[page 63↓]

4  Discussion

Nα‑acetylation, a frequent protein modification in eukaryotes, occurs in the yeast S. cerevisiae mostly by the Nα‑acetyltransferase complex NatA. Previous work had revealed a role for NatA in transcriptional silencing of heterochromatin-like regions in the yeast genome, although the mechanism by which NatA functions in silencing had remained unclear.

In this work, we used different approaches to find silencing components that require Nα‑acetylation by NatA, and both tested silencing proteins directly for Nα‑acetylation and screened genetically for suppressors of the nat1Δ phenotype. With Orc1 and Sir3, we identified two NatA substrates with a direct role in silencing, whose function is partially impaired by the loss of Nα‑acetylation. However, as inferred from their double mutant phenotype, NatA’s role in silencing likely comprises the Nα‑acetylation of more components.

Interestingly, the subunits of NatA are conserved in higher organisms. Since only preliminary data exist on their function to date, future research is required to reveal their role in cellular processes of complex organisms. For this challenge, the identification of NatA functions in yeast provides a useful basis.

4.1 Relevance of Nα -acetylation for Orc1

In this study, we gained genetic and biochemical evidence that Orc1, the biggest subunit of the ORC complex, is a substrate of NatA. Furthermore, we found that Nα‑acetylation of Orc1 was essential for its function in telomeric silencing.

Why was telomeric but not HM silencing affected by the lack of Nα‑acetylation of Orc1? One possible reason is that the semistable manner of telomeric repression makes it more sensitive than stable HM silencing to orc1 mutations. The semistability has been proposed to originate in reduced silencing establishment at telomeres, resulting in the variegated expression of subtelomeric genes (Chien et al. 1993). Another possible explanation for the specific telomeric effect is a difference in structure of the silencing complexes at HM loci and chromosomal ends. Thus, Nα‑acetylation might be of different relevance for the role of Orc1 at the different silenced loci.

How does Nα‑acetylation affect Orc1’s silencing function? So far, the recruitment of Sir1 to the HM silencers has been considered the exclusive task of Orc1 in silencing. However, genetic [page 64↓]data indicate that Orc1 has additional silencing functions at the telomeres. Foremost, telomeric silencing completely depends upon ORC while being unaffected by the deletion of Sir1 (Fox et al. 1997).

Several lines of evidence suggest that Nα‑acetylation of Orc1, although being required for telomeric silencing, has no impact on Orc1’s ability to interact with Sir1: Firstly, Orc1-tethered silencing in a nat1Δ strain was still dependent on Sir1. Secondly, in a two-hybrid assay, Sir1 interacted with Orc1(5-235), whose N-terminus was blocked to acetylation in that it was N‑terminally fused to the Gal4 activation domain. Thirdly, unacetylated orc1 mutants were fully sensitive to α‑factor and did not resemble the typical establishment-defective α‑factor response phenotype of sir1Δ mutants. Finally, the observation that Nα‑acetylation is not required by Orc1 for Sir1 binding is in agreement with the observation that SIR1 overexpression suppressed the nat1Δ silencing defect at HML (Stone et al. 1991).

Thus, our data propose a novel function of Orc1 in silencing that is in addition to Sir1 recruitment. We hypothesize that the Orc1 amino terminus interacts with an as yet unidentified silencing protein that functions primarily in telomeric silencing, and that this interaction requires Nα‑acetylation of Orc1 by NatA. Crystallographic data show that the extreme N‑terminus of Orc1 is exposed on the surface of the protein in a structure distinct from the Sir1 interaction domain (Zhang et al. 2002) (Fig. 4.1), thus rendering it a potential interaction module for another protein. The novel interaction partner may specifically recognize the N-terminus of Orc1 in its acetylated form. Precedence for modification dependent protein interactions comes from bromodomain and chromodomain proteins that preferentially bind specific acetylated or methylated histone residues, respectively (Jenuwein and Allis 2001).

