Histone phosphorylation during sea urchin development

Studies on histone phosphorylation during transitions in chromatin structure occurringin vivoduring spermatogenesis and early embryogenesis in sea urchins are reviewed and evaluated in the light of recent studies on histone phosphorylation occurring during chromatin synthesis in frog egg extractsin vitroand evidence that protein kinases and phosphatases play direct roles in the regulation of cellular structure. Sperm-specific histone variants Sp H1 and Sp H2B are maintained as phosphorylated derivatives N and O/P throughout spermatogenesis and early embryogenesis and egg specific histone variants CS H1 and CS H2A are phosphorylated during early embryogenesis. These developmental correlations provide clues about the roles of histone phosphorylation in control of chromatin structurein vivoand provide a basis for the interpretation of data obtained from in-vitro sperm chromatin remodeling in egg extracts and from biochemical studies on the effects of histone phosphorylation on DNA binding. The potential consequences for chromatin structure of the various histone phosphorylation events observed in sea urchins and frog egg extracts are discussed.     

Role of histone modifications in defining chromatin structure and function

Chromosomes in eukaryotic cell nuclei are not uniformly organized, but rather contain distinct chromatin elements, with each state having a defined biochemical structure and biological function. These are recognizable by their distinct architectures and molecular components, which can change in response to cellular stimuli or metabolic requirements. Chromatin elements are characterized by the fundamental histone and DNA components, as well as other associated non-histone proteins and factors. Post-translational modifications of histone proteins in particular often correlate with a specific chromatin structure and function. Patterns of histone modifications are implicated as having a role in directing the level of chromatin compaction, as well as playing roles in multiple functional pathways directing the readout of distinct regions of the genome. We review the properties of various chromatin elements and the apparent links of histone modifications with chromatin organization and functional output.     

Hierarchical Chromatin Structure of <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> Revealed by Atomic Force Microscopy

Many structural studies on higher eukaryotic chromatin have been carried out, but chromatin structure in fungi remains unclear. Schizosaccharomyces pombe has been used for investigations of chromosome function; however, the structural details of S. pombe chromatin have not been clarified owing to its small nucleus. We used atomic force microscopy for nano-scale imaging of chromatin isolated from S. pombe. Topographic images indicated that nuclear chromatin contained at least three hierarchical structures: large-scale chromatin fibers, spherical domains in the fibers, and nodules in the domains. The average diameters of the domain and the nodule were 363 ± 85.2 nm and 46.2 ± 9.30 nm. Each structure comprising the hierarchy was similar to higher eukaryotic chromatin thus far observed, despite definite differences in chromatin organization at the nucleosomal level. The presence of histone H1 suggested that there might be an alternative to compensate for histone H1 lacking in S. pombe.     

Association by guilt: identification of DLX5 as a target for MeCP2 provides a molecular link between genomic imprinting and Rett syndrome

Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder affecting almost exclusively girls. Although mutations in methyl-CpG-binding protein (MeCP2) are known to be associated with RTT, gene expression patterns are not significantly altered in MeCP2-deficient cells. A recent study1 identified MeCP2-mediated histone modification and formation of a higher-order chromatin loop structure specifically associated with silent chromatin at the Dlx5-Dlx6 locus in normal cells, and its absence thereof in RTT patients. This altered expression of Dlx5 through loss of silent chromatin loop formation provides a molecular mechanism underlying RTT and proposes a novel role for MeCP2 in chromatin organization and imprinting.     

Histone Octamer Helical Tubes Suggest that an Internucleosomal Four-Helix Bundle Stabilizes the Chromatin Fiber

A major question in chromatin involves the exact organization of nucleosomes within the 30-nm chromatin fiber and its structural determinants of assembly. Here we investigate the structure of histone octamer helical tubes via the method of iterative helical real-space reconstruction. Accurate placement of the x-ray structure of the histone octamer within the reconstructed density yields a pseudoatomic model for the entire helix, and allows precise identification of molecular interactions between neighboring octamers. One such interaction that would not be obscured by DNA in the nucleosome consists of a twofold symmetric four-helix bundle formed between pairs of H2B-α3 and H2B-αC helices of neighboring octamers. We believe that this interface can act as an internucleosomal four-helix bundle within the context of the chromatin fiber. The potential relevance of this interface in the folding of the 30-nm chromatin fiber is discussed.     

Multimerization of Drosophila sperm protein Mst77F causes a unique condensed chromatin structure

Despite insights on the cellular level, the molecular details of chromatin reorganization in sperm development, which involves replacement of histone proteins by specialized factors to allow ultra most condensation of the genome, are not well understood. Protamines are dispensable for DNA condensation during Drosophila post-meiotic spermatogenesis. Therefore, we analyzed the interaction of Mst77F, another very basic testis-specific protein with chromatin and DNA as well as studied the molecular consequences of such binding. We show that Mst77F on its own causes severe chromatin and DNA aggregation. An intrinsically unstructured domain in the C-terminus of Mst77F binds DNA via electrostatic interaction. This binding results in structural reorganization of the domain, which induces interaction with an N-terminal region of the protein. Via putative cooperative effects Mst77F is induced to multimerize in this state causing DNA aggregation. In agreement, overexpression of Mst77F results in chromatin aggregation in fly sperm. Based on these findings we postulate that Mst77F is crucial for sperm development by giving rise to a unique condensed chromatin structure.     

