Tubulin-chromatin interactions: evidence for tubulin-binding sites on chromatin and isolated oligonucleosomes

Abstract

The interaction of tubulin with chromatin has been studied using a radiolabeled tubulin binding assay and velocity sedimentation analysis on isokinetic sucrose gradients. Soluble chromatin was prepared by mild micrococcal nuclease digestion of rat liver nuclei and tubulin was purified from rat brain by temperature-dependent assembly-disassembly and phosphocellulose chromatography. The tubulin-binding assay is based on the ability of chromatin to precipitate quantitatively at physiological ionic strength allowing separation of free tubulin from chromatin-bound tubulin. The binding of tubulin to unfractionated soluble chromatin was rapid, reversible and saturable. Saturation of binding sites was obtained using tubulin concentrations ranging from 0.5 to 400 micrograms/ml, in the presence of a high concentration (2.5 mg/ml) of another acidic protein, bovine serum albumin. The Scatchard and Hill plots showed that tubulin bound to a single class of non-interacting sites and yielded values of (0.5-0.6) X 10(7) M-1 for an apparent Ka and a maximal binding capacity of 0.8 nmol tubulin/mg DNA, i.e. about 1 molecule of tubulin/10 nucleosomes. Similar binding parameters were obtained when binding experiments were performed with insoluble chromatin in 0.15 M NaCl. Velocity sedimentation analysis of tubulin-chromatin complexes revealed that tubulin bound to all classes of chromatin oligomers, irrespective of the length of the nucleosomal chain. Tubulin-trinucleosome complexes formed from isolated trinucleosome in the presence of an excess of tubulin were separated from free reactants. It was found that 10-15% of the starting oligonucleosomal species reacted with tubulin, in a stoichiometry of about 0.8 molecule of tubulin/nucleosome. Given the characteristics of the binding and the expected cellular free tubulin concentration, the tubulin-chromatin interaction could possibly take place in vivo, when the nuclear membrane breaks down during the first steps of mitosis.