Inhibition of multicellular development switches cell death of Dictyostelium discoideum towards mammalian-like unicellular apoptosis.

The multicellular development of the single celled eukaryote Dictyostelium discoideum is induced by starvation and consists of initial aggregation of the isolated amoebae, followed by their differentiation into viable spores and dead stalk cells. These stalk cells retain their structural integrity inside a stalk tube that support the spores in the fruiting body. Terminal differentiation into stalk cells has been shown to share several features with programmed cell death (Cornillon et al. (1994), J. Cell Sci. 107, 2691-2704). Here we report that, in the absence of aggregation and differentiation, D. discoideum can undergo another form of programmed cell death that closely resembles apoptosis of most mammalian cells, involves loss of mitochondrial transmembrane potential, phosphatidylserine surface exposure, and engulfment of dying cells by neighboring D. discoideum cells. This death has been studied by various techniques (light microscopy and scanning or transmission electron microscopy, flow cytometry, DNA electrophoresis), in two different conditions inhibiting D. discoideum multicellular development. The first one, corresponding to an induced unicellular cell death, was obtained by starving the cells in a "conditioned" cell-free buffer, prepared by previous starvation of another D. discoideum cell population in potassium phosphate buffer (pH 6.8). The second one, corresponding to death of D. discoideum after axenic growth in suspension, was obtained by keeping stationary cells in their culture medium. In both cases of these unicellular-specific cell deaths, microscopy revealed morphological features known as hallmarks of apoptosis for higher eukaryotic cells and apoptosis was further corroborated by flow cytometry. The occurrence in D. discoideum of programmed cell death with two different phenotypes, depending on its multicellular or unicellular status, is further discussed.

Inner nuclear membrane protein LBR preferentially interacts with DNA secondary structures and nucleosomal linker.

The lamin B receptor (LBR) is an integral protein of inner nuclear membrane whose nucleoplasmic amino-terminal domain contributes to the attachment of the membrane to chromatin. Here we analyzed the interactions of a recombinant GST protein containing the amino-terminal domain of the protein with in vitro reconstituted nucleosomes and short DNA fragments. Data show that the LBR amino-terminal domain (AT) binds linker DNA but does not interact with the nucleosome core. Titration and competition studies revealed that the interaction between LBR AT and DNA is saturable, of high affinity (K(D) approximately 4 nM), independent of DNA sequence, and enhanced by DNA curvature and supercoiling. In this respect, LBR amino-terminal domain binding to nucleosomes is similar to that of histone H1 and non histone proteins HMG1/2 which both bind preferentially to linker DNA and present a significant affinity for DNA secondary structures.

LBR, a chromatin and lamin binding protein from the inner nuclear membrane, is proteolyzed at late stages of apoptosis.

Chromatin condensation and apposition to the nuclear envelope is an important feature of the execution phase of apoptosis. During this process, lamin proteins that are located between the inner nuclear membrane and heterochromatin are proteolyzed by the apoptosis-specific protease caspase 6. We have investigated the fate of nuclear membranes during apoptosis by studying the lamin B receptor (LBR), a transmembrane protein of the inner nuclear membrane. LBR interacts through its nucleoplasmic amino-terminal domain with both heterochromatin and B-type lamins, and is phosphorylated throughout the cell cycle, but on different sites in interphase and mitosis. We report here that: (i) the amino-terminal domain of LBR is specifically cleaved during apoptosis to generate an approximately 20 kDa soluble fragment; (ii) the cleavage of LBR is a late event of apoptosis and occurs subsequent to lamin B cleavage; (iii) the phosphorylation of LBR during apoptosis is similar to that occurring in interphase. As the association of condensed chromatin with the inner nuclear membrane persists until the late stages of apoptosis, we suggest that the chromatin binding protein LBR plays a major role in maintaining this association.

Octamer displacement and redistribution in transcription of single nucleosomes.

Single nucleosomes were assembled on a 357bp DNA fragment containing a 5S RNA gene from sea urchin and a promoter for SP6 RNA polymerase, and were fractionated as a function of their positions by gel electrophoresis. Transcribed nucleosome positions were detected by observing band disappearance in gels, which in turn provided evidence for the displacement of the histone octamer upon transcription. Differential band disappearance showed that nucleosomes closer to the promoter were harder to transcribe, and transcription was blocked when the nucleosome proximal boundary was at the start site. Nucleosomes located at discrete positions were also eluted from the gel bands and transcribed. In this case, new bands appeared as a consequence of octamer redistribution. Such redistribution occurred over all untranscribed positions, as well as over transcribed positions close enough to the promoter. Similar conclusions were derived from another previously investigated fragment containing a Xenopus 5S RNA gene.

Chromatin reconstitution on small DNA rings. IV. DNA supercoiling and nucleosome sequence preference.

Nucleosome formation on inverted repeats or on some alternations of purines and pyrimidines can be inhibited in vitro by DNA supercoiling through their supercoiling-induced structural transitions to cruciforms or Z-form DNA, respectively. We report here, as a result of study of single nucleosome reconstitutions on a DNA minicircle, that a physiological level of DNA supercoiling can also enhance nucleosome sequence preference. The 357 base-pair minicircle was composed of a promoter of phage SP6 RNA polymerase joined to a 256 base-pair fragment containing a sea urchin 5 S RNA gene. Nucleosome formation on the promoter was found to be enhanced on a topoisomer with in vivo superhelix density when compared to topoisomers of lower or higher superhelical densities, to the nicked circle, or to the linear DNA. In contrast, nucleosomes at other positions appeared to be insensitive to supercoiling. This observation relied on a novel procedure for the investigation of nucleosome positioning. The reconstituted circular chromatin was first linearized using a restriction endonuclease, and the linear chromatin so obtained was electrophoresed as nucleoprotein in a polyacrylamide gel. The gel showed well-fractionated bands whose mobilities were a V-like function of nucleosome positions, with the nucleosome near the middle migrating less. This behavior is similar to that previously observed for complexes of sequence-specific DNA-bending proteins with circularly permuted DNA fragments, and presumably reflects the change in the direction of the DNA axis between the entrance and the exit of the particle. Possible mechanisms for such supercoiling-induced modulation of nucleosome formation are discussed in the light of the supercoiling-dependent susceptibility to cleavage of the naked minicircle with S1 and Bal31 nucleases; and a comparison between DNase I cleavage patterns of the modulated nucleosome and of another, non-modulated, overlapping nucleosome.

Chromatin reconstitution on small DNA rings. III. Histone H5 dependence of DNA supercoiling in the nucleosome.

Mononucleosomes were reconstituted on small DNA rings in the presence of histone H5 and relaxed to an equilibrium using calf thymus topoisomerase I. DNA products, when compared to the equilibria observed with the same minicircles in the absence of histones, showed that a linking number reduction of 1.6 to 1.7 was associated with this reconstitution, in contrast with the 1.1 to 1.2 figure reported in our recent study of the H5-free nucleosome. Gel electrophoretic properties and electron microscopic visualization of the nucleosomes suggest a correlation between this increase and a further wrapping of the DNA around the histone core from less than 1.5 turns of the superhelix in the absence of H5, to close to two turns in its presence. Implications for DNA topology in chromatin are discussed.