Chromatin assembly during S phase: contributions from histone deposition, DNA replication and the cell division cycle

During S phase of the eukaryotic cell division cycle, newly replicated DNA is rapidly assembled into chromatin. Newly synthesised histones form complexes with chromatin assembly factors, mediating their deposition onto nascent DNA and their assembly into nucleosomes. Chromatin assembly factor 1, CAF-1, is a specialised assembly factor that targets these histones to replicating DNA by association with the replication fork associated protein, proliferating cell nuclear antigen, PCNA. Nucleosomes are further organised into ordered arrays along the DNA by the activity of ATP-dependent chromatin assembly and spacing factors such as ATP-utilising chromatin assembly and remodelling factor ACF. An additional level of controlling chromatin assembly pathways has become apparent by the observation of functional requirements for cyclin-dependent protein kinases, casein kinase II and protein phosphatases. In this review, we will discuss replication-associated histone deposition and nucleosome assembly pathways, and we will focus in particular on how nucleosome assembly is linked to DNA replication and how it may be regulated by the cell cycle control machinery.     

Chromosome stabilizing structures in mitotic Indian muntjac (Muntiacus muntjak) cells1

Summary A new technique which removes all membranes, cytoskeletal elements, organelles, but preserves intact metaphase, anaphase and telophase configurations is combined with scanning electron microscopy (SEM) as an approach for direct visualization of chromosomal behavior in late mitosis. With this approach we are able to confirm the presence of a centromeric ring which stabilizes the centromeres during the cell cycle and present evidence for a lattice-like sheet of interchromatidic fibers in late mitosis.Supported by the National Institute of Health Grant CA32572 to L. D. Hodge.     

Functional characterization of CP148, a novel key component for centrosome integrity in Dictyostelium

The Dictyostelium centrosome consists of a layered core structure surrounded by a microtubule-nucleating corona. A tight linkage through the nuclear envelope connects the cytosolic centrosome with the clustered centromeres within the nuclear matrix. At G2/M the corona dissociates, and the core structure duplicates, yielding two spindle poles. CP148 is a novel coiled coil protein of the centrosomal corona. GFP-CP148 exhibited cell cycle-dependent presence and absence at the centrosome, which correlates with dissociation of the corona in prophase and its reformation in late telophase. During telophase, GFP-CP148 formed cytosolic foci, which coalesced and joined the centrosome. This explains the hypertrophic appearance of the corona upon strong overexpression of GFP-CP148. Depletion of CP148 by RNAi caused virtual loss of the corona and disorganization of interphase microtubules. Surprisingly, formation of the mitotic spindle and astral microtubules was unaffected. Thus, microtubule nucleation complexes associate with centrosomal core components through different means during interphase and mitosis. Furthermore, CP148 RNAi caused dispersal of centromeres and altered Sun1 distribution at the nuclear envelope, suggesting a role of CP148 in the linkage between centrosomes and centromeres. Taken together, CP148 is an essential factor for the formation of the centrosomal corona, which in turn is required for centrosome/centromere linkage.     

Histone chaperones: 30 years from isolation to elucidation of the mechanisms of nucleosome assembly and disassembly

Some three decades have passed since the discovery of nucleosomes in 1974 and the first isolation of a histone chaperone in 1978. While various types of histone chaperones have been isolated and functionally analyzed, the elementary processes of nucleosome assembly and disassembly have been less well characterized. Recently, the tertiary structure of a hetero-trimeric complex composed of the histone chaperone CIA/ASF1 and the histone H3-H4 dimer was determined, and this complex was proposed to be an intermediate in nucleosome assembly and disassembly reactions. In addition, CIA alone was biochemically shown to dissociate the histone (H3-H4)₂ tetramer into two histone H3-H4 dimers. This activity suggested that CIA regulates the semi-conservative replication of nucleosomes. Here, we provide an overview of prominent histone chaperones with the goal of elucidating the mechanisms that preserve and modify epigenetic information. We also discuss the reactions involved in nucleosome assembly and disassembly. Received 5 July 2007; received after revision 8 September 2007; accepted 13 September 2007 M. Eitoku, L. Satoa: These authors contributed equally to this work.     

How to halve ploidy: lessons from budding yeast meiosis

Maintenance of ploidy in sexually reproducing organisms requires a specialized form of cell division called meiosis that generates genetically diverse haploid gametes from diploid germ cells. Meiotic cells halve their ploidy by undergoing two rounds of nuclear division (meiosis I and II) after a single round of DNA replication. Research in Saccharomyces cerevisiae (budding yeast) has shown that four major deviations from the mitotic cell cycle during meiosis are essential for halving ploidy. The deviations are (1) formation of a link between homologous chromosomes by crossover, (2) monopolar attachment of sister kinetochores during meiosis I, (3) protection of centromeric cohesion during meiosis I, and (4) suppression of DNA replication following exit from meiosis I. In this review we present the current understanding of the above four processes in budding yeast and examine the possible conservation of molecular mechanisms from yeast to humans.     

