Dynamique de la Chromatine

Publications de l’équipe

Année de publication : 2006

Jill A Mello, Jonathan G Moggs, Geneviève Almouzni (2006 May 6)

Analysis of DNA repair and chromatin assembly in vitro using immobilized damaged DNA substrates.

Methods in molecular biology (Clifton, N.J.) : 477-87 En savoir plus
Résumé

Significant advances have been made in identifying a complex network of proteins that could play a role in the repair of DNA damage in the context of chromatin. Insights into this process have been obtained by combining damaged DNA substrates with mammalian cell-free systems that contain both DNA repair and chromatin assembly activities. The methods described in this chapter provide a powerful approach for the detection of proteins recruited during the recognition and repair of DNA lesions, including repair proteins and chromatin associated factors. Substrates for the recruitment assay consist of DNA containing damage that is immobilized on magnetic beads. A human cell-free system that supports both DNA repair and chromatin assembly is incubated with the immobilized DNA-damaged substrates, and proteins associated with the DNA are then isolated and subjected to analysis. We present here protocols for preparing bead-linked DNA substrates containing different types of lesions, for the reaction of the damaged DNA with cell-free systems, and for the subsequent analysis of proteins that are recruited to the immobilized damaged DNA substrates.

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Sophie E Polo, Danièle Roche, Geneviève Almouzni (2006 Mar 15)

New histone incorporation marks sites of UV repair in human cells.

Cell : 481-93 En savoir plus
Résumé

Chromatin organization is compromised during the repair of DNA damage. It remains unknown how and to what extent epigenetic information is preserved in vivo. A central question is whether chromatin reorganization involves recycling of parental histones or new histone incorporation. Here, we devise an approach to follow new histone deposition upon UV irradiation in human cells. We show that new H3.1 histones get incorporated in vivo at repair sites. Remarkably we find that H3.1, which is deposited during S phase, is also incorporated outside of S phase. Histone deposition is dependent on nucleotide excision repair (NER), indicating that it occurs at a postrepair stage. The histone chaperone chromatin assembly factor 1 (CAF-1) is directly involved in the histone deposition process in vivo. We conclude that chromatin restoration after damage cannot rely simply on histone recycling. New histone incorporation at repair sites both challenges epigenetic stability and possibly contributes to damage memory.

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Alejandra Loyola, Tiziana Bonaldi, Danièle Roche, Axel Imhof, Geneviève Almouzni (2006 Feb 7)

PTMs on H3 variants before chromatin assembly potentiate their final epigenetic state.

Molecular cell : 309-16 En savoir plus
Résumé

Histone posttranslational modifications (PTMs) and sequence variants regulate genome function. Although accumulating evidence links particular PTM patterns with specific genomic loci, our knowledge concerning where and when these PTMs are imposed remains limited. Here, we find that lysine methylation is absent prior to histone incorporation into chromatin, except at H3K9. Nonnucleosomal H3.1 and H3.3 show distinct enrichments in H3K9me, such that H3.1 contains more K9me1 than H3.3. In addition, H3.3 presents other modifications, including K9/K14 diacetylated and K9me2. Importantly, H3K9me3 was undetectable in both nonnucleosomal variants. Notably, initial modifications on H3 variants can potentiate the action of enzymes as exemplified with Suv39HMTase to produce H3K9me3 as found in pericentric heterochromatin. Although the set of initial modifications present on H3.1 is permissive for further modifications, in H3.3 a subset cannot be K9me3. Thus, initial modifications impact final PTMs within chromatin.

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Année de publication : 2005

Annabelle Gérard, Stéphane Koundrioukoff, Vincent Ramillon, Jean-Christophe Sergère, Niels Mailand, Jean-Pierre Quivy, Geneviève Almouzni (2005 Dec 2)

The replication kinase Cdc7-Dbf4 promotes the interaction of the p150 subunit of chromatin assembly factor 1 with proliferating cell nuclear antigen.

EMBO reports : 817-23 En savoir plus
Résumé

The coordination of chromatin assembly with DNA replication, which is essential for genomic stability, requires the combined activation of histone deposition with the firing of replication origins. We report here the direct interaction of chromatin assembly factor 1 (CAF1), a key factor involved in histone deposition, with the replication kinase Cdc7-Dbf4. We isolated a complex containing both the largest subunit of CAF1 (p150) and the Cdc7-Dbf4 kinase specifically in S phase and thus prove the existence of this interaction in vivo. We then show that the Cdc7-Dbf4 kinase efficiently phosphorylates p150. This event induces a change in p150 oligomerization state, which promotes binding to proliferating cell nuclear antigen (PCNA). Conversely, CAF1 recruitment is reduced in a PCNA/DNA loading assay using Cdc7-depleted extracts. Our data define p150 as a new target for this kinase with implications for the coordination between DNA replication and CAF1 functions.

