ROLE OF H1 IN CHROMATIN FOLDING - A THERMODYNAMIC STUDY OF CHROMATIN RECONSTITUTION BY DIFFERENTIAL SCANNING CALORIMETRY

In a series of related papers, we have recently presented the results of a thermodynamic approach to the conformational transitions of bulk chromatin induced in vitro by different structure-perturbing agents, s uch as the intercalating dye ethidium bromide or the ionic strength. I n all these studies, we took advantage of the capability of differenti al scanning calorimetry to detect the changes in the melting behavior of the structural domains of chromatin (the linker and the core partic le) associated with the order-disorder transitions. This technique als o revealed that the higher-order structure undergoes a catastrophic de condensation process in the course of the transformation of rat hepato cytes as well as of cultured cells. Therefore, several questions arose concerning the biological function (if any) of the changes in the deg ree of condensation of bulk chromatin, as well as the mechanism of tra nsition and the nature of the modulating agents. In this paper, we rep ort a thermodynamic analysis of the reconstitution of Ill-depleted cal f thymus chromatin with the purpose of establishing (1) the binding mo de of H1 and (2) the energetics and cooperativity of the transition fr om the unfolded to the condensed state. When H1 is progressively extra cted from calf thymus nuclei by high-salt treatment, the endotherm at 107 degrees C, characteristic of the core particles interacting within condensed domains, converts into the thermal transition at 90 degrees C, resulting from the denaturation of noninteracting core particles. Binding of H1 fully restores the thermal profile of native chromatin. Analysis of H1 association shows that binding occurs at independent si tes with K-A = (3.67 +/- 0.60) x 10(4) M(-1) and each site comprising 180 +/- 10 bp. The experimental dependence of the fraction of condense d chromatin on R, the moles of bound H1 per nucleosome mole, was compa red with a simple thermodynamic model for the conformational change. T his analysis yields a value of -5 kcal per nucleosome mole for the int eraction free energy of nucleosomes within the ordered state. The proc ess of condensation, is not, however, a highly cooperative (all-or-non e) one, as expected from a consideration of the solenoidal model for t he 30 nm fiber. Rather, nucleation of the helical state involves the f ace-to-face interaction between consecutive core particles, and the gr owth is largely determined by the mergence and rearrangement of neighb oring clusters of helically arrayed nucleosomes.