Dynamique de la Chromatine

Publications de l’équipe

Année de publication : 1993

A P Wolffe, G Almouzni, K Ura, D Pruss, J J Hayes (1993 Jan 1)

Transcription factor access to DNA in the nucleosome.

Cold Spring Harbor symposia on quantitative biology : 225-35 En savoir plus
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Année de publication : 1991

G Almouzni, M Méchali, A P Wolffe (1991 Feb 1)

Transcription complex disruption caused by a transition in chromatin structure.

Molecular and cellular biology : 655-65 En savoir plus
Résumé

Chromatin structure is known to influence class III gene expression in vitro. We describe the active transcription of Xenopus class III genes following replication and assembly into chromatin by using Xenopus egg extracts. Changes in the structure of this active chromatin dependent on the presence of exogeneous Mg2+ ATP or on the addition of a mixture of histones H2A and H2B are shown to lead to the selective repression of Xenopus 5S RNA genes. Preexisting transcription complexes on 5S DNA are disrupted following the reorganization of a « disordered » histone-DNA complex into a structure consisting of physiologically spaced nucleosomes. Thus, we demonstrate that chromatin structural transitions can have dominant and specific effects on transcription.

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Année de publication : 1990

G Almouzni, D J Clark, M Méchali, A P Wolffe (1990 Oct 11)

Chromatin assembly on replicating DNA in vitro.

Nucleic acids research : 5767-74 En savoir plus
Résumé

Replicating single-stranded DNA is preferentially assembled into chromatin in Xenopus egg extracts relative to non-replicating double-stranded DNA. We have examined the molecular basis of this phenomenon. Single-stranded DNA itself is not a favored template for nucleosome assembly in comparison to double-stranded DNA. Complementary strand synthesis is required for the rapid assembly of nucleosomes. We present evidence that the assembly of chromatin on replicating DNA is a two step phenomenon. The first step involves the replication of DNA and the assembly of an intermediate structure, the second step involves the sequestration of histones H2A/H2B onto DNA. Histones H2A/H2B are preferentially sequestered onto replicated DNA in comparison to non-replicated DNA incubated in the extract.

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M Méchali, G Almouzni, Y Andéol, J Moreau, S Vriz, M Leibovici, J Hourdry, J Géraudie, T Soussi, M Gusse (1990 Mar 1)

Genes and mechanisms involved in early embryonic development in Xenopus laevis.

The International journal of developmental biology : 51-9 En savoir plus
Résumé

Our laboratory is studying genes involved in the regulation of the balance between cell growth and differentiation during embryonic development in Xenopus. We have analyzed the developmental expression of the proto-oncogenes c-myc, and KiRas 2B, the proliferating cell nuclear antigen (PCNA), and the tumor suppressor gene p53. These genes, usually expressed during cell proliferation, are expressed in the oocyte in large quantities, but the majority of their maternal RNAs are degraded by the gastrula stage. The expression of c-myc and the localization of the protein indicate that c-myc has the characteristics expected for a gene involved in the regulation of the mid-blastula transition, when zygotic expression is turned on in the embryo. Its expression during late development or during regeneration indicates that it enables the cells to remain competent for cycling during organogenesis. In vitro systems that reproduce the principal cellular functions during early development are used as model systems to understand the mechanisms involved in early embryogenesis.

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G Almouzni, M Méchali, A P Wolffe (1990 Feb 1)

Competition between transcription complex assembly and chromatin assembly on replicating DNA.

The EMBO journal : 573-82 En savoir plus
Résumé

We have used a Xenopus egg extract to show that a competition exists between the assembly of transcription complexes and nucleosomes on replicating 5S DNA. This competition results in the establishment of a transcriptionally repressed state for 5S DNA that is dependent on core histones but not on the precise positioning of the cores. The repression is selective, since satellite I DNA is not significantly repressed under these conditions. We demonstrate that the efficiency of chromatin assembly compared with transcription complex assembly is an important variable in determining gene activity.