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Sophie E Polo, Geneviève Almouzni (2005 Nov 22)

Chromatin assembly: a basic recipe with various flavours.

Current opinion in genetics & development : 104-11 En savoir plus
Résumé

Packaging of eukaryotic genomes into chromatin is a hierarchical mechanism, starting with histone deposition onto DNA to produce nucleosome arrays, which then further fold and ultimately form functional domains. Recent studies provide interesting insight into how nucleosome assembly is coordinated with histone and DNA metabolism and underline the combined contribution of histone chaperones and chromatin remodelers. How these factors operate at a molecular level is a matter of current investigation. New data highlight the importance of histone dimers as deposition entities for de novo nucleosome assembly and identify dedicated machineries involved in histone variant deposition.

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Y Nakatani, D Ray-Gallet, J-P Quivy, H Tagami, G Almouzni (2005 Aug 25)

Two distinct nucleosome assembly pathways: dependent or independent of DNA synthesis promoted by histone H3.1 and H3.3 complexes.

Cold Spring Harbor symposia on quantitative biology : 273-80 En savoir plus
Résumé

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Gaudeline Wagner, Aurélien Bancaud, Jean-Pierre Quivy, Cédric Clapier, Geneviève Almouzni, Jean-Louis Viovy (2005 Aug 12)

Compaction kinetics on single DNAs: purified nucleosome reconstitution systems versus crude extract.

Biophysical journal : 3647-59 En savoir plus
Résumé

Kinetics of compaction on single DNA molecules are studied by fluorescence videomicroscopy in the presence of 1), Xenopus egg extracts and 2), purified nucleosome reconstitution systems using a combination of histones with either the histone chaperone Nucleosome Assembly Protein (NAP-1) or negatively charged macromolecules such as polyglutamic acid and RNA. The comparison shows that the compaction rates can differ by a factor of up to 1000 for the same amount of histones, depending on the system used and on the presence of histone tails, which can be subjected to post-translational modifications. Reactions with purified reconstitution systems follow a slow and sequential mechanism, compatible with the deposition of one (H3-H4)(2) tetramer followed by two (H2A-H2B) dimers. Addition of the histone chaperone NAP-1 increases both the rate of the reaction and the packing ratio of the final product. These stimulatory effects cannot be obtained with polyglutamic acid or RNA, suggesting that yNAP-1 impact on the reaction cannot simply be explained in terms of charge screening. Faster compaction kinetics and higher packing ratios are reproducibly reached with extracts, indicating a role of additional components present in this system. Data are discussed and models proposed to account for the kinetics obtained in our single-molecule assay.

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Manel Esteller, Geneviève Almouzni (2005 Apr 14)

How epigenetics integrates nuclear functions. Workshop on epigenetics and chromatin: transcriptional regulation and beyond.

EMBO reports : 624-8 En savoir plus
Résumé

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Dominique Ray-Gallet, Annabelle Gérard, Sophie Polo, Geneviève Almouzni (2005 Apr 7)

[Variations on the topic of the « histone code »].

Médecine sciences : M/S : 384-9 En savoir plus
Résumé

Histones are the fundamental structural proteins intimately associated with eukaryotic DNA to form a highly ordered and condensed nucleoproteic complex termed chromatin. They are the targets of various posttranslational modifications including acetylation, methylation, phosphorylation and ubiquitination that modulate the structure/function of chromatin. The combinatorial nature of histone modifications is hypothesized to define a « histone code » that considerably extends the information potential of the genetic code, giving rise to epigenetic information. Moreover, most core histones consist of several nonallelic variants that can mark specific loci and could play an important role in establishment and maintenance of epigenetic memory. Here we will briefly present our current knowledge about histone posttranslational modifications and their implications in the regulation of epigenetic information. We will next describe core histone variants, insisting on their mode of incorporation into chromatin to discuss their epigenetic function and inheritance.

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Angela Taddei, Danièle Roche, Wendy A Bickmore, Geneviève Almouzni (2005 Jan 21)

The effects of histone deacetylase inhibitors on heterochromatin: implications for anticancer therapy?

EMBO reports : 520-4 En savoir plus
Résumé

Histone acetylation regulates many chromosome functions, such as gene expression and chromosome segregation. Histone deacetylase inhibitors (HDACIs) induce growth arrest, differentiation and apoptosis of cancer cells ex vivo, as well as in vivo in tumour-bearing animal models, and are now undergoing clinical trials as anti-tumour agents. However, little attention has been paid to how HDACIs function in these biological settings and why different cells respond in different ways. Here, we discuss the consequences of inhibiting histone deacetylases in cycling versus non-cycling cells, in light of the dynamics of histone acetylation patterns with a specific emphasis on heterochromatic regions of the genome.