Further evidence for the hypothesis that Nα‑acetylation regulates interactions between Orc1 and other proteins comes from our observation of a synthetic lethal interaction between nat1∆ and SUM1‑1, but not sum1∆. Interestingly, the SUM1‑1 mutation confers to the Sum1‑1 protein the ability to interact with ORC, but retains the ability to interact with the histone deacetylase Hst1. Thus, Sum1‑1 binds to the silencers via ORC, recruits Hst1 to the HM silencers and establishes Sir2 independent silencing at the HM loci (Rusche and Rine 2001). The binding of Sum1‑1 to ORC is abrogated by deletion of the amino-terminal 235 amino acids of Orc1, suggesting that Sum1‑1 interacts with the Orc1 N-terminus. Interestingly, the slow growth rate of SUM1‑1 mutants was suppressed by additional orc mutations in an earlier study, which implicated that Sum1-1 interferes with DNA replication through ORC (Rusche and Rine 2001). Therefore, one interpretation of the inviability of nat1 SUM1‑1 strains is that Sum1‑1 interacts better (i.e. stronger and at more genomic locations) with Orc1 in its [page 65↓]unacetylated form, and that this inhibits replication initiation, which is ORC’s essential function. This hypothesis is supported by the observation that the SUM1‑1 nat1∆ synthetic lethality was abrogated by the deletion of the amino-terminal 235 amino acids of Orc1. Thus, we postulate that Nα‑acetylation of Orc1 regulates its ability to interact with Sum1‑1 as well as with other proteins.

N‑terminal protein acetylation has been hypothesized initially to protect proteins from degradation (Jornvall 1975; Hershko et al. 1984). However, several observations indicate that this does not hold true for the influence of NatA on silencing. First, we found that the level of Orc1 protein was indistinguishable between wild-type and nat1∆ strains. Second, the effect of nat1∆ on HML silencing and temperature sensitivity was suppressed by overexpression of the ribosome-bound chaperone Ssb1 (Gautschi et al. 2003), suggesting that nat1∆ caused a defect in protein folding rather than stability. However, SSB1 overexpression did not suppress the telomeric silencing defect of nat1∆ and the orc1 N-terminal mutants, thus supporting the notion that acetylated Orc1 specifically recruits a novel protein to establish silencing rather than affecting Orc1 folding.

Interestingly, Nα‑acetylation affects Orc1’s function in silencing, but not its function in replication initiation. Together with our observation that N‑terminal truncations of up to 100 amino acids from Orc1 have no effect on growth, this confirms the earlier hypothesis of Bell et al. (1995) that the N‑terminus of Orc1 has no function in replication. Notably, in contrast to Orc1 acetylation, NatA activity has an impact on ORC’s replication function, because nat1Δ orc2-1 double mutants were inviable. This suggested that other ORC subunits, or other replication factors, may be Nα‑acetylated by NatA, and that this acetylation may impinge upon their ability to initiate replication, perhaps by affecting their ability to interact with other replication proteins.

Future experiments are required to validate our model of a protein interaction specific to Nα‑acetylated Orc1. One possible approach to identify such an interacting protein is a two‑hybrid screen with Orc1 fused C-terminally to the Gal4 binding domain as bait. Those prey proteins that bind to Orc1 in a wild-type but not in a nat1Δ strain are good candidates for novel interaction partners of Nα‑acetylated Orc1. If these interactions can be confirmed by in vitro and in vivo binding studies, it will be further interesting to determine whether mutations of the candidates mimic the phenotype of the unacetylated orc1 mutants.

Another future task is to specify the role of NatA in replication. Here, one obvious question is the Nα‑acetylation of ORC subunits other than Orc1. As judged their penultimate amino acid, Orc3, Orc4 and Orc6 are potential NatA substrates whose Nα‑acetylation can be investigated [page 66↓] in vivo. Additional candidates for NatA targets in replication are the cell‑cycle genes, which also display genetic interactions to ORC alleles (Loo et al. 1995).