Possible target of Abelson virus phosphokinase in cell transformation

By fusing interphase cells to cells undergoing mitosis, the interphase chromosomes can be visualized. When analyzed in this way, chromosomes of normal mouse cells show characteristic undercondensed centromeric regions. We have found that the centromeric regions of chromosomes from Abelson virus-transformed cells are fully condensed. Abelson virus transforms mouse cells by introducing into them a virally encoded phosphokinase that is expressed constitutively. Thus, we propose that the condensation of centromeric chromatin is a result of overphosphorylation by the Abelson virus phosphokinase, and that the centromeric region is the relevant target of overphosphorylation in transformed cell growth.     

VRK1 and AURKB form a complex that cross inhibit their kinase activity and the phosphorylation of histone H3 in the progression of mitosis

Regulation of cell division requires the integration of signals implicated in chromatin reorganization and coordination of its sequential changes in mitosis. Vaccinia-related kinase 1 (VRK1) and Aurora B (AURKB) are two nuclear kinases involved in different steps of cell division. We have studied whether there is any functional connection between these two nuclear kinases, which phosphorylate histone H3 in Thr3 and Ser10, respectively. VRK1 and AURKB are able to form a stable protein complex, which represents only a minor subpopulation of each kinase within the cell and is detected following nocodazole release. Each kinase is able to inhibit the kinase activity of the other kinase, as well as inhibit their specific phosphorylation of histone H3. In locations where the two kinases interact, there is a different pattern of histone modifications, indicating that there is a local difference in chromatin during mitosis because of the local complexes formed by these kinases and their asymmetric intracellular distribution. Depletion of VRK1 downregulates the gene expression of BIRC5 (survivin) that recognizes H3-T3ph, both are dependent on the activity of VRK1, and is recovered with kinase active murine VRK1, but not with a kinase-dead protein. The H3–Thr3ph–survivin complex is required for AURB recruitment, and their loss prevents the localization of ACA and AURKB in centromeres. The cross inhibition of the kinases at the end of mitosis might facilitate the formation of daughter cells. A sequential role for VRK1, AURKB, and haspin in the progression of mitosis is proposed.     

Anti-DNA antibodies: aspects of structure and pathogenicity

Anti-DNA antibodies contribute to the pathology of systemic lupus erythematosus. Their depositon in tissue lesions could result from localization of preformed immune complexes of antibodies with DNA or nucleosomes, or from cross-reaction of anti-DNA antibodies directly with tissue proteins. Structural analyses contribute to understanding their pathogenic potential. Primary structures of lupus immunoglobulin G double-stranded DNA-binding autoantibodies are determined by immunoglobulin genes with mutated variable region segments, indicative of selection by immunizing antigen. Arginine, lysine and asparagine residues in complementarity-determining region favor DNA binding. Heavy-chain variable regions make major contributions to DNA binding; affinity and specificity of binding are modulated or can be abrogated by the light-chain variable domain. Crytallographic structure is known for a few antibody-DNA complexes and several ligand-free Fab fragments. Computer modeling supplements this limited information. Structural information of lupus antibody interactions with both DNA and cross-reacting molecules will support use of ligands to inhibit tissue deposition of the antibodies and prevent lesion formation in lupus. Received 4 July 2002; accepted 23 July 2002 RID="*" ID="*"Corresponding author.     

Biogenesis of bacterial inner-membrane proteins

All cells must traffic proteins into and across their membranes. In bacteria, several pathways have evolved to enable protein transfer across the inner membrane, the periplasm, and the outer membrane. The major route of protein translocation in and across the cytoplasmic membrane is the general secretion pathway (Sec-pathway). The biogenesis of membrane proteins not only requires protein translocation but also coordinated targeting to the membrane beforehand and folding and assembly into their protein complexes afterwards to function properly in the cell. All these processes are responsible for the biogenesis of membrane proteins that mediate essential functions of the cell such as selective transport, energy conversion, cell division, extracellular signal sensing, and motility. This review will highlight the most recent developments on the structure and function of bacterial membrane proteins, focusing on the journey that integral membrane proteins take to find their final destination in the inner membrane.     

