Dynamique de la Chromatine

Publications de l’équipe

Année de publication : 1999

A Taddei, D Roche, J B Sibarita, B M Turner, G Almouzni (1999 Dec 22)

Duplication and maintenance of heterochromatin domains.

The Journal of cell biology : 1153-66 En savoir plus
Résumé

To investigate the mechanisms that assure the maintenance of heterochromatin regions, we took advantage of the fact that clusters of heterochromatin DNA replicate late in S phase and are processed in discrete foci with a characteristic nuclear distribution. At the light microscopy level, within these entities, we followed DNA synthesis, histone H4 acetylation, heterochromatin protein 1 (Hp1alpha and -beta), and chromatin assembly factor 1 (CAF-1). During replication, Hp1alpha and -beta domains of concentration are stably maintained, whereas heterochromatin regions are enriched in both CAF-1 and replication-specific acetylated isoforms of histone H4 (H4Ac 5 and 12). We defined a time window of 20 min for the maintenance of this state. Furthermore, treatment with Trichostatin A (TSA), during and after replication, sustains the H4Ac 5 and 12 state in heterochromatin excluding H4Ac 8 and 16. In comparison, early replication foci, at the same level, did not display any specific enrichment in H4Ac 5 and 12. These data emphasize the specific importance for heterochromatin of the replication-associated H4 isoforms. We propose that perpetuation of heterochromatin involves self-maintenance factors, including local concentration of Hp1alpha and -beta, and that a degree of plasticity is provided by the cycle of H4 acetylation/deacetylation assisted by CAF-1.

Replier
J G Moggs, G Almouzni (1999 Jun 18)

Assays for chromatin remodeling during DNA repair.

Methods in enzymology : 333-51 En savoir plus
Résumé

Replier
J G Moggs, G Almouzni (1999 Apr 24)

Chromatin rearrangements during nucleotide excision repair.

Biochimie : 45-52 En savoir plus
Résumé

The removal of DNA damage from the eukaryotic genome requires DNA repair enzymes to operate within the complex environment of chromatin. We review the evidence for chromatin rearrangements during nucleotide excision repair and discuss the extent and possible molecular mechanisms of these rearrangements, focusing on events at the nucleosome level of chromatin structure.

Replier
S Björklund, G Almouzni, I Davidson, K P Nightingale, K Weiss (1999 Apr 2)

Global transcription regulators of eukaryotes.

Cell : 759-67 En savoir plus
Résumé

Replier

Année de publication : 1998

E Martini, D M Roche, K Marheineke, A Verreault, G Almouzni (1998 Nov 13)

Recruitment of phosphorylated chromatin assembly factor 1 to chromatin after UV irradiation of human cells.

The Journal of cell biology : 563-75 En savoir plus
Résumé

The subcellular distribution and posttranslational modification of human chromatin assembly factor 1 (CAF-1) have been investigated after UV irradiation of HeLa cells. In an asynchronous cell population only a subfraction of the two large CAF-1 subunits, p150 and p60, were found to exist in a chromatin-associated fraction. This fraction is most abundant during S phase in nonirradiated cells and is much reduced in G2 cells. After UV irradiation, the chromatin-associated form of CAF-1 dramatically increased in all cells irrespective of their position in the cell cycle. Such chromatin recruitment resembles that seen for PCNA, a DNA replication and repair factor. The chromatin-associated fraction of p60 was predominantly hypophosphorylated in nonirradiated G2 cells. UV irradiation resulted in the rapid recruitment to chromatin of phosphorylated forms of the p60 subunit. Furthermore, the amount of the p60 and p150 subunits of CAF-1 associated with chromatin was a function of the dose of UV irradiation. Consistent with these in vivo observations, we found that the amount of CAF-1 required to stimulate nucleosome assembly during the repair of UV photoproducts in vitro depended upon both the number of lesions and the phosphorylation state of CAF-1. The recruitment of CAF-1 to chromatin in response to UV irradiation of human cells described here supports a physiological role for CAF-1 in linking chromatin assembly to DNA repair.

Replier
C Verheggen, S Le Panse, G Almouzni, D Hernandez-Verdun (1998 Sep 10)

Presence of pre-rRNAs before activation of polymerase I transcription in the building process of nucleoli during early development of Xenopus laevis.

