Role of H1 in Chromatin Folding. A Thermodynamic Study of Chromatin Reconstitution by Differential Scanning Calorimetry

Abstract

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, such as the intercalating dye ethidium bromide or the ionic strength. In all these studies, we took advantage of the capability of differential scanning calorimetry to detect the changes in the melting behavior of the structural domains of chromatin (the linker and the core particle) associated with the order-disorder transitions. This technique also revealed that the higher-order structure undergoes a catastrophic decondensation process in the course of the transformation of rat hepatocytes as well as of cultured cells. Therefore, several questions arose concerning the biological function (if any) of the changes in the degree of condensation of bulk chromatin, as well as the mechanism of transition and the nature of the modulating agents. In this paper, we report a thermodynamic analysis of the reconstitution of H1-depleted calf thymus chromatin with the purpose of establishing (1) the binding mode of H1 and (2) the energetics and cooperativity of the transition from the unfolded to the condensed state. When H1 is progressively extracted 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 sites with KA = (3.67 +/- 0.60) x 10(4) M-1 and each site comprising 180 +/- 10 bp. The experimental dependence of the fraction of condensed chromatin on R, the moles of bound H1 per nucleosome mole, was compared with a simple thermodynamic model for the conformational change. This analysis yields a value of -5 kcal per nucleosome mole for the interaction free energy of nucleosomes within the ordered state. The process of condensation, is not, however, a highly cooperative (all-or-none) one, as expected from a consideration of the solenoidal model for the 30 nm fiber. Rather, nucleation of the helical state involves the face-to-face interaction between consecutive core particles, and the growth is largely determined by the mergence and rearrangement of neighboring clusters of helically arrayed nucleosomes.

Chromatin: A tunable spring at work inside chromosomes

“…Moreover, this configuration strongly favours a second compaction step, ensured by the attractive interactions between nucleosomes (or rather chromatosomes) that arise when the nuclesome faces are close enough. We in particular recover in this scheme the two-stage compaction of the 30-nm chromatin fiber observed experimentally [46] [47]. An insight on this interaction-induced compaction can be obtained by setting the effective parameters r nucl and H to 0, thus mimicking the enhanced influence of linker histone at high salt; in this case, P decreases to about 6 nm, indicating that nucleosomes actually stack very closely onto each other and lead to a superstable (and presumably rigid) fiber.…”

The Condensation of Chromatin in Apoptotic Thymocytes Shows a Specific Structural Change

“…Due to its sharp subdivision into two basic supramolecular domains (the linker and the core particle) the polynucleosomal chain lends itself to conformational studies by DSC. Therefore, this technique is a powerful tool for investigating the overall organization of chromatin in situ (10,12,13,(25)(26)(27). The thermal denaturation profile of interphase nuclei shows two major heat absorption peaks (labeled IV and V in Fig.…”

“…In a recent paper, we have shown that the nucleosomes interact weakly in the 30-nm fiber. The experimental value of the interaction free energy is as low in magnitude as Ϫ5 kcal/ nucleosome mole (10), so that even limited biochemical modifications of the histone complement could result in a structural change. In this report we show that the basic conformational feature of chromatin in apoptosis is the tight face-to-face packaging of nucleosomes, which might be related to the increase in the amount of the unacetylated forms of histones H3 and H4 occurring in the course of condensation.…”

SCN− binding to the charged lysines of histones end domains mimics acetylation and shows the major histone–DNA interactions involved in eu and heterochromatin stabilization

“…After staining with Coomassie brilliant blue the amounts of histones were determined by scanning the gels as already reported [Cavazza et al, 1991;Russo et al, 1995]. The same procedure was used to evaluate the fraction of trypsinized histones.…”

“…This correlation was established by exploiting the extraordinary capability of differential scanning calorimetry (DSC) to distinguish between the condensed and the unfolded state of the genome [Nicolini et al, 1983]. Using DSC, we and other groups were indeed able to characterize the thermodynamics of salt induces condensation [Cavazza et al, 1991;Labarbe et al, 1996], to elucidate the mechanism of binding of H1 to core chromatin [Russo et al, 1995], to describe the all-or-none structural transition which occurs inside the chromatin loop [Balbi et al, 1999] and to identify the role of histone acetylation [Gavazzo et al, 1997] and of the nuclear envelope [Spadiliero et al, 2002], respectively, in the modulation of chromatin higher order structure.…”