Protamines: structural complexity, evolution and chromatin patterning

Despite their relatively arginine-rich composition, protamines exhibit a high degree of structural variation. Indeed, the primary structure of these histone H1-related sperm nuclear basic proteins (SNBPs) is not random and is the depository of important phylogenetic information. This appears to be the result of their fast rate of evolution driven by positive selection. The way by which the protein variability participates in the transitions that lead to the final highly condensed chromatin organization of spermatozoa at the end of spermiogenesis is not clearly understood. In this paper we focus on the transient chromatin/nucleoplasm patterning that occurs in either a lamellar step or an inversion step during early and mid-spermiogenesis. This takes place in a small subset of protamines in internally fertilizing species of vertebrates, invertebrates and plants. It involves "complex" protamines that are processed, replaced, or undergo side chain modification (such as phosphorylation or disulfide bond formation) during the histone-to-protamine transition. Characteristic features of such patterning, as observed in TEM photomicrographs, include: constancy of the dominant pattern repeat distance λ(m) despite dynamic changes in developmental morphology, bicontinuity of chromatin and nucleoplasm, and chromatin orientation either perpendicular or parallel to the nuclear envelope. This supports the hypothesis that liquid - liquid phase separation by the mechanism of spinodal decomposition may be occurring during spermiogenesis in these species. Spinodal decomposition involves long wave fluctuations of the local concentration with a low energy barrier and thus differs from the mechanism of nucleation and growth that is known to occur during spermiogenesis in internally fertilizing mammals.     

Nuclear morphogenesis during spermiogenesis in the nudibranch molluscHypselodoris tricolor (Gastropoda,opisthobranchia)

An electron microscope study was carried out on Hypselodoris tricolor spermatids to describe the development of the nuclear morphogenesis and investigate the possible cause(s) of the change in the shape of the spermatid nucleus during spermiogenesis. Three different stages may be distinguished in the course of the nuclear morphogenesis on the basis of the morphology and inner organization of the nucleus. Stage 1 spermatid nuclei are spherical or ovoid in shape and the nucleoplasm finely granular in appearance. Stage 2 nuclei exhibit a disc- or cup-shaped morphology, and the chromatin forms short, thin filaments. During stage 3, a progressive nuclear elongation takes place, accompanied by chromatin rearrangement, first into fibers and then into lamellae, both formations helically oriented. A row of microtubules attached to the nuclear envelope completely surrounds the nucleus. Interestingly, the microtubules always lie parallel to the chromatin fibers adjacent to them. Late stage 3 spermatids show the highest degree of chromatin condensation and lack the manchette at the end of spermiogenesis. Our findings indicate the existence of a clear influence exerted on the chromatin by the manchette microtubules, which appear to be involved in determining the specific pattern of chromatin condensation in Hypselodoris tricolor.     

Characterization of chromatin-condensing proteins during spermiogenesis in a neogastropod mollusc (Murex brandaris)

During the process of chromatin cndensation in the spermiogenesis of the neogastropod mollusc Murex brandaris, the nuclear protein complement undergoes a complex series of changes. These changes lead to the appearance of three small protamines in the ripe sperm nuclei. We have characterized this system electrophoretically and at the compositions with antibodies elicited against a specific spermatozoan protamine. Our results indicate that the complex pattern of chromatin condensation during spermiogenesis in this species (M. brandaris) may be modulated by a series of post-translational (and intranuclear) modifications of DNA-interacting proteins, such as precursors to the sperm protamines. The amino acid composition of each sperm protamine is remarkably simple (lys + arg + gly ≥96 mol%). This system of spermiogenic/spermatozoal proteins in the neogastropod M. brandaris clearly differs from that in patellogastropods and archaeogastropods, and it may be helpful in understanding evolutionary changes in the chromatin condensation pattern during the spermiogenesis of gastropod molluscs. © 1994 Wiley-Liss, Inc.     

Cytopathological effects of protein synthesis inhibitor emetine on HeLa cells and their nucleoli

Eukaryotic cell nucleolus is a highly dynamic structure, which is sensitive to all changes within or outside cell borders. Numerous data are available on changes of the nucleolar structure and functions under different treatments. However, almost nothing is known about the action of translation inhibitors on the nucleolus, although these substances, together with TNF-alpha, are commonly used for apoptosis induction, both for scientific and therapeutic purposes. Emetine is one of such inhibitors. We have shown that emetine suppresses cell viability, decreases mitotic index, and induces apoptosis in HeLa cells. Emetine action is irreversible, and it sensitizes cells to unfavourable external conditions. The emetine action causes redistribution of UBF, one of RNA-polymerase I factor, from the nucleolus to nucleoplasm even after a short exposure, i.e. when the morphology of the nucleus and chromatin still keeps its native pattern. It is important that other nucleolar proteins, such as fibrillarin and B23, are not recognized in the nucleoplasm until the very late stages of apoptotic process. A suggestion is made that changes in UBF localization may be associated with the onset of ribosomal repeat cleavage and migration of rDNA-"free" fragments from the nucleolus to nucleoplasm. It looks likely that these changes can serve as an initial morphological indication of apoptosis.     

