Telomeres and meiosis in health and disease

Beyond their role in replication and chromosome end capping, telomeres are also thought to function in meiotic chromosome pairing, meiotic and mitotic chromosome segregation as well as in nuclear organization. Observations in both somatic and meiotic cells suggest that the positioning of telomeres within the nucleus is highly specific and believed to be dependent mainly on telomere interactions with the nuclear envelope either directly or through chromatin interacting proteins. Although little is known about the mechanism of telomere clustering, some studies show that it is an active process. Recent data have suggested a regulatory role for telomere chromatin structure in telomere movement. This review will summarize recent studies on telomere interactions with the nuclear matrix, telomere chromatin structure and factors that modify telomere chromatin structure as related to regulation of telomere movement.     

Regulatory mechanisms of kinetochore–microtubule interaction in mitosis

Interaction of microtubules with kinetochores is fundamental to chromosome segregation. Kinetochores initially associate with lateral surfaces of microtubules and subsequently become attached to microtubule ends. During these interactions, kinetochores can move by sliding along microtubules or by moving together with depolymerizing microtubule ends. The interplay between kinetochores and microtubules leads to the establishment of bi-orientation, which is the attachment of sister kinetochores to microtubules from opposite spindle poles, and subsequent chromosome segregation. Molecular mechanisms underlying these processes have been intensively studied over the past 10 years. Emerging evidence suggests that the KNL1–Mis12–Ndc80 (KMN) network plays a central role in connecting kinetochores to microtubules, which is under fine regulation by a mitotic kinase, Aurora B. However, a growing number of additional molecules are being shown to be involved in the kinetochore–microtubule interaction. Here I overview the current range of regulatory mechanisms of the kinetochore–microtubule interaction, and discuss how these multiple molecules contribute cooperatively to allow faithful chromosome segregation.     

S-phase checkpoint regulations that preserve replication and chromosome integrity upon dNTP depletion

DNA replication stress, an important source of genomic instability, arises upon different types of DNA replication perturbations, including those that stall replication fork progression. Inhibitors of the cellular pool of deoxynucleotide triphosphates (dNTPs) slow down DNA synthesis throughout the genome. Following depletion of dNTPs, the highly conserved replication checkpoint kinase pathway, also known as the S-phase checkpoint, preserves the functionality and structure of stalled DNA replication forks and prevents chromosome fragmentation. The underlying mechanisms involve pathways extrinsic to replication forks, such as those involving regulation of the ribonucleotide reductase activity, the temporal program of origin firing, and cell cycle transitions. In addition, the S-phase checkpoint modulates the function of replisome components to promote replication integrity. This review summarizes the various functions of the replication checkpoint in promoting replication fork stability and genome integrity in the face of replication stress caused by dNTP depletion.     

Integrase, LEDGF/p75 and HIV replication

HIV integrates a DNA copy of its genome into a host cell chromosome in each replication cycle. The essential DNA cleaving and joining chemistry of integration is known, but there is less understanding of the process as it occurs in a cell, where two complex and dynamic macromolecular entities are joined: the viral pre-integration complex and chromatin. Among implicated cellular factors, much recent attention has coalesced around LEDGF/p75, a nuclear protein that may act as a chromatin docking factor or receptor for lentiviral pre-integration complexes. LEDGF/p75 tethers HIV integrase to chromatin, protects it from degradation, and strongly influences the genome-wide pattern of HIV integration. Depleting the protein from cells and/or over-expressing its integrase-binding domain blocks viral replication. Current goals are to establish the underlying mechanisms and to determine whether this knowledge can be exploited for antiviral therapy or for targeting lentiviral vector integration in human gene therapy. Received 25 November 2007; received after revision 7 January 2008; accepted 10 January 2008     

Microtubule dynamics alter the interphase nucleus

Microtubules are known to drive chromosome movements and to induce nuclear envelope breakdown during mitosis and meiosis. Here we show that microtubules can enforce nuclear envelope folding and alter the levels of nuclear envelope-associated heterochromatin during interphase, when the nuclear envelope is intact. Microtubule reassembly, after chemically induced depolymerization led to folding of the nuclear envelope and to a transient accumulation of condensed chromatin at the site nearest the microtubule organizing center (MTOC). This microtubule-dependent chromatin accumulation next to the MTOC is dependent on the composition of the nuclear lamina and the activity of the dynein motor protein. We suggest that forces originating from simultaneous polymerization of microtubule fibers deform the nuclear membrane and the underlying lamina. Whereas dynein motor complexes localized to the nuclear envelope that slide along the microtubules transfer forces and/or signals into the nucleus to induce chromatin reorganization and accumulation at the nuclear membrane folds. Thus, our study identified a molecular mechanism by which mechanical forces generated in the cytoplasm reshape the nuclear envelope, alter the intranuclear organization of chromatin, and affect the architecture of the interphase nucleus.     