Since both Orc1 and the subunits of NatA are evolutionarily conserved, it will be further interesting to test if Nα‑acetylation plays a role in the function of Orc1 homologs in higher organisms.

4.2 Function of the N-terminal 100 amino acid domain of Orc1

In this study, we found that N-terminal deletions of 50, 100, and in some cases only 28 amino acids from the N‑terminus of Orc1 disturbed silencing at the HM loci and telomeres. Since this phenotype differed clearly from that of the missing Nα‑acetylation, different facets of Orc1’s function in silencing appear to be affected by the two types of mutations.

The N‑terminal 100 amino acids of Orc1 belong to the BAH domain. This domain is found also in a number of other chromatin-associated proteins, such as mammalian DNA (cytosine‑5) methyltransferases, components of the RISC chromatin-remodeling complex and histone deacetylase complexes (Callebaut et al. 1999), and was thus proposed to function as a protein-protein interacting module involved in transcriptional regulation and chromatin-mediated gene silencing.

By sequence homology analysis, the BAH domain of Orc1 had been originally located to amino acids 48 to189 (Callebaut et al. 1999). Crystallographic data of Zhang et al. (2002) redefined the region between amino acids 10 and 190 as the BAH core domain, whose secondary structure is mainly composed of β‑strands (Fig. 4.1). In addition, the so-called H‑domain, a small, non‑conserved helical subdomain between amino acids 100 and 129 (β6 and β7), was identified to be necessary and sufficient for the interaction with Sir1. This interaction is supported by the core domain, since a number of amino acid interactions stabilize the position of the H‑domain with respect to the core (Zhang et al. 2002). Notably, almost all of the amino acids participating in these interactions lie between amino acids 100 and 190, and the loop between amino acids 21 and 35 (connecting β1 and β2) was dispensable for Sir1 binding. This suggested that the region before amino acid 100 was not important for the function of the BAH domain. In contrast to this notion, we found that the two-hybrid interaction of Orc1 with Sir1 required the first 50 amino acids of Orc1, implicating that this region was also relevant for the BAH function.

[page 67↓]

Nevertheless, our data propose that the orc1 N‑terminal deletion mutants had further defects than the loss of Sir1 binding. Strikingly, truncated orc1 mutants did not resemble the α‑factor response phenotype of sir1Δ, and instead formed shmoo clusters, which indicate defects in the maintenance of silent chromatin rather than its establishment (Enomoto and Berman 1998). In contrast to that, an orc1 mutant missing the H‑domain responded to α‑factor similarly to sir1Δ (Zhang et al. 2002). Since the deletion of the N‑terminal 50 amino acids of Orc1 further decreased the α‑factor sensitivity of sir1Δ, we propose that the orc1 mutation caused a structural defect at the HML silencer, in addition to ineffective reestablishment of silencing due to the loss of SIR1. This structural defect might be based on the loss an interacting partner of Orc1 that is distinct from Sir1. This hypothesis is further supported by the telomeric phenotype of the orc1 truncation mutants, which was earlier characterized as a Sir1 independent effect.

Fig. 4.1: The crystal structure of the N-terminal domain of Orc1.
(A) Ribbon presentation. (B) Topology diagram showing the fold of the structure and deletion sites of the orc1 mutants investigated in this study. The BAH core structure is colored blue, the H-domain is shown in magenta, N- and C-terminal helices are shown in red. (adapted from Zhang et al. 2002)

Altogether, our data suggest that deletions within the first 100 amino acids of Orc1 not only have a destabilizing effect on the interaction with Sir1, but also impair the interaction of Orc1 [page 68↓]with (a) further silencing partner(s). We hypothesize that this protein requires Nα‑acetylation as well as an intact N‑terminal structure of Orc1 for its binding (Fig. 4.2).

