Dynamique de la Chromatine

Publications de l’équipe

Année de publication : 2004

Christèle Maison, Geneviève Almouzni (2004 Apr 9)

HP1 and the dynamics of heterochromatin maintenance.

Nature reviews. Molecular cell biology : 296-304 En savoir plus
Résumé

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Sophie E Polo, Stamatios E Theocharis, Jerzy Klijanienko, Alexia Savignoni, Bernard Asselain, Philippe Vielh, Geneviève Almouzni (2004 Apr 3)

Chromatin assembly factor-1, a marker of clinical value to distinguish quiescent from proliferating cells.

Cancer research : 2371-81 En savoir plus
Résumé

Histone synthesis and chromatin assembly are mainly associated with DNA replication and are thus intimately involved in cell cycle regulation. The expression of key components involved in these events in human cells was studied in relation to cell-proliferative status. Among several chromatin assembly factors, chromatin assembly factor (CAF)-1 stood out as the most discriminating marker of the proliferative state. We show, using both immunofluorescence and Western blot analysis, that the expression of both CAF-1 large subunits, p150 and p60, is massively down-regulated during quiescence in several cell lines. Upon exit from the quiescent state, the CAF-1 subunits are re-expressed early, before DNA replication. The amounts of either total or chromatin-associated pools of CAF-1 proteins correlate directly with cell proliferation. Regulation of CAF-1 expression is partly controlled at the RNA level, as shown by quantitative reverse transcription-PCR and Northern blot experiments. Biological material from benign and malignant human breast tumors analyzed by immunocytochemistry and immunohistochemistry exhibits a strong positive correlation between CAF-1 p60 expression and the following proliferation markers: S-phase fraction (r = 0.84, P < 0.0001); Ki-67 (r = 0.94, P < 0.0001); and proliferating cell nuclear antigen (r = 0.95, P = 0.0001). We discuss the advantages of using CAF-1 to assess cell proliferation. High CAF-1 p60 levels are also shown to be associated with various prognostic factors. Our data highlight the precise association of CAF-1 expression with the proliferative state and validate the use of this factor as a useful proliferation marker and prognostic indicator in malignant and benign breast lesions.

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Anja Groth, Dominique Ray-Gallet, Jean-Pierre Quivy, Jiri Lukas, Jiri Bartek, Geneviève Almouzni (2004 Apr 1)

Human Asf1 regulates the flow of S phase histones during replicational stress.

Molecular cell : 301-11 En savoir plus
Résumé

Maintenance of chromosomal integrity requires tight coordination of histone biosynthesis with DNA replication. Here, we show that extracts from human cells exposed to replication stress display an increased capacity to support replication-coupled chromatin assembly. While in unperturbed S phase, hAsf1 existed in equilibrium between an active form and an inactive histone-free pool, replication stress mobilized the majority of hAsf1 into an active multichaperone complex together with histones. This active multichaperone complex was limiting for chromatin assembly in S phase extracts, and hAsf1 was required for the enhanced assembly activity in cells exposed to replication stress. Consistently, siRNA-mediated knockdown of hAsf1 impaired the kinetics of S phase progression. Together, these data suggest that hAsf1 provides the cells with a buffering system for histone excess generated in response to stalled replication and explains how mammalian cells maintain a critical « active » histone pool available for deposition during recovery from replication stresses.

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Alejandra Loyola, Genevieve Almouzni (2004 Mar 17)

Histone chaperones, a supporting role in the limelight.

Biochimica et biophysica acta : 3-11 En savoir plus
Résumé

In eukaryotic cells, highly basic histone proteins are associated with the DNA to form the nucleosome, the fundamental unit of chromatin. Histones are closely escorted by histone chaperones from their point of synthesis up to their delivery site. We will present an overview of the histone chaperones identified to date with their various roles, in an attempt to highlight their importance in cellular metabolism. Nucleoplasmin will illustrate a role in histone storage and Nap-1, a histone translocator. CAF-1 and Hira will provide examples of distinct histone deposition factors coupled to and uncoupled from DNA synthesis, respectively, while Asf1 could act as a histone donor. We then will illustrate with two examples how histone chaperones can be associated with chromatin remodeling activities. Finally, we will discuss how the RbAp46/48 proteins, as escort factors, are part of multiple complexes with various functions. Based on these examples, we will propose a scheme in which the diverse roles of histone chaperones are integrated within an assembly line for chromatin formation and regulation. Finally, we discuss how these chaperones may have more than a supporting role in a histone metabolic pathway.

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Dominique Ray-Gallet, Geneviève Almouzni (2004 Feb 12)

DNA synthesis-dependent and -independent chromatin assembly pathways in Xenopus egg extracts.

Methods in enzymology : 117-31 En savoir plus
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Alisson M de M C Gontijo, Catherine M Green, Geneviève Almouzni (2004 Jan 17)

Repairing DNA damage in chromatin.

