Temperature-induced chromatin changes in ethanol-fixed cells.

Abstract

We studied the chromatin structure of rat thymocytes fixed in 70% ethanol at 0-44 degrees C by flow cytometry and gel electrophoresis. The fluorescence of the DNA-specific dye mithramycin increased by 93% when thymocytes were exposed at 44 degrees C in the fixative compared to cells kept at 0 degrees C. Antibody labeling (X-ANA) of the core histones was 65% lower for the 44 degrees C-treated cells compared to the control cells (0 degree C). The emission anisotropies of the DNA-specific dye Hoechst 33258 bound to chromatin were 0.341 and 0.318 for thymocytes fixed at 0 degree C and 44 degrees C, respectively. Increased mobility of DNA in chromatin of 44 degrees C-treated cells, as revealed by the emission anisotropy of Hoechst 33258, was not due to denaturation of DNA but was probably caused by removal of constraints situated at short intervals (less than or equal to 50 BP) along the DNA helix. The short intervals between these constraints in chromatin fixed at 0 degree C suggests that they were histones. PAGE of 0.5 N H2SO4-extracted histones showed that the 44 degrees C treatment reduced total core histone content by 65% and that the different histones were lost in unequal amounts. The loss was about 75% and 54% for the histone pairs H3/H4 and H2A/H2B, respectively. The amount of H1 was reduced by about 25% on temperature treatment. The temperature-induced change in the chromatin structure of the cells in 70% ethanol was biphasic. A change in the three-dimensional structure of chromatin occurred for temperatures up to 20 degrees C (no histones were released but binding of mithramycin increased by approximately 15%, whereas the binding of X-ANA decreased by the same amount). Sixty-five percent of core histones were released in the second phase (20-44 degrees C), which may explain the further increase and decrease in the binding of mithramycin and X-ANA, respectively.