A good deal of information on the thermodynamic properties of chromatin was derived in the last few years from optical melting experiments. The structural domains of the polynucleosomal chain, the linker, and the core particle denature as independent units. The differential scanning calorimetry profile of isolated chromatin is made up of three endotherms, at approximately 74, 90, and 107 degrees C, having an almost Gaussian shape. Previous work on this matter, however, was mainly concerned with the dependence of the transition enthalpy on external parameters, such as the ionic strength, or with the melting of nuclei from different sources. In this paper we report the structural assignment of the transitions of rat liver nuclei, observed at 58, 66, 75, 92, and 107 degrees C. They are representative of the quiescent state of the cell. The strategy adopted in this work builds on the method developed for the investigation of complex biological macromolecules. The heat absorption profile of the nucleus was related to the denaturation of isolated nuclear components; electron microscopy and electrophoretic techniques were used for their morphological and molecular characterization. The digestion of chromatin by endogenous nuclease mimics perfectly the decondensation of the higher order structure and represented the source of several misinterpretations. This point was carefully examined in order to define unambiguously the thermal profile of native nuclei. The low-temperature transitions, centered around 58 and 66 degrees C, arise from the melting of scaffolding structures and of the proteins associated with heterogeneous nuclear RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Using differential scanning calorimetry and complementary ultrastructural observations, we have characterized the status of chromatin during the transformation of rat hepatocytes in the resistant hepatocyte model of Solt and Farber (1976. Nature (Lond.). 263:701-703). Differential scanning calorimetry affords a measure of the degree of condensation of chromatin in situ and has therefore been used in this work for the purpose of establishing the nature of the structural changes associated with the emergence of successive cellular populations. Since the resistant hepatocyte model generates a series of synchronous phenotypic changes, it was possible to determine unambiguously the content of heterochromatin at each step of the process. The higher-order structure undergoes a partial relaxation in early developing nodules, isolated 16 weeks after initiation; the thermal transition at 90 degrees C, which is characteristic of noninteracting core particles, increases with respect to control hepatocytes. Dramatic changes occur in persistent (46-week) nodules. The 90 degrees C endotherm dominates the thermogram, while the transition at 107 degrees C, corresponding to the denaturation of the core particle packaged within the heterochromatic domains, disappears. The complete loss of the higher-order structure at this stage of transformation has been further verified by ultrastructural observations on thin nuclear sections. Ten-nm filaments, having a beaded appearance, are scattered throughout the nucleoplasm and clearly result from the decondensation of 30-nm-thick fibers. This catastrophic relaxation process cannot be related to an effective increase in gene activity. Rather, our observations suggest that during transformation chromatin is in a state of high transcriptional competence associated with the alert of general cellular programs. This view is consistent with the finding that in persistent nodules the DNA is extensively hypomethylated with respect to normal liver.

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Differential scanning calorimetry of chromatin isolated from rat liver cells revealed three discrete thermal transitions whose temperatures and melting enthalpies depend on ionic strength in the range 0 to 600 millimolar NaCl. Intact nuclei showed a fourth thermal transition at a lower temperature and different melting enthalpies for the other three transitions still present at temperatures similar to those obtained in isolated chromatin. The data are discussed in terms of the tertiary, quaternary, and quinternary structures of chromatin DNA.

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Extremely large domains of the genome of resting cells (calf thymus) have been visualized in the electron microscope by combining mild extraction procedures with a non-artifactual method of mounting the sample (the phospholipid monolayer technique). The observed chromatin strands, free from distortion, reach contour lengths up to 60 micrometers. After lysis of the nuclei, four classes of fibres may be identified on the basis of their diameters (30, 24, 18 and 11 nm, respectively). The morphology of giant chromatin strands is strikingly regular; long trains of equally sized, arc-shaped segments are observed, their length being, in many cases, multiples of a fixed value. The inflection points delimiting contiguous segments are often associated with laminar fragments of the nuclear envelope or, less frequently, linked to fibrillar elements. It appears that higher-order structures of chromatin in resting cells conform, to a large extent, to a so called ‘drapery-like’ mode, according to which a continuous strand runs between contiguous anchorage sites placed on the nuclear envelope. Because of the presence of regularly spaced inflection points, this organization is much more ordered than expected. Spontaneous unwinding of the fibres at low ionic strength, limited nuclease digestion, and relaxation in the presence of ethidium bromide, have been used as probes of the conformation. All these experiments rule out its identification with a single-strand helix. The final ordered state is attained by folding the basic 11 nm strand and by winding up this configuration on itself. This leads to a coiled-coil or ‘rope-like’ model. The 11 nm strand is ‘punctuated’ by sharp kinks. Roughly, it may be assimilated to a chain of semirigid, freely joined elements. As a consequence, local flexibility is greatly enhanced, so allowing the assembly mode described.