Aggregation of small oligonucleosomal chains into 300-A globular particles.

Abstract

Chicken erythrocyte oligonucleosomes (trimers to about 20-mers) are able to interact with each other through the very Iysine-rich histones (HI and H5) and form heterogeneous globular particles with a mean diameter of about 300 A. These particles assemble spontaneously during micrococcal nuclease digestion of chromatin in the presence of 30 mM NaCl and contain approximately 25 nucleosomes. They are sensitive to ionic strength and unfold at lower salt concentrations but can be reconstituted by restoring the initial salt concentration. Even at 30 mM NaCl, the particles remain dynamic structures, being in equilibrium with their oligonucleosomal components as revealed by the fact that particle stability depends on the concentration of oligonucleosomes. Almost all DNA in nuclei of eukaryotic cells is complexed with histones to form linear arrays of a basic subunit called the nucleosome (1, 2). The nucleosomal chain, which has a diameter of approximately 110 A, is folded in a still unknown fashion into a thicker chromosomal fiber (250-300 A in diameter) (3-5). Two molecular models have been proposed to explain the structure of the chromosomal fiber. In the first, nucleosomes are continuously aligned in a long helix (solenoid) (6-9), whereas in the second nucleosomes are somehow clustered in discrete globular particles (superbeads) (10-13). In any case, many experimental results demonstrate that the very lysine-rich histones (histones of the HI class and histone H5) organize the supranucleosomal packaging through interactions with nucleosomes and probably also with themselves (6,9,14,15).Binding of very lysine-rich histones to DNA is not sufficient to induce chromatin condensation into a thick fiber. At lowNaCl concentrations chromatin appears as a linear chain of nucleosomes, although very lysine-rich histones remain bound to it (6,9,14). Increasing the NaCl concentration leads to a folding of the nucleosome chain into the 250-to 300-A fiber (6,9,14). This transition occurs in a rather narrow salt range (between 20 and 40 mM NaCl), which coincides with the range in which very lysine-rich histones change their binding mode to DNA from a noncooperative to a cooperative interaction (14,16). This suggests that interactions between these histones are important for the maintenance of the 250-to 300-A fiber configuration. In this communication we describe experiments which show that this is indeed the case. Workinq at 30 mM NaCI, an ionic strength at which the 250-to 300-A fiber is already unstable, we found that small oligonucleosomes aggregate into compact globular structures with a diameter (about 300A) very similar to the thickness of the native chromosomal fiber.The folded and unfolded states of these structures are found to be in equilibrium under these rather destabilizing conditions.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 6443The formation ...

Assembly of oligonucleosomes into a limit series of multimeric higher-order chromatin structures

“…The pvesence of an assembly barrier has alio been supoested by Jorcano et a1 (18). These author5 showed that particles containinp about 25 nucleosomes were fnrmpd hv an aaareaation of oliaanuclkosomes in the sire ranme of trimers UD to .…”

“…It has been peported t h a t t h e H1, H5 molecules accumulate on -, and s t a b i l i s e t h e m a t e r i a l present w i t h i n , t h e f a s t sedimentina peak of t o t a l n u c l e a r e x t r a c t s (11,12,14,18,20,21). Therefore we t r i e d t o q i a n t i t a t e t h e amount of l y s i n e -r i c h h i s t o n e s present on our assembly products.…”

“…One must, however, be careful when considering the in vitro observation of these supranucleosomal particles as representative for the chromatin conformation in vivo. Indeed, the aggregation of small oligonucleosomes with lysine-rich histones into larger structures has been reported [18, 191. Such an aggregation could also account for the observation of discrete supranucleosomal structures.…”

“…Consequently some oligonucleosomes will aggregate into larger structures with the aid of additional H1 and H5 molecules (arising from other oligo-or mononucleosomes and reflecting the H5 preference for higher-order structures [26]. These aggregates survive exposure to low ionic strength [18,261. Therefore their oligonucleosomal fractions will be slightly depleted of H1 and H5 molecules compared to our fractions.…”

Chicken erythrocyte nucleus contains two classes of chromatin that differ in micrococcal nuclease susceptibility and solubility at physiological ionic strength.

“…Chromatin soluble in digestion buffer (ionic strength "70 mM) but insoluble at physiological ionic strength (I chromatin) was broken down to mononucleosomes that were insoluble in digestion buffer (Fig. 2) Jorcano et at (29) have recently reported a study on chicken erythrocyte bulk chromatin. Extensive nucleolysis ofbulk chromatin at 370C resulted in two main fractions: mono-and dinucleosomes that did not aggregate and were deficient in the lysine-rich histones H1 and H5 and oligonucleosomes (3 c R < 8) that contained an excess of lysine-rich histones and were capable of associating into globular particles containing 25-29 nucleosomes.…”