The Journal of cell biology : 1167-80 En savoir plus
Résumé

During the early development of Xenopus laevis, we followed in individual nuclei the formation of a nucleolus by examining simultaneously its structural organization and its transcriptional competence. Three distinct situations were encountered with different frequencies during development. During the first period of general transcriptional quiescence, the transcription factor UBF of maternal origin, was present in most nuclei at the ribosomal gene loci. In contrast, fibrillarin, a major protein of the processing machinery, was found in multiple prenucleolar bodies (PNBs) whereas nucleolin was dispersed largely in the nucleoplasm. During the second period, for most nuclei these PNBs had fused into two domains where nucleolin concentrated, generating a structure with most features expected from a transcriptionally competent nucleolus. However, RNA polymerase I-dependent transcription was not detected using run-on in situ assays whereas unprocessed ribosomal RNAs were observed. These RNAs were found to derive from a maternal pool. Later, during a third period, an increasing fraction of the nuclei presented RNA polymerase I-dependent transcription. Thus, the structural organization of the nucleolus preceded its transcriptional competence. We conclude that during the early development of X. laevis, the organization of a defined nucleolar structure, is not associated with the transcription process per se but rather with the presence of unprocessed ribosomal RNAs.

Replier
S Lorain, J P Quivy, F Monier-Gavelle, C Scamps, Y Lécluse, G Almouzni, M Lipinski (1998 Aug 26)

Core histones and HIRIP3, a novel histone-binding protein, directly interact with WD repeat protein HIRA.

Molecular and cellular biology : 5546-56 En savoir plus
Résumé

The human HIRA gene has been named after Hir1p and Hir2p, two corepressors which together appear to act on chromatin structure to control gene transcription in Saccharomyces cerevisiae. HIRA homologs are expressed in a regulated fashion during mouse and chicken embryogenesis, and the human gene is a major candidate for the DiGeorge syndrome and related developmental disorders caused by a reduction to single dose of a fragment of chromosome 22q. Western blot analysis and double-immunofluorescence experiments using a specific antiserum revealed a primary nuclear localization of HIRA. Similar to Hir1p, HIRA contains seven amino-terminal WD repeats and probably functions as part of a multiprotein complex. HIRA and core histone H2B were found to physically interact in a yeast double-hybrid protein interaction trap, in GST pull-down assays, and in coimmunoprecipitation experiments performed from cellular extracts. In vitro, HIRA also interacted with core histone H4. H2B- and H4-binding domains were overlapping but distinguishable in the carboxy-terminal region of HIRA, and the region for HIRA interaction was mapped to the amino-terminal tail of H2B and the second alpha helix of H4. HIRIP3 (HIRA-interacting protein 3) is a novel gene product that was identified from its HIRA-binding properties in the yeast protein interaction trap. In vitro, HIRIP3 directly interacted with HIRA but also with core histones H2B and H3, suggesting that a HIRA-HIRIP3-containing complex could function in some aspects of chromatin and histone metabolism. Insufficient production of HIRA, which we report elsewhere interacts with homeodomain-containing DNA-binding factors during mammalian embryogenesis, could perturb the stoichiometric assembly of multimolecular complexes required for normal embryonic development.

Replier

Année de publication : 1997

P H Gaillard, J G Moggs, D M Roche, J P Quivy, P B Becker, R D Wood, G Almouzni (1997 Oct 8)

Initiation and bidirectional propagation of chromatin assembly from a target site for nucleotide excision repair.

The EMBO journal : 6281-9 En savoir plus
Résumé

To restore full genomic integrity in a eukaryotic cell, DNA repair processes have to be coordinated with the resetting of nucleosomal organization. We have established a cell-free system using Drosophila embryo extracts to investigate the mechanism linking de novo nucleosome formation to nucleotide excision repair (NER). Closed-circular DNA containing a uniquely placed cisplatin-DNA adduct was used to follow chromatin assembly specifically from a site of NER. Nucleosome formation was initiated from a target site for NER. The assembly of nucleosomes propagated bidirectionally, creating a regular nucleosomal array extending beyond the initiation site. Furthermore, this chromatin assembly was still effective when the repair synthesis step in the NER process was inhibited.

Replier
K Ura, H Kurumizaka, S Dimitrov, G Almouzni, A P Wolffe (1997 Apr 15)

Histone acetylation: influence on transcription, nucleosome mobility and positioning, and linker histone-dependent transcriptional repression.

