Dynamique de la Chromatine

Publications de l’équipe

Année de publication : 2007

Anja Groth, Armelle Corpet, Adam J L Cook, Daniele Roche, Jiri Bartek, Jiri Lukas, Geneviève Almouzni (2007 Dec 22)

Regulation of replication fork progression through histone supply and demand.

Science (New York, N.Y.) : 1928-31 En savoir plus
Résumé

DNA replication in eukaryotes requires nucleosome disruption ahead of the replication fork and reassembly behind. An unresolved issue concerns how histone dynamics are coordinated with fork progression to maintain chromosomal stability. Here, we characterize a complex in which the human histone chaperone Asf1 and MCM2-7, the putative replicative helicase, are connected through a histone H3-H4 bridge. Depletion of Asf1 by RNA interference impedes DNA unwinding at replication sites, and similar defects arise from overproduction of new histone H3-H4 that compromises Asf1 function. These data link Asf1 chaperone function, histone supply, and replicative unwinding of DNA in chromatin. We propose that Asf1, as a histone acceptor and donor, handles parental and new histones at the replication fork via an Asf1-(H3-H4)-MCM2-7 intermediate and thus provides a means to fine-tune replication fork progression and histone supply and demand.

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Leanne De Koning, Armelle Corpet, James E Haber, Geneviève Almouzni (2007 Nov 5)

Histone chaperones: an escort network regulating histone traffic.

Nature structural & molecular biology : 997-1007 En savoir plus
Résumé

In eukaryotes, DNA is organized into chromatin in a dynamic manner that enables it to be accessed for processes such as transcription and repair. Histones, the chief protein component of chromatin, must be assembled, replaced or exchanged to preserve or change this organization according to cellular needs. Histone chaperones are key actors during histone metabolism. Here we classify known histone chaperones and discuss how they build a network to escort histone proteins. Molecular interactions with histones and their potential specificity or redundancy are also discussed in light of chaperone structural properties. The multiplicity of histone chaperone partners, including histone modifiers, nucleosome remodelers and cell-cycle regulators, is relevant to their coordination with key cellular processes. Given the current interest in chromatin as a source of epigenetic marks, we address the potential contributions of histone chaperones to epigenetic memory and genome stability.

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Aline V Probst, Geneviève Almouzni (2007 Sep 6)

Pericentric heterochromatin: dynamic organization during early development in mammals.

Differentiation; research in biological diversity : 15-23 En savoir plus
Résumé

Constitutive heterochromatin in mammals is essentially found at centromeres, which are key chromosomal elements that ensure proper chromosome segregation. These regions are considered to be epigenetically defined, given that it is not sequence composition but chromatin organization that defines centromere function. How such an epigenetically defined domain, like the centromere, can be established during development and maintained during somatic cell life are fundamental questions. This review discusses the most recent insights into centromeric heterochromatin organization and replication. We further highlight the plasticity of this domain by describing the large-scale re-organization that occurs during development.

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Petra Hajkova, Katia Ancelin, Tanja Waldmann, Nicolas Lacoste, Ulrike C Lange, Francesca Cesari, Caroline Lee, Genevieve Almouzni, Robert Schneider, M Azim Surani (2007 Jul 27)

Chromatin dynamics during epigenetic reprogramming in the mouse germ line.

Nature : 877-81 : DOI : 10.1038/nature06714 En savoir plus
Résumé

A unique feature of the germ cell lineage is the generation of totipotency. A critical event in this context is DNA demethylation and the erasure of parental imprints in mouse primordial germ cells (PGCs) on embryonic day 11.5 (E11.5) after they enter into the developing gonads. Little is yet known about the mechanism involved, except that it is apparently an active process. We have examined the associated changes in the chromatin to gain further insights into this reprogramming event. Here we show that the chromatin changes occur in two steps. The first changes in nascent PGCs at E8.5 establish a distinctive chromatin signature that is reminiscent of pluripotency. Next, when PGCs are residing in the gonads, major changes occur in nuclear architecture accompanied by an extensive erasure of several histone modifications and exchange of histone variants. Furthermore, the histone chaperones HIRA and NAP-1 (NAP111), which are implicated in histone exchange, accumulate in PGC nuclei undergoing reprogramming. We therefore suggest that the mechanism of histone replacement is critical for these chromatin rearrangements to occur. The marked chromatin changes are intimately linked with genome-wide DNA demethylation. On the basis of the timing of the observed events, we propose that if DNA demethylation entails a DNA repair-based mechanism, the evident histone replacement would represent a repair-induced response event rather than being a prerequisite.