S-phase-coupled apoptosis in tumor suppression

DNA replication is essential for accurate transmission of genomic information from parental to daughter cells. DNA replication is licensed once per cell division cycle. This process is highly regulated by both positive and negative regulators. Over-replication, under-replication, as well as DNA damage in a cell all induce the activation of checkpoint control pathways such as ATM/ATR, CHK kinases, and the tumor suppressor protein p53, which provide “damage controls” via either DNA repairs or apoptosis. This review focuses on accumulating evidence, with the emphasis on recently discovered Killin, that S-phase checkpoint control is crucial for a mammalian cell to make a life and death decision in order to safeguard genome integrity.     

The equilibrium of ubiquitination and deubiquitination at PLK1 regulates sister chromatid separation

PLK1 regulates almost every aspect of mitotic events, including mitotic entry, spindle assembly, chromosome alignment, sister chromatid segregation, metaphase-anaphase transition, cytokinesis, etc. In regulating the chromosome alignment and sister chromatid segregation, PLK1 has to be localized to and removed from kinetochores at the right times, and the underlying mechanism that regulates PLK1 both spatially and temporally only became clearer recently. It has been found that deubiquitination and ubiquitination of PLK1 are responsible for its localization to and dissociation from the kinetochores, respectively. The equilibrium of this ubiquitination and deubiquitination plays an important role in regulating proper chromosome alignment and timely sister chromatid segregation. Here, we summarize and discuss the recent findings in investigating the spatial and temporal regulation of PLK1 during chromosome alignment and sister chromatid segregation.     

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.     

Putting CENP-A in its place

The centromere is the chromosomal region that directs kinetochore assembly during mitosis in order to facilitate the faithful segregation of sister chromatids. The location of the human centromere is epigenetically specified. The presence of nucleosomes that contain the histone H3 variant, CENP-A, are thought to be the epigenetic mark that indicates active centromeres. Maintenance of centromeric identity requires the deposition of new CENP-A nucleosomes with each cell cycle. During S-phase, existing CENP-A nucleosomes are divided among the daughter chromosomes, while new CENP-A nucleosomes are deposited during early G1. The specific assembly of CENP-A nucleosomes at centromeres requires the Mis18 complex, which recruits the CENP-A assembly factor, HJURP. We will review the unique features of centromeric chromatin as well as the mechanism of CENP-A nucleosome deposition. We will also highlight a few recent discoveries that begin to elucidate the factors that temporally and spatially control CENP-A deposition.     

Partial complementation of a DNA ligase I deficiency by DNA ligase III and its impact on cell survival and telomere stability in mammalian cells

DNA ligase I (LigI) plays a central role in the joining of strand interruptions during replication and repair. In our current study, we provide evidence that DNA ligase III (LigIII) and XRCC1, which form a complex that functions in single-strand break repair, are required for the proliferation of mammalian LigI-depleted cells. We show from our data that in cells with either dysfunctional LigI activity or depleted of this enzyme, both LigIII and XRCC1 are retained on the chromatin and accumulate at replication foci. We also demonstrate that the LigI and LigIII proteins cooperate to inhibit sister chromatid exchanges but that only LigI prevents telomere sister fusions. Taken together, these results suggest that in cells with dysfunctional LigI, LigIII contributes to the ligation of replication intermediates but not to the prevention of telomeric instability.     

Regulation of end-binding protein EB1 in the control of microtubule dynamics

The regulation of microtubule dynamics is critical to ensure essential cell functions, such as proper segregation of chromosomes during mitosis or cell polarity and migration. End-binding protein 1 (EB1) is a plus-end-tracking protein (+TIP) that accumulates at growing microtubule ends and plays a pivotal role in the regulation of microtubule dynamics. EB1 autonomously binds an extended tubulin-GTP/GDP-Pi structure at growing microtubule ends and acts as a molecular scaffold that recruits a large number of regulatory +TIPs through interaction with CAP-Gly or SxIP motifs. While extensive studies have focused on the structure of EB1-interacting site at microtubule ends and its role as a molecular platform, the mechanisms involved in the negative regulation of EB1 have only started to emerge and remain poorly understood. In this review, we summarize recent studies showing that EB1 association with MT ends is regulated by post-translational modifications and affected by microtubule-targeting agents. We also present recent findings that structural MAPs, that have no tip-tracking activity, physically interact with EB1 to prevent its accumulation at microtubule plus ends. These observations point out a novel concept of “endogenous EB1 antagonists” and emphasize the importance of finely regulating EB1 function at growing microtubule ends.     

