Duplication and Maintenance of Heterochromatin Domains

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 (Hp1α and -β), and chromatin assembly factor 1 (CAF-1). During replication, Hp1α and -β 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 Hp1α and -β, and that a degree of plasticity is provided by the cycle of H4 acetylation/deacetylation assisted by CAF-1.

Nucleosome assembly and epigenetic inheritance

“…In addition, DNMT1 directly interacts with histone H3K9 methyltransferase G9a, and recruits G9a to the replication foci (Estève et al, 2006). Using high resolution imaging techniques, Taddei et al (1999) provided evidences that CAF-1 persists on newly replicated DNA after its synthesis for a period of more than 20 min, in consistence with the in vitro finding that CAF-1 assembles nucleosome post-replication (Shibahara and Stillman, 1999). In addition, a similar phenomenon was also observed on PCNA by fluorescent bleaching experiment (Sporbert et al, 2002).…”

“…When synthesized in cytoplasm, H4 is acetylated at K5 and K12 (Sobel et al, 1995;Benson et al, 2006), these acetyl-groups are removed within 20 min after chromatin assembly (Taddei et al, 1999). In DT40 cells, HAT1 mediates H4 acetylation at these two sites (Barman et al, 2006).…”

Chromatin and DNA Replication

“…Cite this article as Cold Spring Harb Perspect Biol 2013;5:a010207 of heterochromatin if not removed (Taddei et al 1999).…”

“…The chromatin marks associated with nascent histones H3 and H4 are transient and rapidly removed during chromatin maturation as detected in heterochromatin regions (Taddei et al 1999). The removal of H4K5 and K12 diacetylation is critical for the association of HP1 and the maintenance of silenced heterochromatin (Taddei et al 1999), as well as proper centromere function (Ekwall et al 1997;Taddei et al 2001).…”

Keeping chromatin quiet

“…[40][41][42][43][44][45] Once assembled into nucleosomes, they become rapidly deacetylated by a mechanism that is poorly understood. 39,43,46 Typical acetylation marks of new histones, for instance acetylation on H4, lysine 12 or on H3 at lysines 14 and/or 18, persist upon SMARCAD1 depletion. 9,40,42,43,45 SMARCAD1 is therefore a prime candidate for mediating deacetylation of newly deposited histones.…”