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Sophie E Polo, Geneviève Almouzni (2007 Jul 18)

DNA damage leaves its mark on chromatin.

Cell cycle (Georgetown, Tex.) : 2355-9 En savoir plus
Résumé

DNA organization into chromatin has a major influence on the cellular response to DNA damage. Recent studies in various systems ranging from yeast to human cells stress the importance of chromatin not simply as a barrier to DNA repair processes but also as an active contributor to the DNA damage response. Indeed, modulations of chromatin organization involving various degrees of rearrangements, such as histone modifications and even nucleosome displacement, can promote efficient repair and also participate in checkpoint signaling. Here, we survey recent progress in delineating how chromatin rearrangements provide crosstalk with the DNA damage response. In particular, we highlight new data on histone dynamics at damage sites and discuss their functional importance for the stable propagation of specific chromatin states.

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Dominique Ray-Gallet, Jean-Pierre Quivy, Herman W W Silljé, Erich A Nigg, Geneviève Almouzni (2007 Apr 5)

The histone chaperone Asf1 is dispensable for direct de novo histone deposition in Xenopus egg extracts.

Chromosoma : 487-96 En savoir plus
Résumé

Histone chaperones that escort histones during their overall lifetime from synthesis to sites of usage can participate in various tasks. Their requirement culminates in the dynamic processes of nucleosome assembly and disassembly. In this context, it is important to define the exact role of the histone chaperone Asf1. In mammals, Asf1 interacts with two other chaperones, CAF-1 and HIRA, which are critical in DNA synthesis-coupled and synthesis-uncoupled nucleosome assembly pathways, respectively. A key issue is whether Asf1 is able or not to deposit histones onto DNA by itself in both pathways. Here, to delineate the precise role of Asf1 in chromatin assembly, we used Xenopus egg extracts as a powerful system to assay de novo chromatin assembly pathways in vitro. Following characterization of both Xenopus Asf1 and p60 (CAF-1), we used immunodepletion strategies targeting Asf1, HIRA, or CAF-1. Strikingly, the depletion of Asf1 led to the simultaneous depletion of HIRA and consequently impaired the DNA synthesis-independent nucleosome assembly pathway. The rescue of nucleosome assembly capacity in such extracts was effective when adding HIRA along with H3/H4 histones, yet addition of Asf1 along with H3/H4 histones did not work. Moreover, nucleosome assembly coupled to DNA repair was not affected in these Asf1/HIRA-depleted extracts, a pathway impaired by CAF-1 depletion. Thus, these data show that Asf1 is not directly involved in de novo histone deposition during DNA synthesis-independent and synthesis-dependent pathways in egg extracts. Based on our results, it becomes important to consider the implications for Asf1 function during early development in Xenopus.

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Alejandra Loyola, Geneviève Almouzni (2007 Mar 5)

Marking histone H3 variants: how, when and why?

Trends in biochemical sciences : 425-33 En savoir plus
Résumé

DNA in eukaryotic cells is compacted into chromatin, a regular repeated structure in which the nucleosome represents the basic unit. The nucleosome not only serves to compact the genetic material but also provides information that affects nuclear functions including DNA replication, repair and transcription. This information is conveyed through numerous combinations of histone post-translational modifications (PTMs) and histone variants. A recent challenge has been to understand how and when these combinations of PTMs are imposed and to what extent they are determined by the choice of a specific histone variant. Here we focus on histone H3 variants and the PTMs that they carry before and after their assembly into chromatin. We review and discuss recent knowledge about how the choice and initial modifications of a specific variant might affect PTM states and eventually the final epigenetic state of a chromosomal domain.

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Elaine M Dunleavy, Alison L Pidoux, Marie Monet, Carolina Bonilla, William Richardson, Georgina L Hamilton, Karl Ekwall, Paul J McLaughlin, Robin C Allshire (2007 Feb 28)

A NASP (N1/N2)-related protein, Sim3, binds CENP-A and is required for its deposition at fission yeast centromeres.

