In this article, we provide evidence that Np95 is a new protein involved in replication through PH. The results presented here show that Np95 is part of this unit and are consistent with a role of Np95 during replication of PH for several reasons. First, Np95 is specifically enriched in heterochromatin only at the time of PH replication in mid-S phase. Second, Np95 is part of the pHDB for it colocalizes with BrdU (this article) and with PCNA (Miura et al., 2001
) in the ring-like structures that correspond to PH replication. Third, it colocalizes with active replication foci during PH DNA replication (mid-S phase) and not during the replication of centromeric heterochromatin and other chromatin structures (late S phase). Fourth, but most important, functional ablation of Np95 reduces both mid- and late S-phase replication more than 35% with respect to controls, in a 10-min BrdU pulse experiment.
A strong reduction of Np95 inside the cells does not completely inhibit DNA replication nor hamper the binding of PCNA to the DNA, indicating that the effect of the reduction of Np95 is more likely to be a slowing down rather than a block of DNA replication and that the impairment of heterochromatin replication does not depend on the stability of the replication machinery. Np95, moreover, does not seem to be a common part of the DNA synthesizing machinery. Thus, the slowing down effect on heterochromatin replication in RNAi experiments must be a consequence of DNA replication–linked nuclear events not directly connected to the replication machinery.
Np95 is a histone-binding protein involved in histone epigenetic modifications. The results we obtained, together with previously published data, are consistent with a model in which Np95 binds to histone H3 (Citterio et al., 2004
), recruits HDAC-1 (Unoki et al., 2004
), and deacetylates the tails of heterochromatic histone H4 (this article). The marked increase of heterochromatic H4Ac-K12 we observe in the absence of Np95 reinforces the thought that Np95 has a role in heterochromatin replication. H4-K12, in fact, has been shown to be acetylated in heterochromatic areas only during histone deposition and for a time window of 20 min afterward (Taddei et al., 1999
). Interestingly, we also observe an acetylation of H4-K8 and H4-K16 at the periphery of the DAPI spots at the time of heterochromatin replication with a distribution that recalls the ring-like one of Np95, suggesting that Np95 should control deacetylation of NH2
lysines of H4 at the level of the pHDB. At this stage of the study, however, we cannot distinguish if the acetylation of H4 lysines occurs at the time of heterochromatin replication or in other moments of the cell cycle, nor we can define if this event involves the newly deposited histones, the parental ones, or both. Importantly, heterochromatic acetylation of H4-K12 has been observed only at the time of heterochromatin replication, and an acetylated form of lysine 8 of H4 has been found in the chromatin assembly complex (CAC), which contains the three subunits of CAF-1 and that is a key intermediate for the de novo nucleosome assembly pathway as it is able to promote DNA replication–dependent chromatin assembly (Verreault et al., 1996
). A very short time might exist, therefore, in which newly deposited histone H4 tails are acetylated at K8 and K16, and the high concentration of Np95 in the pHDB unit, right at the time of heterochromatin replication, guarantees an efficient recruitment of HDAC-1 for the deacetylation of these residues. Further experiments will be necessary to conclusively prove this hypothesis.
tetramers lacking the amino-terminal domains of both histones can be stably bound to CAF-1, which can efficiently promote their assembly in vitro during SV40 DNA replication (Shibahara et al., 2000
). This indicates that the acetylation-deacetylation of newly synthesized H4 is not a limiting step for histone deposition. Thus, the impairment of heterochromatin replication in RNAi-Np95 experiments cannot be due to a histone deposition failure as a consequence of the modified acetylation pattern of NH2
tail lysines of H4.
We show here that an increased level of the RNA of repetitive pericentromeric major satellite sequences in the absence of Np95. This can be interpreted as a direct consequence of the deacetylation of histone H4 NH2
tails, as the deacetylation of both H4AcK8 and H4AcK16 is required for gene silencing, including the transcriptional repression of heterochromatic satellite DNA sequences, and is necessary for proper chromatin maturation in a postreplicative phase. Biamonti and coworkers (Rizzi et al., 2004
) have shown that heat-shock stress bodies, formed on the pericentromeric heterochromatic regions of specific human chromosomes, are enriched in histone H4AcK8 and K16 isoforms and that heat shock triggers the transient accumulation of heterogeneous RNA molecules containing the subclass of satellite III sequences found in human PH. Small RNA produced by satellite sequences are involved in the RNAi machinery that is required for heterochromatin formation (Wassenegger, 2005
). Deacetylation of H4 in PH, therefore, could be involved in establishing and maintaining the silent state of heterochromatin after replication, and this could be a first event during heterochromatin formation. The impairment of satellite silencing blocks many processes associated with the formation of heterochromatin, including DNA and histone methylation (Kanellopoulou et al., 2005
). Np95 has a ubiquitin ligase activity with a H3 specificity in vitro and “ex-vivo” (Citterio et al., 2004
). An interesting possibility is that Np95 could bind to chromatin, deacetylate the key lysines of H4, and ubiquitinate H3, and these could be epigenetic marks needed for the addition of further PH epigenetic marks, such as H3-K9 methylation. Experiments are in progress to address this issue.
Thus, Np95 might be implicated in the process that controls a very initial postreplicative step that leads to the establishment of silent PH, a precondition for the formation of this chromatin district. No variations are observed in the RNA level of minor satellites, which correspond to centromeric heterochromatin, once more suggesting a role of Np95 only for PH. We cannot yet distinguish if the increased transcription of pericentromeric repeats is due to an enhanced transcriptional rate, to an increased stability of the RNA owing to the inhibition of enzymes involved in the RNAi process (Dicer, for example; Wassenegger, 2005
), or both. Nevertheless, in both cases we would expect an impairment of PH formation.
Proper replication and formation of PH are important for the correct segregation of chromosomes and for the maintenance of genome stability. In yeast, heterochromatin is specifically required for cohesion between sister centromeres (Bernard et al., 2001
). In murine cells, forced accumulation of 120-nt centromeric transcripts leads to defects in chromosome segregation and sister-chromatid cohesion, changes in hallmark centromeric epigenetic markers, and mislocalization of centromere-associated proteins essential for centromere function (Bouzinba-Segard et al., 2006
). Although more experiments are needed to deeply investigate the role of major satellites in chromosome segregation and stability, our results can, at least partially, explain the higher genome instability that has been seen in Np95 +/− and −/− ES mouse cells (Muto et al., 2002
Preliminary experiments conducted to study the effect of overexpression of Np95 in the cells (Papait, Pistou, Cogliati, Pecoraro, Babbio, and Bonapace, unpublished results) show that high levels of this protein inside the cells cause profound modifications of PH. Because Np95 and ICBP90 have been found overexpressed in numerous cancers, higher levels of this protein inside the cell could affect proper chromosome organization and thereby chromosome stability and segregation.
Although our results do not formally prove that the deacetylation of H4 lysines is responsible for the observed reduction of heterochromatin replication, a conceivable hypothesis is that the absence of Np95 is preventing the initial deacetylation step necessary for the correct formation of PH and that this event would impair and retard the transfer of replicated, but yet unformed (or at least not yet deacetylated), heterochromatin from the pHDB back into the heterochromatic core, as proposed by the pHDB model (Quivy et al., 2004
In conclusion, Np95 is a new important protein for PH replication, and it has a key role in the regulation of the deacetylation of lysine residues of the NH2 tails of histone H4 and in the transcriptional control of heterochromatic major satellites, a process that occurs during heterochromatin replication. Further experiments will be needed to investigate more deeply how the regulation of the deacetylation of H4 tails and the transcriptional control of major satellites by Np95 are involved in heterochromatin replication and formation.