Nucleosome positioning and nucleosome stacking: two faces of the same coin

Nucleosomes are regularly spaced along eukaryotic genomes. In the emerging model, known as "statistical positioning", this spacing is due to steric repulsion between nucleosomes and to the presence of nucleosome excluding barriers on the genome. However, new experimental evidence recently challenged the "statistical positioning" model (Z. Zhang et al., Science, 2011, 332(6032), 977-980). We propose here that the regular spacing can be better explained by adding attractive interactions between nucleosomes. In our model those attractions are due to the fact that nucleosomes are stacked in regular chromatin fibers. In a self-reinforcing mechanism, regular nucleosome spacing promotes in turn nucleosome stacking. We first show that this model can precisely account for the nucleosome spacing observed in Saccharomyces cerevisiae. We then use a simple toy model to show that attraction between nucleosomes can fasten the formation of the chromatin fiber.     

Chromatin organization during spermiogenesis in Octopus vulgaris. I: Morphological structures

In the process of the chromatin remodeling that occurs during spermiogenesis in some animal species, it is possible to distinguish between two separate aspects: the chromatin condensation pattern itself (granular, fibrillar, or lamellar), and the architecture of this pattern, that is to say, its arrangement within the nucleus. In the cephalopod Octopus vulgaris these two aspects are clearly differentiated. The condensation pattern develops from 25 nm fibers to fibers with a tubular aspect and with a progressively increasing diameter (40-60 nm and then to 80 nm), to end finally in the form of very thin fibers (3-5 nm) product of the coalescence and dissolution of the major fibers. The main directive force that governs this process lies in the global change that occurs in the proteins that interact with all (or the major part) of the genomic DNA. The condensation pattern by itself in this species does not present a fixed order: most of the fibers appear without any predominant spatial direction in the spermiogenic nuclei. However, as the nuclei elongate, the chromatin fibers arrange in parallel following the elongation axis. This parallel disposition of the chromatin fibers appears to be mediated by two specific areas, each of which we call a "polar nuclear matrix" (PNM). These matrices differentiate in the basal and apical nuclear poles adjacent to the centriolar implantation fosse and the acrosome, respectively. The areas that constitute the PNM have the following characteristics: (a) they are the only areas where DNA is found anchored to the nuclear membrane; (b) they are the zones from which the chromatin condensation pattern (fibers/tubules) begins; and (c) they are most probably the points through which the mechanical forces originating from nuclear elongation are transmitted to chromatin, causing the chromatin fibers/tubules to adopt an almost perfectly parallel disposition. Finally, we discuss the importance of the architecture of the chromatin condensation pattern, as it is one of the determining factors of the spatial organization of the mature sperm genome and chromosome positioning.     

Three distinct stages of apoptotic nuclear condensation revealed by time-lapse imaging, biochemical and electron microscopy analysis of cell-free apoptosis

During apoptotic execution, chromatin undergoes a phase change from a heterogeneous, genetically active network to an inert highly condensed form that is fragmented and packaged into apoptotic bodies. We have previously used a cell-free system to examine the roles of caspases or other proteases in apoptotic chromatin condensation and nuclear disassembly. But so far, the role of DNase activity or ATP hydrolysis in this system has not yet been elucidated. Here, in order to better define the stages of nuclear disassembly in apoptosis, we have characterized the apoptotic condensation using a cell-free system and time-lapse imaging. We demonstrated that the population of nuclei undergoing apoptosis in vitro appears to follow a reproducible program of nuclear condensation, suggesting the existence of an ordered biochemical pathway. This enabled us to define three stages of apoptotic chromatin condensation: stage 1 ring condensation; stage 2 necklace condensation; and stage 3 nuclear collapse/disassembly. Electron microscopy revealed that neither chromatin nor detectable subnuclear structures were present inside the stage 1 ring-condensed structures. DNase activity was not essential for stage 1 ring condensation, which could occur in apoptotic extracts depleted of all detectable DNase activity. However, DNase(s) were required for stage 2 necklace condensation. Finally, we demonstrated that hydrolyzable ATP is required for stage 3 nuclear collapse/disassembly. This requirement for ATP hydrolysis further distinguished stage 2 from stage 3. Together, these experiments provide the first steps towards a systematic biochemical characterization of chromatin condensation during apoptosis.     