Molecular cell : 1029-44 En savoir plus
Résumé

A defining feature of centromeres is the presence of the histone H3 variant CENP-A(Cnp1). It is not known how CENP-A(Cnp1) is specifically delivered to, and assembled into, centromeric chromatin. Through a screen for factors involved in kinetochore integrity in fission yeast, we identified Sim3. Sim3 is homologous to known histone binding proteins NASP(Human) and N1/N2(Xenopus) and aligns with Hif1(S. cerevisiae), defining the SHNi-TPR family. Sim3 is distributed throughout the nucleoplasm, yet it associates with CENP-A(Cnp1) and also binds H3. Cells defective in Sim3 function have reduced levels of CENP-A(Cnp1) at centromeres (and increased H3) and display chromosome segregation defects. Sim3 is required to allow newly synthesized CENP-A(Cnp1) to accumulate at centromeres in S and G2 phase-arrested cells in a replication-independent mechanism. We propose that one function of Sim3 is to act as an escort that hands off CENP-A(Cnp1) to chromatin assembly factors, allowing its incorporation into centromeric chromatin.

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Anja Groth, Walter Rocha, Alain Verreault, Geneviève Almouzni (2007 Feb 27)

Chromatin challenges during DNA replication and repair.

Cell : 721-33 En savoir plus
Résumé

Inheritance and maintenance of the DNA sequence and its organization into chromatin are central for eukaryotic life. To orchestrate DNA-replication and -repair processes in the context of chromatin is a challenge, both in terms of accessibility and maintenance of chromatin organization. To meet the challenge of maintenance, cells have evolved efficient nucleosome-assembly pathways and chromatin-maturation mechanisms that reproduce chromatin organization in the wake of DNA replication and repair. The aim of this Review is to describe how these pathways operate and to highlight how the epigenetic landscape may be stably maintained even in the face of dramatic changes in chromatin structure.

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Sophie E Polo, Geneviève Almouzni (2007 Jan 11)

[Chromatin dynamics during the repair of DNA lesions].

Médecine sciences : M/S : 29-31 En savoir plus
Résumé

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Année de publication : 2006

Aline V Probst, Fátima Santos, Wolf Reik, Geneviève Almouzni, Wendy Dean (2006 Dec 18)

Structural differences in centromeric heterochromatin are spatially reconciled on fertilisation in the mouse zygote.

Chromosoma : 403-15 En savoir plus
Résumé

In mammals, paternal and maternal pronuclei undergo profound chromatin reorganisation upon fertilisation. How these events are orchestrated within centromeric regions to ensure proper chromosome segregation in the following cellular divisions is unknown. In this study, we followed the dynamic unfolding of the centromeric regions, i.e. the centric and pericentric satellite repeats, by DNA fluorescent in situ hybridization (FISH) during the first cell cycle up to the two-cell stage. The distinct chromatin from female and male gametes both undergo rapid remodelling and reach a zygotic organisation in which the satellites occupy restricted spatial domains surrounding the nucleolar precursor body. A transition from this zygotic to a somatic cell-like organisation takes place during the two-cell stage. Using 3D immuno-FISH, we find that, whereas maternal pericentric regions are marked with H3K9me3, H4K20me3 and HP1beta, paternal ones only showed HP1beta marking. Thus, despite different chromatin features, male and female pronuclei organise their centromeric regions in the same way within the nuclei to align chromosomes on the metaphase plate and segregate them appropriately. Our findings highlight the importance of ensuring a proper centromere function while preserving the distinction of parental genome origin during the return to totipotency in the zygote.

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Irina Panteleeva, Stéphanie Boutillier, Violaine See, Dave G Spiller, Caroline Rouaux, Geneviève Almouzni, Delphine Bailly, Christèle Maison, Helen C Lai, Jean-Philippe Loeffler, Anne-Laurence Boutillier (2006 Nov 21)

HP1alpha guides neuronal fate by timing E2F-targeted genes silencing during terminal differentiation.

The EMBO journal : 3616-28 En savoir plus
Résumé

A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cycle that requires the silencing of genes that drive mitosis. Here, we describe that the alpha isoform of the heterochromatin protein 1 (HP1) protein family exerts such silencing on several E2F-targeted genes. Among the different isoforms, HP1alpha levels progressively increase throughout differentiation and take over HP1gamma binding on E2F sites in mature neurons. When overexpressed, only HP1alpha is able to ensure a timed repression of E2F genes. Specific inhibition of HP1alpha expression drives neuronal progenitors either towards death or cell cycle progression, yet preventing the expression of the neuronal marker microtubule-associated protein 2. Furthermore, we provide evidence that this mechanism occurs in cerebellar granule neurons in vivo, during the postnatal development of the cerebellum. Finally, our results suggest that E2F-targeted genes are packaged into higher-order chromatin structures in mature neurons relative to neuroblasts, likely reflecting a transition from a ‘repressed’ versus ‘silenced’ status of these genes. Together, these data present new epigenetic regulations orchestrated by HP1 isoforms, critical for permanent cell cycle exit during neuronal differentiation.

