Specific binding of cholesterol to chromatin prepared from mouse spleen cells

Mouse spleen cell suspensions were incubated with tritiated cholesterol for various time intervals. The chromatin of these cells was then isolated, washed with Triton X-100, and fractionated on Sephadex columns. It was found that cholesterol binds specifically to the chromatin. The binding was saturated after 45 min of incubation and also displayed characteristics typical of dose-dependent binding. The number of cholesterol molecules bound per nucleus was estimated to be on the order of 10 000. Oxygenated sterols, such as 25-hydroxycholesterol and 7-ketocholesterol, did not compete for the binding with [3H]cholesterol if added in 20-fold molar excess to the incubation medium of the cells. Chromatographic analyses on Sephadex columns displayed a distinct peak of radioactivity. The protein-sterol complex had an apparent molecular weight of 180 000 +/- 27 000. Using extensive digestion with DNase I (EC 3.1.21.1) it could be concluded that DNA, binding to the complex, did not influence the estimate of the molecular weight, whereas digestion with pronase or treatment with sodium dodecyl sulfate destroyed the complex. Additional experiments using sucrose density gradients (5-20%) showed also, that [3H]cholesterol was bound to chromatin by one or several proteins.