Chromatin changes in cell transformation: progressive unfolding of the higher-order structure during the evolution of rat hepatocyte nodules. A differential scanning calorimetry study

Abstract

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.

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.…”

“…Nuclei were obtained by incubation of the pelleted cells in dissociation medium (DM) (75 mM NaCl, 24 mM Na 2 EDTA, 5 mM NaHSO 3 , and 1 mM phenylmethylsulfonyl fluoride, pH 7.8) containing 0.15% (v/v) Triton X-100, following the procedure already employed for calf thymus (12); in the digestion experiments nuclei were incubated at 37°C for 3 h with 50 units/ml micrococcal nuclease (Sigma) according to Russo et al (10). For electron microscopy of thin sections, control, glucocorticoid-treated cells, and nuclei were pelleted at 150 ϫ g for 10 min; fixation, embedding, and sectioning were performed as already reported (13). DNA was isolated by digestion of purified nuclei with proteinase K (Serva) in 10 mM Tris-HCl, 5 mM EDTA, 0.5% SDS, pH 8.0, followed by extraction with phenol and chloroform, and the chain length distribution of the fragments was analyzed on 1.5% agarose gels (14).…”

“…Calorimetry-Differential scanning calorimetry (DSC) experiments were performed as already reported (12,13). Nuclei were isolated from control and from MPS-treated thymocytes as described above.…”

Partially Assembled Nucleosome Structures at Atomic Detail

“…Experimental studies point to progressive unfolding of the chromatin structure in anomalously proliferating (cancer) cells (19,20), which could be associated with PANS formation. Recent genome-wide studies revealed an existence of widespread subnucleosomal structures in dynamic chromatin of living cell (21)(22)(23).…”

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

“…The answer is no. We recall that transition IV results from the melting of the core particles placed within an extended (euchromatin) domain [Balbi et al, 1989]; this subpopulation does not undergo salt induced condensation, as a consequence of the binding of trans active factors and/or of specific interactions with the nuclear matrix [Barboro et al, 1993]. The amount of euchromatin is expected to be related within the number of genes which are at work in a given cell type; since this number is almost the same in all of resting cells, we can explain why DH m IV represents a fixed fraction ($0.3) of the total denaturation enthalpy of nuclear chromatin [Cavazza et al, 1991].…”