Interestingly, the deletion of the region between amino acids 28 and 52 of Orc1 also affected silencing, in contrast to the removal of amino acids 21 to 35 (Zhang et al. 2002). The telomeric phenotype of this mutant points to a special role of this region in the binding of the hypothesized silencing factor, which we propose to act in particular at the telomeres.

Why did the deletion of 10 amino acids from the N‑terminus of Orc1 not effect silencing? Whereas the BAH domain should be still functional in this mutant, Nα‑acetylation by NatA is expected to be interrupted. However, the penultimate amino acid tryptophan makes it to a possible substrate for the Nα‑acetyltransferase NatC. Thus, one conceivable scenario is that the Nα‑acetylation of Orc1(Δ1‑10) by NatC still enables it to interact with the hypothesized silencing factor and therefore to function like full-length Orc1. This could be tested in an IEF gel with Orc1(Δ1‑10) probes derived from wild-type and natC mutant strains.

According to our model, the region comprising the first 100 amino acids of Orc1 has a dual function in silencing: supporting the interaction of the H‑domain with Sir1 and providing the binding site for a further interacting factor. Further studies will be required to test our hypothesis. For example Co-IPs could test Sir1 binding to the truncated Orc1 versions, and ChIP assays can be used to test its association to silencers in the mutant strains. Moreover, it has to be specified what regions of Orc1 are required for the binding of new interacting partners.

Notably, the BAH domain is conserved in all known Orc1 homologs, posing the question of whether this motif mediates a role of Orc1 in transcripitional repression also in higher eukaryotes. Significantly, Orc1 is associated with heterochromatin in Drosophila, where its N‑terminus interacts physically with HP1, a central component of heterochromatin. In addition, the amino terminus of Xenopus Orc1 likewise interacts with HP1 homologs (Pak et al. 1997). Although it is not known at present whether the BAH module plays a direct role in this interaction, these data strongly suggest that not only the replication initiator function, but also the silencing function of ORC is evolutionary conserved.

[page 69↓]

Fig. 4.2: Model of protein interactions at the N-terminus of Orc1.
In addition to Sir1, the Orc1 N-terminus interacts with another, yet unknown protein. This protein requires the Nα‑acetylation of Orc1 for binding and is specifically recruited in the context of telomeric silent chromatin, when Sir1 is absent. In contrast, Sir1 is recruited via the H‑domain of Orc1 specifically to the HM silencers. This binding does not require the Nα‑acetylation of Orc1, but it depends on the integrity of the N‑terminal region of 100 amino acids.

4.3 A model of the role of NatA in silencing

In this study, we sought to reveal by which mechanism NatA functions at silenced loci. We identified Orc1 as a NatA substrate, and showed that its Nα‑acetylation was required particularly for telomeric silencing. Summarizing our data, we propose a novel silencing factor to bind to Orc1 in its Nα‑acetylated form.

In light of the high similarity of between Orc1 and Sir3 N‑termini, it is interesting that we also found Sir3 to be Nα‑acetylated by NatA. One possibility is that acetylated Orc1 and Sir3 both interact with the same hypothesized silencing factor, which is in agreement with the observation that double orc1 sir3 unacetylated mutants showed no additional silencing effects. However, these double mutants showed weaker derepression at the HM loci than nat1Δ, suggesting that further targets of NatA exist that require Nα‑acetylation for their silencing function.

We therefore propose a model, in which the effect of nat1∆ at the HM loci is the sum of several proteins lacking acetylation, among them Orc1 and Sir3. The cumulative effect on these proteins causes Sir3 and perhaps other Sir proteins to lose their ability to bind silenced chromatin, and thus causes derepression. Significantly, missing Nα‑acetylation caused only a partial loss of function in the two substrates here identified. In unacetylated Orc1, Sir1 recruitment was not affected, and unacetylated Sir3 did not completely disrupt silencing as seen in the sir3 null mutant (Rine and Herskowitz 1987).