Biochimie : 1133-47 En savoir plus
Résumé

Understanding how DNA repair processes occur in vivo when access to DNA is hindered by chromatin structural organisation is a current challenge. In general terms, the following sequence of events has to be considered within a chromatin environment: (i) finding a lesion (ii) repairing this lesion, and (iii) full restoration of a functional chromatin locus. In this review, basic principles concerning nucleosome dynamics, both intrinsic properties and those dependent on accessory factors, will be used to discuss the issue of lesion accessibility to damage-detecting proteins within chromatin. In addition, opportunities for damage detection due to chromatin alterations directly linked with transcription and replication processes will be considered. After damage detection, additional processes to enhance accessibility within chromatin may be needed to accommodate downstream repair factors or to allow DNA synthesis, resulting in interdependency between repair and accessibility mechanisms in chromatin. Finally, we will comment on the way in which chromatin assembly factors can participate in the maintenance of chromatin structures during DNA repair.

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Hideaki Tagami, Dominique Ray-Gallet, Geneviève Almouzni, Yoshihiro Nakatani (2004 Jan 14)

Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis.

Cell : 51-61 En savoir plus
Résumé

Deposition of the major histone H3 (H3.1) is coupled to DNA synthesis during DNA replication and possibly DNA repair, whereas histone variant H3.3 serves as the replacement variant for the DNA-synthesis-independent deposition pathway. To address how histones H3.1 and H3.3 are deposited into chromatin through distinct pathways, we have purified deposition machineries for these histones. The H3.1 and H3.3 complexes contain distinct histone chaperones, CAF-1 and HIRA, that we show are necessary to mediate DNA-synthesis-dependent and -independent nucleosome assembly, respectively. Notably, these complexes possess one molecule each of H3.1/H3.3 and H4, suggesting that histones H3 and H4 exist as dimeric units that are important intermediates in nucleosome formation. This finding provides new insights into possible mechanisms for maintenance of epigenetic information after chromatin duplication.

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Année de publication : 2003

Jean-Pierre Quivy, Geneviève Almouzni (2003 Dec 4)

Rad53: a controller ensuring the fine-tuning of histone levels.

Cell : 508-10 En savoir plus
Résumé

Checkpoint proteins are activated in response to genotoxic insults or replication stress to maintain genome integrity. Their function is believed to depend largely on the detection of the DNA damage or defects occurring during replication fork progression.

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Catherine M Green, Geneviève Almouzni (2003 Oct 1)

Local action of the chromatin assembly factor CAF-1 at sites of nucleotide excision repair in vivo.

The EMBO journal : 5163-74 En savoir plus
Résumé

DNA damage and its repair can cause both local and global rearrangements of chromatin structure. In each case, the epigenetic information contained within this structure must be maintained. Using the recently developed method for the localized UV irradiation of cells, we analysed responses that occur locally to damage sites and global events triggered by local damage recognition. We thus demonstrate that, within a single cell, the recruitment of chromatin assembly factor 1 (CAF-1) to UV-induced DNA damage is a strictly local phenomenon, restricted to damage sites. Concomitantly, proliferating cell nuclear antigen (PCNA) locates to the same sites. This localized recruitment suggests that CAF-1 participates directly in chromatin structural rearrangements that occur in the vicinity of the damage. Use of nucleotide excision repair (NER)-deficient cells shows that the NER pathway–specifically dual incision–is required for recruitment of CAF-1 and PCNA. This in vivo demonstration of the local role of CAF-1, depending directly on NER, supports the hypothesis that CAF-1 ensures the maintenance of epigenetic information by acting locally at repair sites.

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Jean-Pierre Quivy, Peter B Becker (2003 Sep 6)

Determination of unknown genomic sequences without cloning.

Methods in molecular biology (Clifton, N.J.) : 373-84 En savoir plus
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Année de publication : 2002

Dominique Ray-Gallet, Jean-Pierre Quivy, Christine Scamps, Emmanuelle M-D Martini, Marc Lipinski, Geneviève Almouzni (2002 Jun 7)

HIRA is critical for a nucleosome assembly pathway independent of DNA synthesis.

Molecular cell : 1091-100 En savoir plus
Résumé

The mammalian HIRA gene encodes a histone-interacting protein whose homolog in Xenopus laevis is characterized here. In vitro, recombinant Xenopus HIRA bound purified core histones and promoted their deposition onto plasmid DNA. The Xenopus HIRA protein, tightly associated with nuclear structures in somatic cells, was found in a soluble maternal pool in early embryos. Xenopus egg extracts, known for their chromatin assembly efficiency, were specifically immunodepleted for HIRA. These depleted extracts were severely impaired in their ability to assemble nucleosomes on nonreplicated DNA, although nucleosome formation associated with DNA synthesis remained efficient. Furthermore, this defect was largely corrected by reintroduction of HIRA along with (H3-H4)(2) tetramers. We thus delineate a nucleosome assembly pathway that depends on HIRA.

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Jill A Mello, Herman H W Silljé, Daniele M J Roche, Doris B Kirschner, Erich A Nigg, Geneviève Almouzni (2002 Mar 19)

Human Asf1 and CAF-1 interact and synergize in a repair-coupled nucleosome assembly pathway.