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Année de publication : 1989

P Brooks, C Dohet, G Almouzni, M Méchali, M Radman (1989 Jun 1)

Mismatch repair involving localized DNA synthesis in extracts of Xenopus eggs.

Proceedings of the National Academy of Sciences of the United States of America : 4425-9 En savoir plus
Résumé

Repair of heteroduplex DNA containing G.T or A.C mismatches or containing two tandem unpaired bases occurred in vitro with Xenopus egg extracts as detected by a physical assay. The repair was accompanied by a mismatch-stimulated and mismatch-localized DNA synthesis. Repaired molecules, separated from unrepaired molecules, showed a 20- to 100-fold increase in DNA synthesis in the region of the mismatch compared to regions distant from the mismatch. The remaining unrepaired heteroduplex DNA included molecules that also displayed mismatch-stimulated DNA synthesis in the mismatch-proximal regions. These may represent intermediates in the repair process. The patterns of DNA synthesis suggest that repair begins at some distance from the mismatch and that as much as 1 kilobase or more can be involved in the mismatch-stimulated synthesis.

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Année de publication : 1988

G Almouzni, M Méchali (1988 Dec 20)

Xenopus egg extracts: a model system for chromatin replication.

Biochimica et biophysica acta : 443-50 En savoir plus
Résumé

A cell-free system derived from Xenopus eggs enables in vitro reproduction of the steps occurring during eukaryotic DNA replication. With a circular single-stranded DNA template, extracts obtained from high-speed centrifugation perform complementary DNA strand synthesis coupled to chromatin assembly. Nucleosomes are formed on the newly replicated DNA and the overall reaction mimics the events occurring during chromosomal replication on the lagging strand at the replication fork. ATP is necessary at all steps examined individually, including RNA priming, elongation of DNA strands and chromatin assembly. Although not required for nucleosome formation, ATP is involved in the correct spacing of nucleosomes and the stability of the assembled chromatin. Replication of double-stranded DNA was observed only with extracts obtained from low-speed centrifugation using demembraned sperm nuclei as substrate. Nuclei are reconstituted around the DNA and then undergo a series of events characteristic of a cell cycle. In contrast, neither DNA elongation or chromatin assembly require formation of the nucleus, and both are independent of the cell cycle.

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G Almouzni, M Méchali (1988 Dec 20)

Assembly of spaced chromatin involvement of ATP and DNA topoisomerase activity.

The EMBO journal : 4355-65 En savoir plus
Résumé

Undiluted extracts from eggs or oocytes of Xenopus laevis support the assembly of chromatin with physiologically spaced nucleosomes. Micrococcal nuclease and DNase I digestion experiments show that nucleosome formation as well as supercoiling of circular DNA concomitant to assembly do not require ATP or Mg2+. However these factors are essential for the stability and the physiological spacing of the assembled chromatin. gamma-S-ATP can substitute for ATP in this process. With topoisomers of defined linking number topological interconversions proceed by steps of unity, both in vitro as well as in vivo, indicating that topoisomerase I is dominantly acting in this process. Novobiocin sensitivity occurred only with diluted extracts and was unrelated to an inhibition of topoisomerase II. Finally, nucleosome assembly occurs efficiently on linear DNA although the assembled DNA is less stable than with circular DNA. From these results we propose that mature chromatin is formed in a two-step reaction. In the first step, nucleosome deposition occurs independently of ATP and Mg2+. Thus, nucleosome formation can be uncoupled from their spacing. In this step, topoisomerase activity is involved in the relaxation of the topological constraints generated by chromatin assembly rather than in the process of assembly itself. The second step, requiring ATP and Mg2+, generates properly spaced chromatin.

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G Almouzni, S Mousseron-Grall, M Méchali (1988 Sep 12)

Oligonucleotide site-directed mutagenesis in Xenopus egg extracts.

