Partial denaturation of small chromatin fragments: direct evidence for the radial distribution of nucleosomes in folded chromatin fibers

A. Bermdez 0 S. Bartolom 0 J.-R. Daban ) 0 0 Departament de Bioquimica i Biologia Molecular, Facultat de Ciencies, Universitat Autonoma de Barcelona , 08193-Bellaterra, Barcelona , Spain Antonio Bermdez, Salvador Bartolom and Joan-Ramon Daban* - Partial denaturation of small chromatin fragments: direct evidence for the radial distribution of nucleosomes in folded chromatin fibers SUMMARY To examine the internal structure of chromatin fibers, we have developed procedures for partial denaturation of small chromatin fragments (8-30 nucleosomes) from chicken erythrocytes. Electron micrographs of samples prepared under conditions that cause nucleosome dissociation show rods and loops projecting from short compact fibers fixed by glutaraldehyde in 1.7 mM Mg2+. According to previous studies in our laboratory, these images correspond to the top view of partially denatured fibers. Our results indicate that rods and loops consist of extended duplex DNA of different lengths. DNA in loops is nicked, as demonstrated by experiments performed in the presence of high concentrations of ethidium bromide. Length measurements indicate that the radial projections of DNA are produced by unfolding of nucleosomal units. Loops are formed by DNA from denatured nucleosomes in internal positions of the fiber; DNA from denatured Eukaryotic chromatin is organized according to various hierarchical levels of DNA folding (Wolffe, 1995; Koshland and Strunnikov, 1996). The fundamental structural unit of chromatin, the nucleosome, contains two turns of DNA (about 165 bp) wrapped around a histone octamer (Richmond et al., 1984; Arents et al., 1991; Arents and Moudrianakis, 1993; Luger et al., 1997), and has associated with it a single molecule of histone H1 (Ramakrishnan et al., 1993; Pruss et al., 1996; Travers and Muyldermans, 1996; Crane-Robinson, 1997). Nucleosomes are connected by linker DNA and form fibers of 30-40 nm in diameter (Thoma et al., 1979; Bradbury and Baldwin, 1986; Gerchman and Ramakrishnan, 1987; Koch et al., 1988; Athey et al., 1990; Woodcock, 1994; Zlatanova et al., 1994) which are probably involved in the packaging of DNA in interphase and metaphase chromosomes (Pienta and Coffey, 1984; Rattner and Lin, 1985; Manuelidis, 1990; Saitoh and Laemmli, 1993; Belmont and Bruce, 1994). The structure of the folded chromatin fiber has been difficult to study (Widom, 1989; Woodcock and Horowitz, 1995; van Holde and Zlatanova, 1995, 1996). Using transmission electron microscopy, the location of nucleosomes in terminal positions form rods. Our micrographs show clearly a radial distribution of DNA loops and rods projecting from fibers. Rods are orthogonal to the surface of the chromatin fragments. Considering that the high ionic strength used in this study (0.8-2.0 M NaCl) neutralizes the electrostatic repulsions between rods and fiber, this observation suggests that rods are extensions of nucleosomes radially organized inside the fiber. The position of the entry points of DNA loops into the fiber could be influenced by constraint on loops, but our results showing that the arc that separates these points in dinucleosome loops is relatively short suggest that consecutive nucleosomes are relatively close to each other in the folded fiber. nucleosomes in the fiber is only clearly seen when chromatin has an extended conformation at low ionic strength. Several laboratories have observed that under these conditions chromatin appears as a zigzag chain of nucleosomes in which linker DNA is completely extended (Thoma et al., 1979; Losa et al., 1984; Woodcock et al., 1984). Studies performed using scanning force microscopy have indicated that nucleosomes form an irregular three-dimensional zigzag in chromatin fibers at low ionic strength (Leuba et al., 1994). This nucleosome distribution is in agreement with the results obtained with relatively extended chromatin fibers observed in frozen hydrated sections of nuclei (Horowitz et al., 1994; Woodcock, 1994). However, at higher ionic strength or in the presence of divalent cations the fiber is highly packaged and appears relatively irregular and segmented (Finch and Klug, 1976; Woodcock et al., 1984; Widom, 1986; Williams et al., 1986; Zlatanova et al., 1994) or even discontinuous (Zentgraf and Franke, 1984; Subirana et al., 1985). To overcome the difficulties encountered in the study of the organization of nucleosomes in these compact structures of high molecular mass, several laboratories have examined the structure of small chromatin fragments (Bartolom et al., 1994) and complexes containing few nucleosomes (Yao et al., 1990, 1991; Bednar et al., 1995). The results obtained in all these microscopy studies, together with indirect data obtained using other techniques (McGhee et al., 1983; Widom and Klug, 1985; Bradbury and Baldwin, 1986; Koch et al., 1987; Williams and Langmore, 1991; Graziano et al., 1994) have led to the suggestion of various models for folded chromatin fibers. At present, there are two kinds of models for the organization of nucleosomes in the folded chromatin fiber. (i) In the solenoidal models linker DNA is folded and consecutive nucleosomes form a helix (Finch and Klug, 1976; McGhee et al., 1983; Butler, 1984; Widom and Klug, 1985; Bartolom et al., 1994; Graziano et al., 1994; Daban and Bermdez, 1998). (ii) In the crossed-linker models (Staynov, 1983; Subirana et al., 1985; Williams et al., 1986; Bordas et al., 1986; Koch, 1989; Athey et al., 1990), and in the variable zigzag nucleosomal ribbon models (Woodcock et al., 1993; Leuba et al., 1994), consecutive nucleosomes are roughly in opposite positions with respect to the fiber axis and linker DNA is extended into the fiber interior. We have previously shown, using transmission electron microscopy (Bartolom et al., 1994), that in the presence of 1.7 mM Mg2+ rotary-shadowed chromatin fragments from chicken erythrocytes containing from about 6 to 35 nucleosomes are circular structures with approximately the same diameter (33 nm). Our results from unidirectional shadowing experiments showed that the height of these structures increases with the number of nucleosomes. These height measurements, and the results showing that the electrophoretic mobility of small chromatin fragments in the presence of 1.7 mM Mg2+ decreases only slightly with molecular mass (Bartolom et al., 1995), led us to the conclusion that under these conditions chromatin fragments are highly packed and form short folded fibers. This high compactness favours the vertical placement of the folded fibers on the carbon film of the electron microscopy grid (Bartolom et al., 1994). In this work, we have taken advantage of the possibility to obtain images corresponding to the top view of short folded fibers to study directly the distribution of nucleosomes within the fiber. Following an experimental approach similar to that of Paulson and Laemmli (1977) for the study of metaphase chromosomes, we have developed different (...truncated)