EMBO reports : 329-34 En savoir plus
Résumé

The efficient assembly of newly replicated and repaired DNA into chromatin is essential for proper genome function. Based on genetic studies in Saccharomyces cerevisiae, the histone chaperone anti-silencing function 1 (Asf1) has been implicated in the DNA repair response. Here, the human homologs are shown to function synergistically with human CAF-1 to assemble nucleosomes during nucleotide excision repair in vitro. Furthermore, we demonstrate that hAsf1 proteins can interact directly with the p60 subunit of hCAF-1. In contrast to hCAF-1 p60, the nuclear hAsf1 proteins are not significantly associated with chromatin in cells before or after the induction of DNA damage, nor specifically recruited to damaged DNA during repair in a bead-linked DNA assay. A model is proposed in which the synergism between hAsf1 and CAF-1 for nucleosome formation during DNA repair is achieved through a transient physical interaction allowing histone delivery from Asf1 to CAF-1.

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Christèle Maison, Delphine Bailly, Antoine H F M Peters, Jean-Pierre Quivy, Danièle Roche, Angela Taddei, Monika Lachner, Thomas Jenuwein, Geneviève Almouzni (2002 Feb 19)

Higher-order structure in pericentric heterochromatin involves a distinct pattern of histone modification and an RNA component.

Nature genetics : 329-34 En savoir plus
Résumé

Post-translational modification of histone tails is thought to modulate higher-order chromatin structure. Combinations of modifications including acetylation, phosphorylation and methylation have been proposed to provide marks recognized by specific proteins. This is exemplified, in both mammalian cells and fission yeast, by transcriptionally silent constitutive pericentric heterochromatin. Such heterochromatin contains histones that are generally hypoacetylated and methylated by Suv39h methyltransferases at lysine 9 of histone H3 (H3-K9). Each of these modification states has been implicated in the maintenance of HP1 protein-binding at pericentric heterochromatin, in transcriptional silencing and in centromere function. In particular, H3-K9 methylation is thought to provide a marking system for the establishment and maintenance of stably repressed regions and heterochromatin subdomains. To address the question of how these two types of modifications, as well as other unidentified parameters, function to maintain pericentric heterochromatin, we used a combination of histone deacetylase inhibitors, RNAse treatments and an antibody raised against methylated branched H3-K9 peptides. Our results show that both H3-K9 acetylation and methylation can occur on independent sets of H3 molecules in pericentric heterochromatin. In addition, we identify an RNA- and histone modification-dependent structure that brings methylated H3-K9 tails together in a specific configuration required for the accumulation of HP1 proteins in these domains.

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B Ladoux, J P Quivy, P S Doyle, G Almouzni, J L Viovy (2002 Feb 13)

Direct imaging of single-molecules: from dynamics of a single DNA chain to the study of complex DNA-protein interactions.

Science progress : 267-90 En savoir plus
Résumé

Recent years have seen significant advances in the characterization and manipulation of individual molecules. The combination of single-molecule fluorescence and micromanipulation enables one to study physical and biological systems at new length scales, to unravel qualitative mechanisms, and to measure kinetic parameters that cannot be addressed by traditional biochemistry. DNA is one of the most studied biomolecules. Imaging single DNA molecules eliminates important limitations of classical techniques and provides a new method for testing polymer dynamics and DNA-protein interactions. Here we review some applications of this new approach to physical and biological problems, focusing on videomicroscopy observations of individual DNA chains extended in a shear flow. We will first describe data obtained on the stretching, relaxation and dynamics of a single tethered polymer in a shear flow, to demonstrate that the deformation of sheared tethered chains is partially governed by the thermally driven fluctuations of the chain transverse to the flow direction. Next, we will show how single-molecule videomicroscopy can be used to study in real time DNA folding into chromatin, a complex association of DNA and proteins responsible for the packaging of DNA in the nucleus of an eukaryotic cell.

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Catherine M Green, Geneviève Almouzni (2002 Jan 19)

When repair meets chromatin. First in series on chromatin dynamics.

EMBO reports : 28-33 En savoir plus
Résumé

In eukaryotic cells, the inheritance of both the DNA sequence and its organization into chromatin is critical to maintain genome stability. This maintenance is challenged by DNA damage. To fully understand how the cell can tolerate genotoxic stress, it is necessary to integrate knowledge of the nature of DNA damage, its detection and its repair within the chromatin environment of a eukaryotic nucleus. The multiplicity of the DNA damage and repair processes, as well as the complex nature of chromatin, have made this issue difficult to tackle. Recent progress in each of these areas enables us to address, both at a molecular and a cellular level, the importance of inter-relationships between them. In this review we revisit the ‘access, repair, restore’ model, which was proposed to explain how the conserved process of nucleotide excision repair operates within chromatin. Recent studies have identified factors potentially involved in this process and permit refinement of the basic model. Drawing on this model, the chromatin alterations likely to be required during other processes of DNA damage repair, particularly double-strand break repair, are discussed and recently identified candidates that might perform such alterations are highlighted.

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