Orientation of nucleosomes in the thirty-nanometer chromatin fiber

Abstract

We have measured the linear dichroism of Mg2+-stabilized and dimethylsuberimidate cross-linked 30-nm chromatin fibers, using electric fields to produce orientation. The limiting dichroism of Mg2+-stabilized fibers at low monovalent ion concentration is -0.09, with indistinguishable results for avian erythrocyte and calf thymus chromatin. Correction of the apparent dichroism signal for an absorbance change that becomes significant at high electric field yields the dichroism of -0.09 and reveals that the fibers reach saturation of their orientation by about 15 kV/cm. Calf thymus chromatin fibers cross-linked at 100 mM NaCl concentration have a dichroism of +0.05, with a dependence of orientation on field similar to that observed for Mg2+-stabilized fibers. Comparative sedimentation studies of Mg2+-stabilized fibers and fibers in 100 mM cross-linking buffer revealed a 25% sedimentation coefficient increase accompanying the dichroism increase from -0.09 to +0.05. We interpret the results to mean that the nucleosomal disk diameters form an angle of about 30 degrees to the chromatin fiber axis in Mg2+-stabilized fibers at low monovalent ion concentration. When 100 mM NaCl is added, the fiber becomes more compact, and the disk diameter angles increase by 8 degrees, to about 38 degrees. Cross-linking itself may also contribute slightly to fiber compaction. The results are consistent with a large longitudinal compressibility of the 30-nm fiber, a feature required for the bendability necessary when the fiber is further coiled to form structures such as chromosomes. Our results indicate that compression is accommodated by small changes in the angular orientation of the nucleosomal disks.