Localizing Replication Sites and Nuclear Proteins

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 (see Figure 1). At the light microscopy level, within these entities, we followed DNA synthesis, histone H4 acetylation, heterochromatin protein 1 (Hp1α and -ß), and chromatin assembly factor 1 (CAF-1). During replication, Hp1α and -ß 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 Hp1 and -ß, and that a degree of plasticity is provided by the cycle of H4 acetylation/deacetylation assisted by CAF-1. The approach of "pulse-chase" to locate both a DNA synthesis event and a nuclear protein should help to establish how a protein of interest can be dynamically involved in complexes associated with replication foci.