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Duplication and maintenance of heterochromatin domains.

Taddei A, Roche D, Sibarita JB, Turner BM, Almouzni G - J. Cell Biol. (1999)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Research section, UMR 144 et 218 du Centre National de la Recherche Scientifique (CNRS), 75248 Paris cedex 05, France.

ABSTRACT
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.

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Domains rich in Hp1α and -β can be found at late replicating foci. Visualization of DNA replication sites in asynchronous populations of mouse (L929) and human (HeLa) cells was performed by in vivo BrdU pulse labeling and detected by immunofluorescence (red). Hp1α and -β were localized using specific mAbs (green). DNA was visualized by DAPI counterstaining (blue). Images of cells harboring replication foci patterns characteristic of either early (top) or late (bottom) S phase are shown. Bars, 10 μm.
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Figure 1: Domains rich in Hp1α and -β can be found at late replicating foci. Visualization of DNA replication sites in asynchronous populations of mouse (L929) and human (HeLa) cells was performed by in vivo BrdU pulse labeling and detected by immunofluorescence (red). Hp1α and -β were localized using specific mAbs (green). DNA was visualized by DAPI counterstaining (blue). Images of cells harboring replication foci patterns characteristic of either early (top) or late (bottom) S phase are shown. Bars, 10 μm.

Mentions: Image acquisition was performed using a Leica TCS-4D confocal scanning microscope, equipped with an Acousto-Optical Tuneable Filter (AOTF), with a 100× NA 1.4 plan-apochromat oil immersion objective. Single optical sections are presented. FITC and Texas red were excited by the argon-krypton laser at 488 and 568 nm, respectively. Red and green fluorescence were separated by a 580-nm long-pass dichroic beam-splitter. A 520-nm band-pass filter and a 590-nm long-pass filter were used to minimize cross-talk and stop laser scattered light. DAPI staining, shown in Fig. 1, was acquired with the UV laser. Before acquiring a double staining z series, the intensity of excitation wavelengths and the power of photodetectors were adjusted to avoid cross-talk. The fluorescence signals from both fluorochromes were recorded simultaneously in one scan, and saved separately on two channels to be processed independently.


Duplication and maintenance of heterochromatin domains.

Taddei A, Roche D, Sibarita JB, Turner BM, Almouzni G - J. Cell Biol. (1999)

Domains rich in Hp1α and -β can be found at late replicating foci. Visualization of DNA replication sites in asynchronous populations of mouse (L929) and human (HeLa) cells was performed by in vivo BrdU pulse labeling and detected by immunofluorescence (red). Hp1α and -β were localized using specific mAbs (green). DNA was visualized by DAPI counterstaining (blue). Images of cells harboring replication foci patterns characteristic of either early (top) or late (bottom) S phase are shown. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2168099&req=5

Figure 1: Domains rich in Hp1α and -β can be found at late replicating foci. Visualization of DNA replication sites in asynchronous populations of mouse (L929) and human (HeLa) cells was performed by in vivo BrdU pulse labeling and detected by immunofluorescence (red). Hp1α and -β were localized using specific mAbs (green). DNA was visualized by DAPI counterstaining (blue). Images of cells harboring replication foci patterns characteristic of either early (top) or late (bottom) S phase are shown. Bars, 10 μm.
Mentions: Image acquisition was performed using a Leica TCS-4D confocal scanning microscope, equipped with an Acousto-Optical Tuneable Filter (AOTF), with a 100× NA 1.4 plan-apochromat oil immersion objective. Single optical sections are presented. FITC and Texas red were excited by the argon-krypton laser at 488 and 568 nm, respectively. Red and green fluorescence were separated by a 580-nm long-pass dichroic beam-splitter. A 520-nm band-pass filter and a 590-nm long-pass filter were used to minimize cross-talk and stop laser scattered light. DAPI staining, shown in Fig. 1, was acquired with the UV laser. Before acquiring a double staining z series, the intensity of excitation wavelengths and the power of photodetectors were adjusted to avoid cross-talk. The fluorescence signals from both fluorochromes were recorded simultaneously in one scan, and saved separately on two channels to be processed independently.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Research section, UMR 144 et 218 du Centre National de la Recherche Scientifique (CNRS), 75248 Paris cedex 05, France.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus