Thermodynamics of condensation of nuclear chromatin. A differential scanning calorimetry study of the salt-dependent structural transitions.

We present a detailed thermodynamic investigation of the conformational transitions of chromatin in calf thymus nuclei. Differential scanning calorimetry was used as the leading method, in combination with infrared spectroscopy, electron microscopy, and techniques for the molecular characterization of chromatin components. The conformational transitions were induced by changes in the counterion concentration. In this way, it was possible to discriminate between the interactions responsible for the folding of the higher order structure and for the coiling of nucleosomal DNA. Our experiments confirm that the denaturation of nuclear chromatin at physiological ionic strength occurs at the level of discrete structural domains, the linker and the core particle, and we were able to rule out that the actual denaturation pattern might be determined by dissociation of the nucleohistone complex and successive migration of free histones toward native regions, as recently suggested. The sequence of the denaturation events is (1) the conformational change of the histone complement at 66 degrees C, (2) the unstacking of the linker DNA at 74 degrees C, and (3) the unstacking of the core particle DNA, that can be observed either at 90 or at 107 degrees C, depending on the degree of condensation of chromatin. Nuclear chromatin unfolds in low-salt buffers, and can be refolded by increasing the ionic strength, in accordance with the well-known behavior of short fragments. The process is athermal, therefore showing that the stability of the higher order structure depends on electrostatic interactions. The transition between the folded conformation and the unfolded one proceeds through an intermediate condensation state, revealed by an endotherm at 101 degrees C. The analysis of the thermodynamic parameters of denaturation of the polynucleosomal chain demonstrates that the wrapping of the DNA around the histone octamer involves a large energy change. The most striking observation concerns the linker segment, which melts a few degrees below the peak temperature of naked DNA. This finding is in line with previous thermal denaturation investigations on isolated chromatin at low ionic strength, and suggests that a progressive destabilization of the linker occurs in the course of the salt-induced coiling of DNA in the nucleosome.