[page 70↓]

Interestingly, the N-terminus of Sir3 interacts in two-hybrid assays with Abf1 (Gasser and Cockell 2001), and it is well conceivable that this interaction depends on Nα-acetylation of Sir3. This may provide an explanation for the missing effect of nat1Δ at HMR-E lacking the Abf1 binding site.

It would be in line with our model if some of the NatA targets in silencing acted not directly at the silenced loci, but rather would influence silencing indirectly by regulating silencing factors. To date, there exist only preliminary data on the regulation of members of the silencing complex. One example for such a regulation is the Sir3 hyperphosphorylation in response to mating pheromone, heat shock and starvation, which increases silencing (Stone and Pillus 1996). This modification requires an activated MAP kinase cascade, although the detailed mechanism of Sir3 regulation remains unclear (Ai et al. 2002; Ray et al. 2003).

How can further NatA substrates that require Nα‑acetylation for their silencing function be identified? Besides testing individual silencing components for Nα‑acetylation, genetic screens provide one possibility to reveal new genes linked to the silencing function of NatA. In light of the results of the multicopy-suppressor screen performed in this study, it is important to strictly focus the screen on those NatA substrates that act in silencing. In other words, when mating ability is used as a sensor for HM silencing, no genes involved in other aspects of mating should be isolated. This could be realized, for instance, by screening for mutants which reestablish silencing in a nat1Δ background. In addition, the screen has to be sensitive enough to detect slightest improvements of HM silencing, since single components have to be isolated out of an orchestra of components that constitute the nat1Δ phenotype.

To complete the picture of NatA’s silencing function, it is also necessary to uncover its involvement at the other silenced loci. Silencing of subtelomeric reporter genes was completely disrupted by the deletion of NAT1, and unacetylated orc1 and sir3 mutants displayed the same effect (Stone et al. 2000). At the moment it is not clear, whether the Nα‑acetylation of Orc1 and Sir3 alone constitutes the role of NatA at telomeres. Further experiments will be required to determine the structural integrity of the perinuclear foci in unacetylated orc1 and sir3 mutants and whether the association of other components, such as Rap1, is likewise affected.

In this study, we present evidence that NatA also has a function in rDNA silencing. Future work has to reveal which silencing components at the rDNA require Nα‑acetylation. Interestingly, both NatA substrates identified here are capable of binding to the rDNA array. Although Sir3 is not required for the repression of rDNA reporter genes (Bryk et al. 1997; Smith and Boeke 1997), it locates in the nucleolus of aging cells (Kennedy et al. 1997). [page 71↓]Notably, the N‑terminus of Sir3 was shown to contain efficient information for nucleolar targeting (Gotta et al. 1998). Given its high homology to the N‑terminus of Orc1, both proteins might be recruited to the rDNA locus by the same mechanism, and a role of the already hypothesized common interaction partner in this process is conceivable. Alternatively, ORC can bind to the rDNA array via the ACS sites that are part of the NTS2 of each repeat (Huang and Moazed 2003). In order to verify a role of Nα‑acetylated Orc1 and Sir3 in rDNA silencing, the unacetylated mutants need to be tested for a rDNA phenotype. An interesting observation with regard to the mechanism of NatA’s role in rDNA silencing was that GFP-tagged Sir2 was still located in the nucleolus of nat1Δ cells (data not shown). This indicated that the integrity of the silencing-mediating RENT complex was not dependent on NatA.

4.4 Nα-acetylation as a conserved eukaryotic protein modification

In this study, we provide evidence that Nα‑acetylation participates in the regulation of chromatin structure in yeast, since the silencing proteins Orc1 and Sir3 depended on this modification to function properly. Thus, we propose that Nα‑acetylation can be classified as a chromatin regulatory mechanism comparable to acetylation or methylation of ε-N‑lysines.

