Duplication and maintenance of heterochromatin domains - PubMed (original) (raw)

Duplication and maintenance of heterochromatin domains

A Taddei et al. J Cell Biol. 1999.

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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Figures

Figure 1

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.

Figure 7

Figure 7

Dynamics of H4 acetylation in heterochromatin regions is restricted to the time of their DNA replication. A, Sites of DNA synthesis in an asynchronous population of HeLa cells were first labeled in vivo by BrdU pulse (red), and chased for 90 min either in the presence or absence of TSA (50 ng/ml). Different acetylated isoforms of H4 were localized using specific pAbs (K5, K8, K12, and K16, green). B, Sites of DNA synthesis in an asynchronous population of HeLa cells were first labeled in vivo by BrdU pulse (red), and chased for 14 h to allow late replicating cells, labeled during the pulse, to exit mitosis, before prolonged incubation in the absence or presence of TSA (50 ng/ml) for 90 min. Different acetylated isoforms of H4 were localized using specific pAbs (green). Digital merge of both signals, corresponding to single optical sections, are shown in each case. Bars, 10 μm. Note that these images were processed differently to permit visualization, because the intensities recorded were very different + or − TSA. Enlarged inserts (top left of each images) correspond to selected foci.

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