A haploid affair: core histone transitions during spermatogenesis.

The process of meiosis reduces a diploid cell to four haploid gametes and is accompanied by extensive recombination. Thus, the dynamics of chromatin during meiosis are significantly different than in mitotic cells. As spermatogenesis progresses, there is a widespread reorganization of the haploid genome followed by extensive DNA compaction. It has become increasingly clear that the dynamic composition of chromatin plays a critical role in the activities of enzymes and processes that act upon it. Therefore, an analysis of the role of histone variants and modifications in these processes may shed light upon the mechanisms involved and the control of chromatin structure in general. Histone variants such as histone H3.3, H2AX, and macroH2A appear to play key roles in the various stages of spermiogenesis, in addition to the specifically modulated acetylation of histone H4 (acH4), ubiquitination of histones H2A and H2B (uH2A, uH2B), and phosphorylation of histone H3 (H3p). This review will examine recent discoveries concerning the role of histone modifications and variants during meiosis and spermatogenesis.     

RNF8-dependent histone ubiquitination during DNA damage response and spermatogenesis

Histone ubiquitination regulates the chromatin structure that is important for many biological processes. Recently, ubiquitination of histones was observed during the DNA damage response (DDR), and this modification is controlled by really interesting new gene (RING) domain E3 ligase, RNF8. Together with the E2 conjugating enzyme UBC13, RNF8 catalyzes ubiquitination of the histones H2A and H2AX during the DDR, thus facilitating downstream recruitment of DDR factors, such as p53 binding protein 1 (53BP1) and breast cancer type 1 susceptibility protein (BRCA1), to the damage site. Accordingly, the RNF8 knockout mice display phenotypes associated with failed DDR, including hypersensitivity to ionizing radiation, V(D)J recombination deficiency, and a predisposition to cancer. In addition to the DDR phenotypes, RNF8 knockout mice fail to generate mature sperm during spermatogenesis, resulting in male sterility. The RNF8 knockout mice also have a drastic reduction in histone ubiquitination in the testes. These findings indicate that the role of histone ubiquitination during chromatin remodeling in two different biological events could be linked by an RNF8-dependent mechanism. Here, we review the molecular mechanism of RNF8-dependent histone ubiquitination both in DDR and spermatogenesis.     

Chromatin on a membrane: accessibility of histone H5 for antibodies in the supernucleosomal structure

Histone H5 accessibility for the antibodies in chromatin was studied. Chromatin was immobilised on the nitrocellulose membrane in conditions which provide different levels of its compactization. Antiserum specific to the globular domain of histone H5 was used. It was shown, that for establishing real protection of histone H5 in the supernucleosomal structure it is necessary to use long fibers of chromatin. Their linking to the membrane must occur by a minimum quantity of points. It was established, that histone H5 is 5 times more accessive in the preparations of dispersed chromatin (low ionic strength) then in chromatin with the supernucleosomal organization (physiological ionic strength). We suppose that the small level of accessibility of histone H5 for the antibodies in the compact chromatin can be explained by some disruptions in the supernucleosomal organization. On the contrary, the long equable solenoid of nucleosomes provides complete protection of histone H5. In accordance with the results obtained, the model of ordered packaging of nucleosomes in the solenoid is discussed. In this model the point of entrance and exit of DNA on the nucleosomes, fixed by globular region of histone H5, is localized inside the solenoid.     

Facteurs impliqués dans le remodelage de la chromatine au cours de la spermiogenèse

Round spermatids are post-meiotic cells with a haploid genome contained in a nucleus, with a structure initially similar to that of the somatic cell nucleus. During spermatogenesis, the spermatid nucleus undergoes drastic remodelling during which it first elongates and then condenses into the very specific and tightly packaged structure of the sperm nucleus. During this remodelling dthe histones are replaced by transition proteins, which, in turn, are replaced by protamines, the specific nuclear proteins of the spermatozoa. Immediately prior to their replacement, the histones are hyperacetylated. The first part of our work was to precisely characterise the changes in histone acetylation during murine spermatogenesis. We have shown that the core histones H2A, H2B, H3 and H4 are hyperacetylated in the elongating spermatids. We have also shown that these changes in acetylation are associated with degradation of the enzymes responsible for histone deacetylation, histone deacetylases or HDACs, while histone acetyl transferases are still present in these cells. The histone acetylation pattern was also investigated during human spermatogenesis, revealing that histone hyperacetylation in the nucleus of elongating spermatids, which appears to be conserved during the course of evolution, also occurs during human spermatogenesis. Moreover, our data obtained from the testes of men with severely altered spermatogenesis, including SCO syndromes (Sertoli Cells Only Syndromes), show that a global hyperacetylation of the Sertoli cell nuclei is associated with an absence of meiotic and post-meiotic cells. This suggests that the global histone acetylation variations observed during spermatogenesis are part of a signalling pathway involving germ cell — Sertoli cell communication. Altogether, these data provide a basis for a better understanding of the mechanisms and identification of the factors involved in post-meiotic remodelling of chromatin.