Repeating oligonucleosomal units. A new element of chromatin structure

volume 8 Number 31980 Nucleic A c i d s Research Repeating oligonucteosomal units. A new element of chromatin structure A.V.ltkes, B.O.Glotov, L-G.Nikolaev, S.R.Preem and E.S.Severin Institute of Molecular Biology, Academy of Sciences of the USSR, Moscow, 117984, USSR Received 5 December 1979 Supranucleosomal chromatin structure has been analysed by the use of histone H1 polymers crosslinked in nuclei and extended chromatin with bifunctional reagents methyl-4-mercaptobutyrimidate (MMB) and dimethyl suberimidate dlhydrochloride. Almost pure H1 homopolymers were obtained in milligram amounts and examined for the distribution in molecular weights. The H1 homopolymer molecules both from nuclei and chromatin have been found to be integer multiples of an elementary structure (called "clisone") consisting of 12 histone H1 molecules. This finding strongly suggests that nucleosomal chains of chromatin are not uniform but rather organized as repeating oligonucleosomal units each consisting of 12 nucleosomes. Correlation between oligonucleosomal structures in nuclei and chromatin implies that a linearized nucleosomal chain retains the information on chromatin superstructure. The relation of the disclosed 12-nucleosome units to superbeads (nucleomeres) and other structures is discussed. INTRODUCTION Over the past years the large body of evidence has been accumulated supporting that the first level of D M package in 1 2 chromatin is a nucleosomal chain (for review see ' ). Elementary unite of this chain, nuclecsomes, are compact deoxyribonucleoo protein particles containing about 200 base pairs of DKA wound on a protein core of eight "inner" histones (2 x H2A, H2B, H3, H4), and histone H1. While core histones are likely to be responsible for the "static" package of DhA in stable so called core particles (histone octamer complexed with I4O base pairs of DNA ' ), histone H1 due to the particular location in a nucleosomal chain and the ability to crosslink distant regions of Q DKA is supposed to contribute the supranucleosomal chromatin structure and govern the dynamics of this structure. © IRL Press limited. 1 Falconbera Court, London W1V 6FG, U.K. 507 ABSTRACT Nucleic Acids Research Numerous electron microscopic studies on chromatin fibrils revealed that one of the dominant structural levels of chromatin at "physiological" salt concentrations was a fibril ("thick fiber) of 250-300 A in diameter 1 0 " 1 2 just three times that of a nucleosome. TheBe fibril6 were also di3cernable in metaphaee chromosomes ' . Analysis of electron microscopic images of chromatin thick fibers led to a conclusion that nucleosomal chains can be solenoidally packed in a 300 A fibril. According to the proposed model, the solenoid has a pitch of 110 A and the diameter of 300 A, 6-7 nucleoeomes forming one turn of the solenoid. A more detailed speculative model of the thick fiber structure has also been proposed 17 Estimation of the number of nucleosomes in a superbead by mild nuclease treatment of chromatin gave a value of 6-10, and later 1 8 of 12. The uncertainty could arise not only from the real heterogeneity of superbeads but also from comparatively low specificity of nuclease action. It is important, that according to the current conception the thick fiber chromatin structure is the level immediately next to that of nucleosoraal chains. The fine structure of a superbead chain is believed to be determined by local changes in the nucleoacme structure, e.g. by variable length of internucleoeomal spacer DKA. It should be stressed that there are actually no data explaining the discrete nature of chromatin structure at the level of superbeads. In particular, there are no indications of periodicity that could fit the size of a euperbead. Therefore we have postulated that oligonucleosomal organization of chromatin includes periodic structural features sufficient to determine the supranucleosomal structure of chrcir.atin. On the assumption of definite independence of hypothetical 508 An alternative chromatin structure is known to be a chain of large subunits called nucleomeres or superbeads . These units are more or less spherical with an approximate diameter of 200-300 A and coneist of several nucleosomes. They are connected by thinner regions to give a superbead chain. Nucleic Acids Research repeating oligonucleosomal units one may expect the existence of periodic, with respect to a linear nucleosomal chain, distortions (changes) in the pattern of histone-histone interactions in native chromatin. To study interactions between the oligonucleosomal units we analysed hiatones crosslinked into polymers within intact nuclei and extended chromatin with reversible and irreversible bifunctional reagents. MATERIALS AMD METHODS Chemicals employed were of analytical and reagent grade additionally purified, if necessary. Isolation and chemical modification of nuclei and chromatin aa well as preparation of polymer protein fractions (see below) were conducted at 0_4°C. Isolation_of_calf_thvmus_nuclei 100 g of frozen calf thymus glands was scissor-minced into pieces and homogenized in 400 ml of 0.25 M sucrose, 10 mM 0.003% (v/v) B-mercaptoethanol, 10 mM TEA*-HC1, pH 7.9. The homogenate was filtered through two layers of cheese-cloth, centrifuged for 5 min at 800 x g, and the nuclear pellet obtained was resuspended in the same buffer, but with B-mercaptoethanol ommited, up to a concentration of 10 mg*ml in DKA. ?regaration_of_extended_chromatin a) Calf thymus nuclei in 0.25 M sucrose, 3 mil MgClg, 0.003:* B-mercaptoethanol, 10 mM TEA-HC1, pH 7.9, (buffer A ) , were centrifuged for 5 min at 800 xg, resuspended in 10 mM KaCl, 3 mM Ka o 3DTA*, 0.155* (v/v) Triton X-100, 10 mM TEA-HC1, pH 7-9, *- _ -1 up to a concentration of 10 mg-ml in DNA and thereafter incubated for 40 min under slow magnetic stirring, b) Calf thymus nuclei in buffer A with 0.2% (v/v) Triton X-100 and 0.5 mM PMSF* (Pierce) added were dialysed overnight against 1 mM Na 2 EDTA, 0.5 mM PMSP, 10 mM Tris-HCl, pH 7.9. Suspension of the lysed nuclei was slightly homogenized with a 509 The results obtained in this approach did support that the level of chromatin structure immediately next to the nucleoeomal one was apparently determined by the discrete repeating oligonucleosomal units. Nucleic Acids Research syringe and then sedimented through a cushion of 6 0 * (w/v) sucrose, 10 mM KaCl, 10 mM Na 2 EDTA, 2.5 mM Tris-HCl, pH 7.9, for 1.5 h at 26000 rpm in an SW 27 rotor (Beckman). Chromatin pellet was resuapended in 10 mfc. Tris-HCl, pH 8.0, 0.5 mM PMSP, and then dialysed against 10 mM ftaCl, 1 mM Ka 2 EDTA, 10 mM TEA-HC1, pH 7.9, for 24 h. Crosslinking 2f_Eroteins_in_nuclei_and_chroniatin1_Iaolation_of In case of suberimidate, treatments with HoO^ and iodoacetamide were emitted. Thereafter HC10. was added to a final concentration of 3-5% (w/v) and selective precipitation was allowed to proceed for 30 min. After centrifugation at 8 (...truncated)