Nielsen, 1982;Nielsen et al, 1985).…”

Final Report of the Safety Assessment of Urea1

“…No change in the size of DNA fragments produced was observed in the presence of Urea. The activity of the nuclease only had a 50% inhibition in 7 M Urea (Nielsen et al 1985).…”

“…The original structures of native and NaCltreated partially dehistonized nucleohistones can be mostly restored after Urea perturbation (Chang and Li 1974). Nielsen et al (1985) studied the effects of Urea on chromatin which contained all histones (H1, H2A, H2B, H3, and H4) isolated from mouse Ehrlich ascites tumor cells. Flow linear dichroism measurements were performed on a Jasco J-500 spectropolarimeter at a flow gradient of 1800 s −1 .…”