Regulation of the cell cycle following DNA damage in normal and Ataxia telangiectasia cells

A proportion of the population is exposed to acute doses of ionizing radiation through medical treatment or occupational accidents, with little knowledge of the immedate effects. At the cellular level, ionizing radiation leads to the activation of a genetic program which enables the cell to increase its chances of survival and to minimize detrimental manifestations of radiation damage. Cytotoxic stress due to ionizing radiation causes genetic instability, alterations in the cell cycle, apoptosis, or necrosis. Alterations in the G1, S and G2 phases of the cell cycle coincide with improved survival and genome stability. The main cellular factors which are activated by DNA damage and interfere with the cell cycle controls are: p53, delaying the transition through the G1-S boundary; p21^WAF1/CIPI, preventing the entrance into S-phase; proliferating cell nuclear antigen (PCNA) and replication protein A (RPA), blocking DNA replication; and the p53 variant protein p53as together with the retinoblastoma protein (Rb), with less defined functions during the G2 phase of the cell cycle. By comparing a variety of radioresistant cell lines derived from radiosensitive ataxia talangiectasia cells with the parental cells, some essential mechanisms that allow cells to gain radioresistance have been identified. The results so far emphasise the importance of an adequate delay in the transition from G2 to M and the inhibition of DNA replication in the regulation of the cell cycle after exposure to ionizing radiation.     

Sex chromosome inactivation in germ cells: emerging roles of DNA damage response pathways

Sex chromosome inactivation in male germ cells is a paradigm of epigenetic programming during sexual reproduction. Recent progress has revealed the underlying mechanisms of sex chromosome inactivation in male meiosis. The trigger of chromosome-wide silencing is activation of the DNA damage response (DDR) pathway, which is centered on the mediator of DNA damage checkpoint 1 (MDC1), a binding partner of phosphorylated histone H2AX (γH2AX). This DDR pathway shares features with the somatic DDR pathway recognizing DNA replication stress in the S phase. Additionally, it is likely to be distinct from the DDR pathway that recognizes meiosis-specific double-strand breaks. This review article extensively discusses the underlying mechanism of sex chromosome inactivation.     

Chromatin structure contribution to the synaptonemal complex formation

Meiosis is a key cellular and molecular process for sexual reproduction contributing to the genetic variability of organisms. This process takes place after DNA replication and consists in a double cellular division, giving rise to four haploid daughter cells or gametes. Meiotic recombination between homologous chromosomes, in the meiotic prophase I, is mediated by a tripartite structure named Synaptonemal Complex (SC). The SC is a peptidic scaffold in which the chromatin of homologous chromosomes is organized during the pachytene stage, holding chromosomes together until the meiotic recombination and genetic exchange have taken place. The role of chromatin structure in formation of the SC and the meiotic recombination at meiotic prophase I remain largely unknown. In this review we address the epigenome contribution to the SC formation at meiotic prophase I, with particular attention on the chromatin structure modifications occurring during the sub-stages of meiotic prophase I. Received 18 September 2008; received after revision 10 October 2008; accepted 24 October 2008     

Different roles of the human Orc6 protein in the replication initiation process

In eukaryotes, binding of the six-subunit origin recognition complex (ORC) to DNA provides an interactive platform for the sequential assembly of pre-replicative complexes. This process licenses replication origins competent for the subsequent initiation step. Here, we analyze the contribution of human Orc6, the smallest subunit of ORC, to DNA binding and pre-replicative complex formation. We show that Orc6 not only interacts with Orc1–Orc5 but also with the initiation factor Cdc6. Biochemical and imaging experiments reveal that this interaction is required for licensing DNA replication competent. Furthermore, we demonstrate that Orc6 contributes to the interaction of ORC with the chaperone protein HMGA1a (high mobility group protein A1a). Binding of human ORC to replication origins is not specified at the level of DNA sequence and the functional organization of origins is poorly understood. We have identified HMGA1a as one factor that might direct ORC to AT-rich heterochromatic regions. The systematic analysis of the interaction between ORC and HMGA1a revealed that Orc6 interacts with the acidic C-terminus of HMGA1a and also with its AT-hooks. Both domains support autonomous replication if targeted to DNA templates. As such, Orc6 functions at different stages of the replication initiation process. Orc6 can interact with ORC chaperone proteins such as HMGA1a to facilitate chromatin binding of ORC and is also an essential factor for pre-RC formation.