Differential scanning calorimetry of nuclei reveals the loss of major structural features in chromatin by brief nuclease treatment.

Abstract

Differential scanning calorimetry revealed that chromatin melts in four distinct transitions in intact HeLa nuclei at 60°C, 76C, 88°C, and 105°C. Calorimetry of whole cells was characterized by the same four transitions along with another at 65°C, which was probably RNA. Isolated chromatin, however, melted in only two transitions at 72°C and 85°C. Very brief digestion of HeLa nuclei with either micrococcal nuclease or DNase I resulted in the conversion of a structure that melted at 105°C to one that melted at 880C. Further digestion with micrococcal nuclease to the level of the mononucleosome did not result in any further substantial changes in either enthalpy or melting temperatures. In contrast, further DNase I digestion that resulted in cleavage within the nucleosome produced a pronounced shift in melting temperatures to two broad transitions at 62C and 78°C.Eukaryotic DNA exists as long continuous molecules but is condensed on the order of 1000-fold to fit within interphase nuclei (1). The lowest level of folding is that of the nucleosome, a repeating subunit consisting of two each of the core histones H2A, H2B, H3, and H4, along with about 200 base pairs of DNA and one molecule of histone H1. Histone H1 is believed to effect further condensation to a 300-A fiber that has been postulated to be a solenoid (2).Studies of chromatin structure above the level of the solenoid have been hampered due to the tendency of chromatin fibers to form aggregates and turbid suspensions at physiological salt conditions. The use of differential scanning microcalorimetry in studies of chromatin structure has the advantage that samples need not be optically clear as is required for spectral studies (3,4 (7). These workers correlated the intensities of the endotherms observed with the relative amounts of DNA and RNA present in several samples.The results of our study suggest that differential scanning calorimetry can be used to characterize the native chromatin structures in nuclei and that nuclei are to be preferred over isolated chromatin and whole cells. The thermodynamically significant chromatin structures of whole cells were shown to be retained in the nucleus, but a major structure was destroyed by brief micrococcal nuclease treatment. MATERIALS AND METHODSCell Culture. HeLa cells, strain S3, were maintained in suspension culture at cell densities of 2-8 x 105 per ml in Joklik's modified spinner medium supplemented with 5% calf serum. Cells were harvested at cell densities of 5-6 x 105 per ml.Isolation of Nuclei. All steps were carried out at 0-40C. (pH 7.5). NaCl, MgCl2, and CaCl2 were added to final concentrations of 150 mM, 1 mM, -and 1 mM, respectively, and the chromatin was incubated for 1 hr at room temperature. Insoluble chromatin was centrifuged for 10 min at 8000 x g and the pellet was used for calorimetric scanning.Gel Electrophoresis of DNA Digestion Fragments. Following digestion by micrococcal nuclease or DNase I, nuclei were pelleted and dissolved in 5 ml of 1 M NaCl/0.6% NaDodSO4 and then extracted ...

Differential scanning calorimetry of chicken erythrocyte nuclei

“…Thus, the first peak (i.e. that at lowest temperature) has been assigned to nuclear matrix proteins and to hnRNA-associated proteins by Balbi et al [5], and to RNA [3] or proteins not usually considered part of chromatin structure [4] There is general agreement that the fourth transition, i.e. that with a mid-point higher than 95 "C, is likely to be due to a particular structural organization of DNA in the intact nucleus or in chromatin carefully prepared without shearing.…”

Effects of tacrine upon murine neuroblastoma cells

“…Thermal transitions on N2A preparations observed at 74~ and 85~ regard, according to the literature (Touchette and Cole, 1985;Touchette et al, 1986), the melting point of DNA and of the DNA-binding proteins. All these findings appear to be relevant results that could open, at least potentially, new pharmacological perspectives on applications of THA besides the AD therapy.…”

“…Differential scanning calorimetry (DSC) was carried out using a Perkin Elmer DSC4 and following the method as described elsewhere (Touchette and Cole, 1985;Touchette et al, 1986).…”

“…High concentration of THA (100 ~tM) was utilized only to maximize the interaction effects due to the low sensitivity of this measurement. The transition at 65~ has been ascribed to ribosomal RNA (Touchette and Cole, 1985). …”

Microcalorimetric Study of Iodized and Noniodized Cells and C-Phycocyanin of Spirulina platensis

“…[1][2][3][4][5] The importance of these studies is that the structural transformations of biopolymers in dilute solutions do not always coincide with the same changes in situ and in vivo. And it is not surprising because many biopolymers function in living cells as supramolecular complexes.…”