However, in contrast to ε-N-acetylation, which readily can be removed by deacetylases (Dutnall and Pillus 2001), Nα‑acetylation is irreversible, and Nα‑deacetylases are hitherto unknown. This raises the question of how the modification can be removed in order to alter protein function upon demand. One possibility is that amino‑terminal proteolysis may remove Nα‑acetylation, as is proposed for histone methylation, which also is irreversible (Jenuwein and Allis 2001). Regulated ubiquitin‑based protein processing (Palombella et al. 1994) is a conceivable mechanism for the purpose, since the mutation of a putative ubiquitin‑specific protease was demonstrated to specifically enhance PEV in Drosophila (Henchoz et al. 1996). Another possibility is the removal of the Nα‑acetylated amino acid. Interestingly, acylamino acid‑releasing enzymes (AARE) have been identified in eukaryotes and an archeon (Ishikawa et al. 1998; Yamauchi et al. 2003), which catalyze the amino‑terminal hydrolysis of Nα‑acylpeptides to release Nα‑acetylated amino acids. Although AAREs act specifically on short nascent chains of 2-5 amino acids (Krishna and Wold 1992), related enzymes might perform this reaction on Nα‑acetylated proteins. A further alternative to modulate the function of Nα‑acetylated silencing proteins may be their removal from chromatin, much like preexisting histone modifications are eliminated by histone replacement during replication. Interestingly, [page 72↓]methylated H3 histones are postulated to be exchanged during transcription for the unmethylated histone variant H3.3, which promotes the generation of active chromatin (Jenuwein and Allis 2001; Ahmad and Henikoff 2002).

We have not investigated whether any of these several theoretical mechanisms regulate the silencing activity of Orc1 or Sir3. In light of our data, we suggest that Nα‑acetylation may provide a stable, long-term epigenetic mark for maintaining chromatin states.

Intriguingly, Nα‑acetylation can be found in all kingdoms of life and especially in eukaryotes. The high level of evolutionary conservation suggests that this protein modification is capable of acting in fundamental cellular processes. In higher organisms, homologs of the NatA subunits have been linked to developmental and differentiation processes. Mouse mNAT1 is expressed in the developing brain and is regulated by physiological levels of functional N‑methyl‑D‑aspartate (NMDA) receptor in developing neurons (Sugiura et al. 2001). The mNAT1 homolog tubedown‑1 is expressed highly in developing tissues and down‑regulated upon differentiation (Gendron et al. 2000). Furthermore, a tubedown‑1 variant, Tbdn100, was isolated in a transcription regulatory complex, suggesting that it may be a transcriptional co‑regulator (Willis et al. 2002). Interestingly, the human homolog NATH also shows high expression in parts of the human brain and is overexpressed in malignant cells, for instance in papillary thyroid carcinomas and several leukemia and carcinoma cell lines (Fluge et al. 2002). It therefore has been hypothesized that NatA overexpression might simply correlate with high transcriptional activity. Even though this may be the case, overdosed Nα‑acetylation itself could result in deregulated gene expression and tumorgenesis, as overexpression of NAT1 impaired the stability of chromosomes in yeast (Ouspenski et al. 1999). In light of our findings, it is tempting to speculate that NatA acetylation regulates cell proliferation by modifying ORC function in replication or in the control of gene expression. Thus, it will be interesting to identify chromatin factors in higher eukaryotes whose function depend on Nα‑acetylation by NatA.

The implication of NatA homologs in cellular differentiation processes provokes the important question whether Nα‑acetylation plays a role in carcinogenesis. Interestingly, inappropriate regulation of chromatin structure (Singh et al. 2000) and notably mutated histone acetylating and deacetylating enzymes (Borrow et al. 1996; Vaziri et al. 2001) have been revealed to be tumor generating factors. In this respect, to enlighten the work of human Nα‑acetylating complexes is a challenging future task, which can be facilitated by a comprehensive knowledge of the mechanisms in yeast.

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