To investigate the mechanisms that assure the maintenance of heterochromati
n regions, we took advantage of the fact that clusters of heterochromatin D
NA replicate late in S phase and are processed in discrete foci with a char
acteristic nuclear distribution. At the light microscopy level, within thes
e entities, we followed DNA synthesis, histone H4 acetylation, heterochroma
tin protein 1 (Hp1 alpha and -beta), and chromatin assembly factor 1 (CAF-1
). During replication, Hp1 alpha and -beta domains of concentration are sta
bly 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. Furt
hermore, treatment with Trichostatin A (TSA), during and after replication,
sustains the H4Ac 5 and 12 state in heterochromatin excluding H4Ac 8 and 1
6. In comparison, early replication foci, at the same level, did not displa
y any specific enrichment in H4Ac 5 and 12. These data emphasize the specif
ic importance for heterochromatin of the replication-associated H4 isoforms
. We propose that perpetuation of heterochromatin involves self-maintenance
factors, including local concentration of Hp1 alpha and -beta, and that a
degree of plasticity is provided by the cycle of H4 acetylation/deacetylati
on assisted by CAF-1.