The EMBO journal : 2096-107 En savoir plus
Résumé

We demonstrate using a dinucleosome template that acetylation of the core histones enhances transcription by RNA polymerase III. This effect is not dependent on an increased mobility of the core histone octamer with respect to DNA sequence. When linker histone is subsequently bound, we find both a reduction in nucleosome mobility and a repression of transcription. These effects of linker histone binding are independent of core histone acetylation, indicating that core histone acetylation does not prevent linker histone binding and the concomitant transcriptional repression. These studies are complemented by the use of a Xenopus egg extract competent both for chromatin assembly on replicating DNA and for RNA polymerase III transcription. Incorporation of acetylated histones and lack of linker histones together facilitate transcription by >10-fold in this system; however, they have little independent effect on transcription. Thus core histone acetylation significantly facilitates transcription, but this effect is inhibited by the assembly of linker histones into chromatin.

Replier
S Bellier, M F Dubois, E Nishida, G Almouzni, O Bensaude (1997 Mar 1)

Phosphorylation of the RNA polymerase II largest subunit during Xenopus laevis oocyte maturation.

Molecular and cellular biology : 1434-40 En savoir plus
Résumé

Xenopus laevis oogenesis is characterized by an active transcription which ceases abruptly upon maturation. To survey changes in the characteristics of the transcriptional machinery which might contribute to this transcriptional arrest, the phosphorylation status of the RNA polymerase II largest subunit (RPB1 subunit) was analyzed during oocyte maturation. We found that the RPB1 subunit accumulates in large quantities from previtellogenic early diplotene oocytes up to fully grown oocytes. The C-terminal domain (CTD) of the RPB1 subunit was essentially hypophosphorylated in growing oocytes from Dumont stage IV to stage VI. Upon maturation, the proportion of hyperphosphorylated RPB1 subunits increased dramatically and abruptly. The hyperphosphorylated RPB1 subunits were dephosphorylated within 1 h after fertilization or heat shock of the matured oocytes. Extracts from metaphase II-arrested oocytes showed a much stronger CTD kinase activity than extracts from prophase stage VI oocytes. Most of this kinase activity was attributed to the activated Xp42 mitogen-activated protein (MAP) kinase, a MAP kinase of the ERK type. Making use of artificial maturation of the stage VI oocyte through microinjection of a recombinant stable cyclin B1, we observed a parallel activation of Xp42 MAP kinase and phosphorylation of RPB1. Both events required protein synthesis, which demonstrated that activation of p34(cdc2)off kinase was insufficient to phosphorylate RPB1 ex vivo and was consistent with a contribution of the Xp42 MAP kinase to RPB1 subunit phosphorylation. These results further support the possibility that the largest RNA polymerase II subunit is a substrate of the ERK-type MAP kinases during oocyte maturation, as previously proposed during stress or growth factor stimulation of mammalian cells.

Replier

Année de publication : 1996

P H Gaillard, E M Martini, P D Kaufman, B Stillman, E Moustacchi, G Almouzni (1996 Sep 20)

Chromatin assembly coupled to DNA repair: a new role for chromatin assembly factor I.

Cell : 887-96 En savoir plus
Résumé

DNA repair in the eukaryotic cell disrupts local chromatin organization. To investigate whether the resetting of nucleosomal arrays can be linked to the repair process, we developed model systems, with both Xenopus egg extract and human cell extracts, to follow repair and chromatin assembly in parallel on circular DNA templates. Both systems were able to carry out nucleotide excision repair of DNA lesions. We observed that UV-dependent DNA synthesis occurs simultaneously with chromatin assembly, strongly indicating a mechanistic coupling between the two processes. A complementation assay established that chromatin assembly factor I (CAF1) is necessary for this repair associated chromatin formation.

Replier

Année de publication : 1995

N Landsberger, M Ranjan, G Almouzni, D Stump, A P Wolffe (1995 Jul 1)

The heat shock response in Xenopus oocytes, embryos, and somatic cells: a regulatory role for chromatin.

