Subunit associations among chromatin particles

Nucleic Acids Research Subunit associations among chromatin particles Ailsa M. Campbell1 and Rosalind I. Cotter 2 * Dept. of Biochemistry, University of Glasgow, Glasgow G12 8QQ, UKand 2 Searle Research Laboratories, Lane End Road, High Wycombe, Bucks, HP12 4HL, UK Received 22 August 1977 The self-association of oligonucleosomal chromatin particles in solution has been studied by light scattering and sedimentation. In the absence of magnesium ions no association is observed. In the presence of 70mM sodium or 2mM magnesium ions mono, di, tri and tetranucleosomes self-associate only if they contain bound histone 1. This association leads to the formation of compact aggregates and is continuous and non-cooperative. The relevance to higher order arrangements of nucleosomes is discussed. INTRODUCTION The infrastructure of chromatin is now generally accepted to be a row of nucleosomal particles spaced along the DNA strand at intervals of 140 to 200 base pairs " , with histone HI being probably associated with the linker DNA ' between 140 base pair core particles which contain histones H2A, H2B, H3 and H4. Nuclease digestion of chromatin generates unit and oligomeric nucleosomes 7-9 which have been subject to conformational analysis both with and without HI ' ' . The packing of the subunits into higher order structures has been discussed in terms of a helix , or solenoid , or superbead having about 6 nucleosomes per turn. We have studied small oligomeric chromatin subparticles and have considered the effects of histone 1 on their association with a view to examining its influence on chromatin superstructure. The nature of the associations, whether continuous or discrete, cooperative or non-cooperative, have been investigated and the shapes of the superstructures are discussed. EXPERIMENTAL Chromatin was released from chicken erythrocyte nuclei isolated according to Shaw et al_-16 by gently spinning in 0.1M sucrose, lOmM Tris-HCl pH 7.5. Subunits of chromatin were prepared by micrococcal nuclease (130 units/ © Information Retrieval Limited 1 Falconberg Court London W1V5FG England 3877 ABSTRACT Nucleic Acids Research ml) digestion f o r 10 minutes at 37°C of chromatin (6-10 mg/ml) in the presence of 0.7mM CaCl 2 . The digest was fractionated on a 10% w/v-45S w/v sucrose gradient in lOmM Tris-HCl, 0.7mM EDTA, pH 7.5 run in a T i l 4 zonal rotor at 48,000 rpm f o r 17 hours . HI and H5 were removed from digested chromatin by dialysis against 0.6M NaCl, lOmM Tris-HCl, 0.7mM EDTA, pH 7.5 and zonal centrifugation in the same buffer. 140 base pair core particles were prepared by digestion of Hl-depleted chromatin and p u r i f i e d by zonal c e n t r i - fugation. DNA from oligomer chromatin particles was extracted with pronase and ft lft RESULTS AND DISCUSSION The properties of our preparations of chromatin subunits having their full complement of HI and H5 (Fig. 1) are typically those shown in Table 1. The sedimentation coefficients '* and melting characteristics are com- 3878 phenol and sized on 3.5% polyacrylamide gels alongside restriction enzyme Hae III (Miles) fragments of PM2 DNA. Extracted protein from particles was run on 15% SDS polyacrylamide slab gels 19g and stained in Coomassie blue. blue, fRelative histone contents were cornpared by densitometry of the gel bands. Thermal melts were carried out at 258 nm using a heating rate of $°C/min in lOmM cacodylate, 0.7mM EDTA pH 7.5. Absorbances were corrected for thermal expansion. Light scattering experiments were performed as described before . The refractive increments of DNA and protein were 0.165 ml/g and 0.176 ml/g respectively and the refractive increments of the nucleosomal particles did not vary with concentration over the range studied (up to 1 mg/ml). Samples were filtered through Schleicher and Schull filters of pore size 0.45n. In all cases the disymmetry (the ratio of light scattered at an angle of 45° to light scattered at an angle of 135°) was less than 1.05, indicating the absence of any high molecular weight contaminants. For clarity the data were plotted as the apparent weight average molecular weight (M^.app) versus concentration rather than as the established reciprocal plot^l. The extrapolation is therefore empirical but agrees with the extrapolation obtained from a reciprocal plot. Analytical ultracentrifugation was performed on a Beckman Model E equipped with UV scanner. The buffer used in all experiments was lOmM Tris-HCl, 40mM NaCl, 0.7mM EDTA pH 7.5 with or without the indicated molarity of MgCl 2 . Nucleic Acids Research 0-3 - 200 600 1000 mean number DNA base pairs JH1 H5 H3 H2B H2A H4 195 360 640 mean number 850 140 DNA base pairs Fig. 1: 15% SDS-polyacrylamide slab gel showing relative amounts of histones 1 and 5 on oligonucleosome preparations. parable with other reported values. The weight average molecular weights are consistent with those calculated from the mean number of base pairs of DNA derived from calibrations against Hae III restriction fragments from PM2 DNA 14 22 • (Fig. 2) assuming a molecular weight of 120,000 for nucleosomal protein and 21,000 for HI 1 2 . 3879 Nucleic Acids Research TABLE I n-oer S 20 V HI + H5/ Hypototal »*chroa1c1ty histone*1 Average no. base pairs of DNA core particle 11.2S 210,000 0 26% 82° 140 Dononucleosorae 11.5S 232,000 0.14 261 82° 195 24X 82° 360 dinucleosooe 15S 510,000 0.17 trinucleosome 19S 750,000 0.20 23X 83° 640 tetranucleosome 22S 1,100,000 0.20 261 85° 850 from scan of Coomassie blue stained 151 SDS polyacrylanide gels ("g. i) It can be seen (Fig. 3) that the molecular weights of all the particles are independent of concentration over the range studied. The lack of selfassociation of oligonucleosomes confirms that the effect of histone 1 on the sedimentation coefficient noted by Noll and Kornberg reflects a change in conformation rather than any tendency of the particles to aggregate. X-ray scattering 23 and electron microscopic evidence suggest that the chromatin nucleofilament is a linear structure which folds up after addition of magnesium or sodium ions. However, higher order condensation among mononucleosomes in the absence of EDTA has been visualised in the electron microscope and chromatin depleted of HI can adopt a coiled structure in the absence of divalent ions. We find no evidence of self-association in the absence of divalent cations, but our ionic strength conditions are not comparable and the internal concentration of chromatin in fibres is greatly in excess of the concentration range studied here. Figure 4 shows the effect of magnesium ions on the apparent molecular weight of oligonucleosomes with increasing concentration. It is apparent that the magnesium induces a high degree of association between oligonucleosomes; this self-association however is completely dependent on the presence of histones 1 and 5, thus nucleosomes depleted of these hi (...truncated)