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Année de publication : 2004

Sophie E Polo, Geneviève Almouzni (2004 Aug 15)

Histone metabolic pathways and chromatin assembly factors as proliferation markers.

Cancer letters : 1-9 En savoir plus
Résumé

The structural organization of DNA into chromatin is of key importance to regulate genome function and stability. Maintenance of such an organization is thus crucial to preserve cellular identity. At each cell cycle, during S phase, this is achieved by duplication of chromatin structure in tight coordination with DNA replication. Such a coordinate process requires histone synthesis and their deposition onto DNA by chromatin assembly factors to be efficiently coupled to DNA synthesis. In this review, we highlight the intimate relationship between these chromatin-related events and DNA replication and we show how it is possible to take advantage of this coupling in order to identify cells with high replicative potential such as tumor cells. On the basis of recent data, we discuss the potential use of chromatin-associated factors as new proliferation markers of interest for cancer diagnosis and prognosis.

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Jean-Pierre Quivy, Danièle Roche, Doris Kirschner, Hideaki Tagami, Yoshihiro Nakatani, Geneviève Almouzni (2004 Aug 13)

A CAF-1 dependent pool of HP1 during heterochromatin duplication.

The EMBO journal : 3516-26 En savoir plus
Résumé

To investigate how the complex organization of heterochromatin is reproduced at each replication cycle, we examined the fate of HP1-rich pericentric domains in mouse cells. We find that replication occurs mainly at the surface of these domains where both PCNA and chromatin assembly factor 1 (CAF-1) are located. Pulse-chase experiments combined with high-resolution analysis and 3D modeling show that within 90 min newly replicated DNA become internalized inside the domain. Remarkably, during this time period, a specific subset of HP1 molecules (alpha and gamma) coinciding with CAF-1 and replicative sites is resistant to RNase treatment. Furthermore, these replication-associated HP1 molecules are detected in Suv39 knockout cells, which otherwise lack stable HP1 staining at pericentric heterochromatin. This replicative pool of HP1 molecules disappears completely following p150CAF-1 siRNA treatment. We conclude that during replication, the interaction of HP1 with p150CAF-1 is essential to promote delivery of HP1 molecules to heterochromatic sites, where they are subsequently retained by further interactions with methylated H3-K9 and RNA.

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Mounia Guenatri, Delphine Bailly, Christèle Maison, Geneviève Almouzni (2004 Aug 11)

Mouse centric and pericentric satellite repeats form distinct functional heterochromatin.

The Journal of cell biology : 493-505 En savoir plus
Résumé

Heterochromatin is thought to play a critical role for centromeric function. However, the respective contributions of the distinct repetitive sequences found in these regions, such as minor and major satellites in the mouse, have remained largely unsolved. We show that these centric and pericentric repeats on the chromosomes have distinct heterochromatic characteristics in the nucleus. Major satellites from different chromosomes form clusters associated with heterochromatin protein 1alpha, whereas minor satellites are individual entities associated with centromeric proteins. Both regions contain methylated histone H3 (Me-K9 H3) but show different micrococcal nuclease sensitivities. A dinucleosome repeating unit is found specifically associated with major satellites. These domains replicate asynchronously, and chromatid cohesion is sustained for a longer time in major satellites compared with minor satellites. Such prolonged cohesion in major satellites is lost in the absence of Suv39h histone methyltransferases. Thus, we define functionally independent centromeric subdomains, which spatio-temporal isolation is proposed to be important for centromeric cohesion and dissociation during chromosome segregation.

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Stephane Koundrioukoff, Sophie Polo, Genevieve Almouzni (2004 Jul 29)

Interplay between chromatin and cell cycle checkpoints in the context of ATR/ATM-dependent checkpoints.

DNA repair : 969-78 En savoir plus
Résumé

Maintenance of both genome stability and its structural organization into chromatin are essential to avoid aberrant gene expression that could lead to neoplasia. Genome integrity being threatened by various sources of genotoxic stresses, cells have evolved regulatory mechanisms, termed cell cycle checkpoints. In general, these surveillance pathways are thought to act mainly to coordinate proficient DNA repair with cell cycle progression. To date, this cellular response to genotoxic stress has been viewed mainly as a DNA-based signal transduction pathway. Recent studies, in both yeast and human, however, highlight possible connections between chromatin structure and cell cycle checkpoints, in particular those involving kinases of the ATM and ATR family, known as key response factors activated early in the checkpoint pathway. In this review, based on this example, we will discuss hypotheses for chromatin-based events as potential initiators of a checkpoint response or conversely, for chromatin-associated factors as targets of checkpoint proteins, promoting changes in chromatin structure, in order to make a lesion more accessible and contribute to a more efficient repair response.

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Alejandra Loyola, Genevieve Almouzni (2004 Jun 9)

Bromodomains in living cells participate in deciphering the histone code.

Trends in cell biology : 279-81 En savoir plus
Résumé

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