Ultrastructural studies of spermatogenesis in the anthocerotales. III. Gamete morphogenesis: From spermatogenous cell through midstage spermatid

An ultrastructural examination of spermatogenesis in Phaeoceros has shown nucleoli to be present in spermatogenous cells and to persist until the centrioles become associated with nuclei of young spermatids. At the onset of multilayered structure (MLS) formation, well-defined aggregations of osmiophilic strands begin to form in the nuclei of young spermatids and disappear shortly after chromatin condensation starts in the midstage spermatids. When the centrioles in the young spermatids are orientated perpendicular to the nuclear envelope, the nucleoplasm immediately in front of them is densely stained. Where the spline tubules of the MLS extend over the nucleus, the nuclear envelope is devoid of pores, and the inner nuclear membrane is contacted internally by the local deposition of dense staining nucleoplasm. Chromatin condensation begins with strands extending perpendicularly from the dense staining nucleoplasm beneath the spline and continues with the nuclear beak becoming filled with condensed chromatin. As the MLS lamellae disappear acropetally, the rear portion of the anterior mitochondrion (AM) extends back under the nuclear beak which now narrows to a size that approximates the anterior end of the nucleus of a spermatozoid. By the end of the mid-spermatid stage, the nucleus has coiled approximately one gyre of a helix and the five or six central slpine tubules extend over the plastid which is now located beneath the front end of the AM. Several profiles of endoplasmic reticulum confluent with the nuclear envelope are present. Possible factors which might play a role in determining the morphology of the mid-spermatids are discussed.     

Histone H1 Subtypes Differentially Modulate Chromatin Condensation without Preventing ATP-Dependent Remodeling by SWI/SNF or NURF

Although ubiquitously present in chromatin, the function of the linker histone subtypes is partly unknown and contradictory studies on their properties have been published. To explore whether the various H1 subtypes have a differential role in the organization and dynamics of chromatin we have incorporated all of the somatic human H1 subtypes into minichromosomes and compared their influence on nucleosome spacing, chromatin compaction and ATP-dependent remodeling. H1 subtypes exhibit different affinities for chromatin and different abilities to promote chromatin condensation, as studied with the Atomic Force Microscope. According to this criterion, H1 subtypes can be classified as weak condensers (H1.1 and H1.2), intermediate condensers (H1.3) and strong condensers (H1.0, H1.4, H1.5 and H1x). The variable C-terminal domain is required for nucleosome spacing by H1.4 and is likely responsible for the chromatin condensation properties of the various subtypes, as shown using chimeras between H1.4 and H1.2. In contrast to previous reports with isolated nucleosomes or linear nucleosomal arrays, linker histones at a ratio of one per nucleosome do not preclude remodeling of minichromosomes by yeast SWI/SNF or Drosophila NURF. We hypothesize that the linker histone subtypes are differential organizers of chromatin, rather than general repressors.     

Chromatin reorganization during spermiogenesis of the mollusc Thais hemostoma (Muricidae): implications for sperm nuclear morphogenesis in cenogastropods

Thais is a cenogastropod mollusc belonging to the Muricidae family. The sperm nuclear morphogenesis of Thais develops in two well-defined and peculiar steps. In the first one, the round early spermatidyl nucleus is penetrated by an endonuclear channel, which arranges as a helix at the inner nuclear surface and organizes the condensing chromatin all around. In the second step, the spiral channel stretches, dragging along the associated chromatin and leading to a definitive cylinder-shaped sperm nucleus. Simultaneously with these changes in nuclear shape, the chromatin is sequentially organized in granules, fibres, lamellae, and, finally, in a very condensed structure, whereas the spermiogenic DNA-associated proteins become more basic and simple. The sperm nucleus contains a small group of protamines consisting of only four types of amino acid (lysine, arginine, glycine, and serine). The most remarkable fact on nuclear spermiogenesis in Thais is that, whereas the chromatin condensation process, the nuclear proteins, and the final shape of sperm nucleus are very similar to those in other muricidae studied, the pathway of nuclear morphogenesis is completely different. We propose an independent genetic control for those two spermiogenic events (chromatin condensation and nucleomorphogenesis). Finally we discuss briefly the main traits of nucleomorphogenesis of muricid molluscs.     

Activation of the c-Jun N-terminal kinase pathway by MST1 is essential and sufficient for the induction of chromatin condensation during apoptosis

Chromatin condensation is the most recognizable nuclear hallmark of apoptosis. Cleavage and activation of MST1 by caspases induce chromatin condensation. It was previously reported that, during apoptosis, activated MST1 induced c-Jun N-terminal kinase (JNK) activation and also phosphorylated histone H2B. However, which of these mechanisms underlies MST1's induction of chromatin condensation has yet to be clarified. Here, we report that MST1-mediated activation of JNK is both essential and sufficient for chromatin condensation. MST1 activation did not result in chromatin condensation in mitogen-activate protein kinase kinase 4 (MKK4)/MKK7 double knockout (MKK4/7 DKO) embryonic stem (ES) cells, which genetically lack the ability to activate JNK. On the other hand, constitutively active JNK was able to induce chromatin condensation in MKK4/7 DKO ES cells. In contrast, histone H2B phosphorylation did not correlate with chromatin condensation in wild-type ES cells. Finally, inhibition of JNK as well as inhibitor of caspase-activated DNase blocked chromatin condensation during Fas-mediated apoptosis of Jurkat cells. Taken together, our results indicate that caspase-mediated cleavage of MST1, followed by MST1-mediated activation of the JNK pathway, is the mechanism responsible for inducing chromatin condensation during apoptosis.