Structural domains and conformational changes in nuclear chromatin: a quantitative thermodynamic approach by differential scanning calorimetry

Abstract

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)

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

“…Scan 4, nuclei prepared from thymocytes cultured for 18 h in the absence of MPS; minor alterations in the thermal profile with respect to the control (scan 1) are apparent, pointing to the occurrence of a limited extent of apoptosis after culture in agreement with previous observations (2). As expected, after 18 h of treatment the thermal profile (scan 5) shows several similarities with that of nuclei extensively digested with micrococcal nuclease (scan 6) since both the unfolding of the 30-nm fiber and DNA fragmentation result in the disappearance of endotherm V at 107°C (12,26). Extensively degraded chromatin is, however, further characterized by an appreciable decrease (from 90 to 84°C) in the temperature of endotherm IV (12,26).…”

“…As expected, after 18 h of treatment the thermal profile (scan 5) shows several similarities with that of nuclei extensively digested with micrococcal nuclease (scan 6) since both the unfolding of the 30-nm fiber and DNA fragmentation result in the disappearance of endotherm V at 107°C (12,26). Extensively degraded chromatin is, however, further characterized by an appreciable decrease (from 90 to 84°C) in the temperature of endotherm IV (12,26). The bar corresponds to 3 m. The size marker is ⌽X174/HaeIII.…”

“…Thus, the structural changes of nuclear chromatin can be quantitatively detected. By this approach, we and others have previously characterized the condensation process induced by salts (12,27) or histone H1 (10) in the absence of DNA cleavage as well as the effect of endogenous or exogenous nucleases (26). The DSC analysis of nuclear chromatin from thymocytes undergoing apoptosis led to a clear-cut result (Fig.…”

“…The thermal denaturation profile of interphase nuclei shows two major heat absorption peaks (labeled IV and V in Fig. 1H) at 90 and 107°C that arise from the melting of the core particle DNA when the core particle is placed within an unfolded loop and the higher order structure domains, respectively (12,26). Thus, the structural changes of nuclear chromatin can be quantitatively detected.…”

“…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.…”