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Annabelle Gérard, Sophie E Polo, Danièle Roche, Geneviève Almouzni (2006 Jun 24)

Methods for studying chromatin assembly coupled to DNA repair.

Methods in enzymology : 358-74 En savoir plus
Résumé

In the eukaryotic nucleus, the DNA repair machinery operates on chromatin-embedded DNA substrates. Currently, a favored model for DNA repair into chromatin involves the transient disruption of chromatin organization to facilitate access of the repair machinery to DNA lesions. Importantly, this model implies that, in addition to DNA repair, a subsequent step is necessary to restore a proper chromatin structure. To study this latter step, we describe here methods for simultaneously analyzing chromatin assembly and DNA repair both in vitro and in vivo. Several cell-free systems have been developed that reproduce both DNA repair and nucleosome assembly. These in vitro systems are based on the use of defined damaged DNA. Two complementary assays are routinely used: (i) with circular DNA molecules, one can monitor in a combined analysis both repair synthesis and plasmid supercoiling; (ii) with immobilized damaged DNA, one follows specific protein interactions including histone deposition. In addition, in vivo assays have been designed to monitor the recruitment of chromatin assembly factors onto damaged chromatin either at a global level over the whole cell nucleus or locally at sites of DNA damage. Combination of these approaches provides powerful tools to gain insights into the mechanism by which chromatin organization can be restored after repair of DNA lesions.

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Martin Houlard, Soizik Berlivet, Aline V Probst, Jean-Pierre Quivy, Patrick Héry, Geneviève Almouzni, Matthieu Gérard (2006 Jun 13)

CAF-1 is essential for heterochromatin organization in pluripotent embryonic cells.

PLoS genetics : e181 En savoir plus
Résumé

During mammalian development, chromatin dynamics and epigenetic marking are important for genome reprogramming. Recent data suggest an important role for the chromatin assembly machinery in this process. To analyze the role of chromatin assembly factor 1 (CAF-1) during pre-implantation development, we generated a mouse line carrying a targeted mutation in the gene encoding its large subunit, p150CAF-1. Loss of p150CAF-1 in homozygous mutants leads to developmental arrest at the 16-cell stage. Absence of p150CAF-1 in these embryos results in severe alterations in the nuclear organization of constitutive heterochromatin. We provide evidence that in wild-type embryos, heterochromatin domains are extensively reorganized between the two-cell and blastocyst stages. In p150CAF-1 mutant 16-cell stage embryos, the altered organization of heterochromatin displays similarities to the structure of heterochromatin in two- to four-cell stage wild-type embryos, suggesting that CAF-1 is required for the maturation of heterochromatin during preimplantation development. In embryonic stem cells, depletion of p150CAF-1 using RNA interference results in the mislocalization, loss of clustering, and decondensation of pericentric heterochromatin domains. Furthermore, loss of CAF-1 in these cells results in the alteration of epigenetic histone methylation marks at the level of pericentric heterochromatin. These alterations of heterochromatin are not found in p150CAF-1-depleted mouse embryonic fibroblasts, which are cells that are already lineage committed, suggesting that CAF-1 is specifically required for heterochromatin organization in pluripotent embryonic cells. Our findings underline the role of the chromatin assembly machinery in controlling the spatial organization and epigenetic marking of the genome in early embryos and embryonic stem cells.

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Danièle Roche, Geneviève Almouzni, Jean-Pierre Quivy (2006 Jun 3)

Chromatin assembly of DNA templates microinjected into Xenopus oocytes.

Methods in molecular biology (Clifton, N.J.) : 139-47 En savoir plus
Résumé

The packaging of deoxyribonucleic acid (DNA) into chromatin within the eukaryotic nucleus can affect processes such as DNA replication, transcription, recombination, and repair. Therefore, studies aimed at understanding at the molecular level how these processes are operating have to take into account the chromatin context. We present a method to assemble DNA into chromatin by nuclear microinjection into Xenopus oocytes. This method allows in vivo chromatin formation in a nuclear environment. We provide the experimental procedures for oocyte preparation, DNA injection, and analysis of the assembled chromatin.

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