Investigations on the refolding of chromatin using scanning force microscopy have recently renewed interest in the three-dimensional organization of the higher order structure, so complementing several observations on the molecular functioning of the transcription machinery showing that the unfolding of heterochromatin represents an obligatory step in gene activation and vice versa. A few years ago we (Allera et al. J. Biol. Chem. 272, 10817, 1997) reported several striking observations on the status of chromatin of apoptotic thymocytes. All of the genome was found to be organized into a woven network of 11 nm fibers, arising from the close face to face packing of nucleosomes, a conformation strongly reminiscent of the organization of the columnar phases of core particles described by Levforestier and Livolant (Biophys. J. 73, 1771, 1997). In addition, the onset of apoptosis involved a significant (from 42 to 70%) increase in the unacetylated isoform of histone H4. In this report we show first that H4 acetylation does not directly modulate chromatin higher order structure. Indeed, apoptosis is induced quite efficiently in rat thymocytes by treatment with sodium butyrate, an inhibitor of histone deacetylases, as judged from the observation of the relevant hallmarks. Also the ultrastructure of chromatin was undistinguishable from that isolated from thymocytes treated with methylprednisolone sodium succinate; the percentage of the unacetylated isoform of H4 (32%) was, however, lower than that of control cells (42 %). Ultrastructural observation in combination with image analysis and Fourier filtering confirmed that the 11 nm fiber arises from the face to face packing of nucleosomes. When the fiber is restrained in the network, however, it experiences a considerable elastic stress. Indeed, a basic morphological motif is the presence of interwound regions giving rise to a characteristic figure of eight configuration. When the intercalating agent ethidium bromide (EB) is added to the chromatin solution at a molar ratio r of 0.028 (r=molecules of bound EB/molecules of DNA phosphate) we observe the complete relaxation of the superstructure but not the separation of the core particles, while at the same value or r the linker appears as a straight segment connecting well separated cores in control chromatin. A second relevant morphological feature is the origin of the crosslinking regions in the network. Image analysis clearly shows that these regions correspond to short stretches of the polynucleosomal chain in which the core particles interact edge to edge, and can therefore firmly associate with another stretch through face to face interactions. Our results demonstrate for the first time that the bulk of apoptotic chromatin is negatively supercoiled. A structural and thermadynamic investigation on the mechanism of heterochromatin formation from our laboratory has shown that this process is driven by an all or none phase transition inside the loop (Balbi et al., Biophys. J., 77, 2725, 1999). Since we have shown that apoptotic condensation occurs in the absence of DNA cleavage, it is plausible to assume that also the chromatin transitions in apoptosis are entirely determined by internucleosomal interactions inside a topologically isolated unit.

Condensation of Chromatin in Apoptosis is Supercoiling determined

D'Arrigo C;Mormino M;
2003

Abstract

Investigations on the refolding of chromatin using scanning force microscopy have recently renewed interest in the three-dimensional organization of the higher order structure, so complementing several observations on the molecular functioning of the transcription machinery showing that the unfolding of heterochromatin represents an obligatory step in gene activation and vice versa. A few years ago we (Allera et al. J. Biol. Chem. 272, 10817, 1997) reported several striking observations on the status of chromatin of apoptotic thymocytes. All of the genome was found to be organized into a woven network of 11 nm fibers, arising from the close face to face packing of nucleosomes, a conformation strongly reminiscent of the organization of the columnar phases of core particles described by Levforestier and Livolant (Biophys. J. 73, 1771, 1997). In addition, the onset of apoptosis involved a significant (from 42 to 70%) increase in the unacetylated isoform of histone H4. In this report we show first that H4 acetylation does not directly modulate chromatin higher order structure. Indeed, apoptosis is induced quite efficiently in rat thymocytes by treatment with sodium butyrate, an inhibitor of histone deacetylases, as judged from the observation of the relevant hallmarks. Also the ultrastructure of chromatin was undistinguishable from that isolated from thymocytes treated with methylprednisolone sodium succinate; the percentage of the unacetylated isoform of H4 (32%) was, however, lower than that of control cells (42 %). Ultrastructural observation in combination with image analysis and Fourier filtering confirmed that the 11 nm fiber arises from the face to face packing of nucleosomes. When the fiber is restrained in the network, however, it experiences a considerable elastic stress. Indeed, a basic morphological motif is the presence of interwound regions giving rise to a characteristic figure of eight configuration. When the intercalating agent ethidium bromide (EB) is added to the chromatin solution at a molar ratio r of 0.028 (r=molecules of bound EB/molecules of DNA phosphate) we observe the complete relaxation of the superstructure but not the separation of the core particles, while at the same value or r the linker appears as a straight segment connecting well separated cores in control chromatin. A second relevant morphological feature is the origin of the crosslinking regions in the network. Image analysis clearly shows that these regions correspond to short stretches of the polynucleosomal chain in which the core particles interact edge to edge, and can therefore firmly associate with another stretch through face to face interactions. Our results demonstrate for the first time that the bulk of apoptotic chromatin is negatively supercoiled. A structural and thermadynamic investigation on the mechanism of heterochromatin formation from our laboratory has shown that this process is driven by an all or none phase transition inside the loop (Balbi et al., Biophys. J., 77, 2725, 1999). Since we have shown that apoptotic condensation occurs in the absence of DNA cleavage, it is plausible to assume that also the chromatin transitions in apoptosis are entirely determined by internucleosomal interactions inside a topologically isolated unit.
2003
Istituto per lo Studio delle Macromolecole - ISMAC - Sede Milano
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.14243/92830
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