Nucleic acids research : 8525-39 En savoir plus
Résumé

Addition of M13mp18 single-stranded DNA annealed with an oligonucleotide to a Xenopus egg extract results in a rapid and efficient incorporation of the oligonucleotide in a complete double-stranded supercoiled molecule. Both the efficiency of DNA synthesis and the recovery of complete double-stranded molecules are increased relative to the reaction carried out by the classical technique using the E. coli Klenow DNA polymerase, DNA ligase, dNTPs, ATP and ions. Site specific mutagenesis was assayed by reverting a point mutation in the lacz region of M13mp18. The color assay described by Messing and sequencing of the DNA extracted from isolated plaques was used to check for the reversion. A 2 hr incubation of the heteroduplex carrying the mutagenic oligonucleotide in the Klenow-ligase-dNTP mixture allows a recovery of 6% mutant phage after transformation of competent cells with the reaction products. Using the Xenopus egg extract, 83% mutant phage were recovered after the same incubation time, in reactions entirely performed in parallel. The Xenopus extract is stable and contains all components required for the assay, including all ionic and protein factors; thus the only addition is the annealed DNA. Such an eukaryotic system is therefore an attractive alternative to the reconstituted prokaryotic DNA polymerase-DNA ligase system for site specific mutagenesis.

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G Almouzni, M Méchali (1988 Sep 1)

Removal of RNA by treating agarose and acrylamide gels with RNase in situ.

Trends in genetics : TIG : 270 En savoir plus
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M Méchali, M Gusse, S Vriz, M Taylor, Y Andéol, J Moreau, J Hourdry, M Leibovici, A Brulfert, G Almouzni (1988 Jul 1)

Proto-oncogenes and embryonic development.

Biochimie : 895-8 En savoir plus
Résumé

The role of proto-oncogenes in embryonic development was investigated using one of the most characterized vertebrates, the amphibian Xenopus laevis. Genes which belong to the major proto-oncogene families have been detected in Xenopus genome. The developmental control of the myc gene was assayed using a characterized Xenopus myc probe and specific antibodies. The myc gene is highly expressed as a stable maternal mRNA in oocyte, and an unfertilized egg contains 5 X 10(5)-fold the myc RNA content of a proliferative somatic cell. The myc RNA store is evenly distributed in the oocyte and the egg. Fertilization triggers a post-transcriptional control of the gene and the RNA store is progressively degraded to a constitutive value of 10 to 30 myc RNA copies registered per gastrula embryonic cell. The 62K myc protein is accumulated late in oogenesis. This uncoupling of myc expression and cell proliferation appears as a specific developmental regulation of the myc gene, adapted to the series of rapid cell cleavages occurring after fertilization.

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G Almouzni, M Méchali (1988 Mar 1)

Assembly of spaced chromatin promoted by DNA synthesis in extracts from Xenopus eggs.

The EMBO journal : 665-72 En savoir plus
Résumé

A cell-free system from Xenopus eggs mimics the reaction occurring at the eukaryotic replicative fork in vivo when chromatin assembly is coupled to complementary strand synthesis of DNA. DNA synthesis on single-stranded circular DNA promotes supercoiling and the replicated molecule sediments as a discrete nucleoprotein complex. Micrococcal nuclease digestion reveals a characteristic pattern of multiples of 200 bp of DNA. The kinetics of chromatin assembly and DNA synthesis are coincident and both processes occur with a rate comparable with chromosomal replication in vivo in early embryos. The DNA synthesis reaction can be uncoupled from the assembly reaction. Thus, titration of chromatin proteins by preincubation of the extract with double-stranded DNA prevents the supercoiling of replicated DNA without affecting the rate of synthesis. In contrast, chromatin assembly performed on unreplicated double-stranded DNA is a slower process as compared with the assembly coupled to DNA synthesis. Supercoiled molecules are detected after 30 min replication whereas at least 2 h are required to observe the first form I DNA with unreplicated double-stranded DNA. Such a system where chromatin assembly is promoted by DNA synthesis should be valuable for studying the interaction of specific factors with DNA during chromatin assembly at the replicative fork.

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