Developmental biology : 62-74 En savoir plus
Résumé

The heat shock response in Xenopus laevis has been reported to be developmentally regulated at the transcriptional level. We find that the heat shock response of an exogenous Xenopus hsp70 gene introduced into Xenopus oocytes, embryos, and somatic cells is dependent on the transcriptional assay conditions employed. Under conditions of efficient chromatin assembly, transcription from the Xenopus hsp70 gene promoter is repressed in oocytes and embryos, yet the promoter responds to heat shock by activating transcription. Under conditions of inefficient chromatin assembly, the Xenopus hsp70 gene is constitutively active in oocytes and somatic cells. Our results resolve previous controversy concerning the existence of a heat shock response for the hsp70 promoter in oocytes and illustrate the importance of considering chromatin assembly as a contributory factor in reconstructing the developmental control of gene expression.

Replier
G Almouzni, A P Wolffe (1995 Apr 18)

Constraints on transcriptional activator function contribute to transcriptional quiescence during early Xenopus embryogenesis.

The EMBO journal : 1752-65 En savoir plus
Résumé

We have examined the cause of transcriptional quiescence prior to the mid-blastula transition (MBT) in Xenopus laevis. We have found distinct requirements for transcription of class II and class III genes. An artificial increase of the amount of DNA present within the embryo over that found at the MBT allows precocious transcription of tRNA genes, but not of the adenovirus E4 or human cytomegalovirus (CMV) promoters. Thus titration of an inhibitor by exogenous DNA determines class III but not class II gene activation. We demonstrate that the action of the inhibitor depends on the association of core histones with DNA. The addition of exogenous TBP, together with an increase in the amount of DNA within the embryo, allows significant basal transcription of class II genes prior to the MBT, whereas it does not increase transcription of tRNA genes. To examine the activation of transcription above basal levels, we used a defined minimal promoter containing five Gal4 binding sites and the activator Gal4-VP16. Precocious transcriptional activation is directed by Gal4-VP16 prior to the MBT, demonstrating that a functional transcriptional machinery exists at this early developmental stage. Furthermore, since this activation can occur in the absence of exogenous TBP or chromatin titration, a transcription factor that can penetrate chromatin is sufficient for recruitment of this machinery to a promoter. Our results support the hypothesis that the temporal regulation of transcription during early embryogenesis in Xenopus reflects not only a titration of inhibitors by DNA, but also a deficiency in the activity of transcriptional activators prior to the MBT.

Replier

Année de publication : 1994

G Almouzni, S Khochbin, S Dimitrov, A P Wolffe (1994 Oct 1)

Histone acetylation influences both gene expression and development of Xenopus laevis.

Developmental biology : 654-69 En savoir plus
Résumé

We examine the potential role of histone hyperacetylation in gene activation during Xenopus development using Trichostatin A, (TSA), a specific inhibitor of histone deacetylase. We find that TSA is very effective in inducing both core histone hyperacetylation and histone H1(0) gene expression in a Xenopus somatic cell line. In contrast, TSA does not induce histone hyperacetylation or histone H1(0) transcription in Xenopus oocytes. Histone hyperacetylation is developmentally regulated during Xenopus embryogenesis; hyperacetylated histones first accumulate early in gastrulation. The capacity of TSA to induce histone H1(0) gene expression correlates with the induction of histone hyperacetylation. Concentrations of TSA sufficient to induce histone hyperacetylation in Xenopus embryos delay gastrulation and cause diminished midtrunk and posterior formation, suggesting defects in mesoderm formation. Although the constitutive hyperacetylation of the histones does not prevent either the cell division or differentiation sufficient for early morphogenesis it has a role in establishing stable states of differential gene activity during gastrulation.

Replier
M Familari, G Almouzni, A P Wolffe (1994 Apr 20)

Isolation of a potentially functional Y-box protein (MSY-1) processed pseudogene from mouse: evolutionary relationships within the EF1A/dbpB/YB-1 gene family.

Gene : 255-9 En savoir plus
Résumé

A processed pseudogene from Mus musculus, designated psi MSY-2, was obtained with a MSY-1 cDNA (encoding mouse Y-box factor 1) probe. Mouse psi MSY-2 is intronless and has an ORF with an in-frame translational stop. The pseudogene has repeat sequences at the 5′ and 3′ boundaries, suggestive of an origin as a retroposon, and exhibits mutagenesis of CpG residues at a frequency at least tenfold higher than predicted from random mutagenesis. This indicates that ‘repeat-induced point mutagenesis’ or ripping has occurred. We find that the mouse genome contains many DNA sequences with homology to a cDNA encoding the DNA-binding domain of the Y-box proteins. We estimate that there are at least 15 copies per haploid genome.

Replier