Determination of the connectivity of newborn neurons in mammalian olfactory circuits

The mammalian olfactory bulb is a forebrain structure just one synapse downstream from the olfactory sensory neurons and performs the complex computations of sensory inputs. The formation of this sensory circuit is shaped through activity-dependent and cell-intrinsic mechanisms. Recent studies have revealed that cell-type specific connectivity and the organization of synapses in dendritic compartments are determined through cell-intrinsic programs already preset in progenitor cells. These progenitor programs give rise to subpopulations within a neuron type that have distinct synaptic organizations. The intrinsically determined formation of distinct synaptic organizations requires factors from contacting cells that match the cell-intrinsic programs. While certain genes control wiring within the newly generated neurons, other regulatory genes provide intercellular signals and are only expressed in neurons that will form contacts with the newly generated cells. Here, the olfactory system has provided a useful model circuit to reveal the factors regulating assembly of the highly structured connectivity in mammals.     

Nuclear transport and the mitotic apparatus: an evolving relationship

The trafficking of macromolecules between the cytoplasm and the nucleus is controlled by the nuclear pore complexes (NPCs) and various transport factors that facilitate the movement of cargos through the NPCs and their accumulation in the target compartment. While their functions in transport are well established, an ever-growing number of observations have also linked components of the nuclear transport machinery to processes that control chromosome segregation during mitosis, including spindle assembly, kinetochore function, and the spindle assembly checkpoint. In this review, we will discuss this evolving area of study and emerging hypotheses that propose key roles for components of the nuclear transport apparatus in mitotic progression.     

The role of LamininB2 (LanB2) during mesoderm differentiation in Drosophila

In Drosophila, four genes encode for laminin subunits and the formation of two laminin heterotrimers has been postulated. We report the identification of mutations in the Drosophila LamininB2 (LanB2) gene that encodes for the only laminin γ subunit and is found in both heterotrimers. We describe their effects on embryogenesis, in particular the differentiation of visceral tissues with respect to the ECM. Analysis of mesoderm endoderm interaction indicates disrupted basement membranes and defective endoderm migration, which finally interferes with visceral myotube stretching. Extracellular deposition of laminin is blocked due to the loss of the LanB2 subunit, resulting in an abnormal distribution of ECM components. Our data, concerning the different function of both trimers during organogenesis, suggest that these trimers might act in a cumulative way and probably at multiple steps during ECM assembly. We also observed genetic interactions with kon-tiki and thrombospondin, indicating a role for laminin during muscle attachment.     

Scaffolding proteins and their role in viral assembly

Scaffolding proteins are proteins that are required to catalyse, regulate or modulate some step in the assembly of a macromolecular complex. They associate specifically with the nascent protein complex during assembly, but are subsequently removed, and are absent from the mature structure. Scaffolding proteins have been described primarily from viral systems, in particular from the double-stranded DNA bacteriophages, but most likely play a more general role in macromolecular assembly, a fundamental process in all biological systems. Scaffolding proteins may act in a specific fashion, by actively encouraging the formation of correct protein-protein interactions, or more generally by nucleating and promoting assembly. They may also work to ensure the fidelity of the assembly process by preventing the formation of improper interactions, in many ways similar to the role of molecular chaperones in protein folding. In viruses, scaffolding proteins are found both in the form of internal cores and external bracing, and may form elaborate and complex structures. This review will focus on the viral scaffolding proteins, for which an increasing amount of structural and functional information has recently become available.     

Heterochromatin associated with active versus inactive centromeres of mouse replicates at different times

Summary A subline of mouse L-cells carries a dicentric chromosome in which one centromere always separates prematurely. This centromere is not involved in the dynamics of chromosome migration and is considered inactive. By use of anti-BRdU antibody binding to BRdU-treated chromosomes it is shown that the pericentric constitutive heterochromatin associated with the prematurely separating centromere replicates earlier than its counterpart associated with the active centromere and even before several euchromatic regions in the genome. These results point to a possible mechanism by which dicentric chromosomes segregate equationally.     

Heterochromatin associated with active versus inactive centromeres of mouse replicates at different times

A subline of mouse L-cells carries a dicentric chromosome in which one centromere always separates prematurely. This centromere is not involved in the dynamics of chromosome migration and is considered inactive. By use of anti-BRdU antibody binding to BRdU-treated chromosomes it is shown that the pericentric constitutive heterochromatin associated with the prematurely separating centromere replicates earlier than its counterpart associated with the active centromere and even before several euchromatic regions in the genome. These results point to a possible mechanism by which dicentric chromosomes segregate equationally.     

Typical worlds

Hugh Everett III presented pure wave mechanics, sometimes referred to as the many-worlds interpretation, as a solution to the quantum measurement problem. While pure wave mechanics is an objectively deterministic physical theory with no probabilities, Everett sought to show how the theory might be understood as making the standard quantum statistical predictions as appearances to observers who were themselves described by the theory. We will consider his argument and how it depends on a particular notion of branch typicality. We will also consider responses to Everett and the relationship between typicality and probability. The suggestion will be that pure wave mechanics requires a number of significant auxiliary assumptions in order to make anything like the standard quantum predictions.