After DNA replication, recombination or repair, DNA is re-assembled into nucleosomes. This mechanism involves several protein complexes, which function as chaperones and help to integrate histones and DNA into a highly organized chromatin structure. During S-phase of the cell cycle, the parental nucleosomes become temporarily separated. After they pass the replication fork, nucleosomes are newly assembled onto the two DNA daughter strands by integrating pre-existing histones as well as newly synthesized histones.

Chromatin assembly factor I (CAF-I)

In S. cerevisiae, CAF-I is a chromatin assembly factor that delivers histone H3 and H4 to DNA during DNA replication or DNA repair (Gaillard, et al., 1996);(Kamakaka, et al., 1996);(Kaufman, et al., 1997). In order to bind to the replication fork, CAF-I interacts with the proliferating cell nuclear antigen (PCNA) (Verreault, et al., 1996).

CAF-I is an evolutionary conserved heterotrimeric complex with the subunits Cac1, Cac2 and Cac3. Deletion of any of the three CAC genes leads to an increase in ultraviolet (UV) radiation sensitivity, implying a defect in nucleotide excision repair (Game and Kaufman, 1999). Additionally, CAC deletions reduce position dependent gene silencing at the telomeres (Enomoto, et al., 1997);(Kaufman, et al., 1997), the rDNA locus (Smith, et al., 1999) and the mating-type loci (Enomoto and Berman, 1998);(Kaufman, et al., 1998), suggesting a role for CAF-I in heterochromatin formation. As a deletion of the three CAF-I subunits does not result in a G2 arrest and is not lethal for the cell (Kaufman, et al., 1997), it is likely that one or more independent pathways for chromatin assembly exist.

Anti-silencing factor 1 (Asf1)

Asf1 is thought to act in concert with CAF-I as a chromatin assembly factor. It also promotes assembly of nucleosomes in vitro (Tyler, et al., 1999) and the D. melanogaster homologue of Asf1 was shown to interact with histones H3 and H4 that carry the acetylation pattern of newly synthesized histones (Tyler, et al., 2001).

ASF1 was originally identified in a screen for high-dosage disrupters of silencing at the mating-type loci in S. cerevisiae (Le, et al., 1997). When overexpressed, it also leads to reduced silencing at the telomeres (Le, et al., 1997);(Singer, et al., 1998) and at the rDNA locus (Singer, et al., 1998). Asf1 is not essential for the cell, but a deletion causes defects in heterochromatic gene silencing, slow growth due to a lengthened S-phase, sensitivity to DNA damaging and replication blocking agents (Le, et al., 1997);(Tyler, et al., 1999) and an increase in chromosome loss (Le, et al., 1997).

Additionally, Asf1 interacts with the CAF-I subunit Cac2 and increases CAF-I activity in nucleosome assembly in Drosophila as well as in yeast (Mello, et al., 2002);(Tyler, et al., 2001). Inactivation of both CAF-I and Asf1 leads to a synergistic reduction in heterochromatic gene silencing, since double mutants display more severe phenotypes than strains with either single mutant (Tyler, et al., 1999). These results imply that CAF-I and Asf1 are both chromatin assembly factors that integrate histone H3 and H4 into chromatin in partially overlapping pathways. Nevertheless, CAF-I and Asf1 also have distinct roles in chromatin assembly, as they show different interactions with proteins involved in cell cycle checkpoint, non-homologous end joining (NHEJ) or H2A phosphorylation and because mutations in either CAC or ASF1 result in different gross chromosomal rearrangement (GCR) rates (Myung, et al., 2003).

Histone regulation genes (Hir)

Gene products from the HIR (histone regulatory) genes HIR1 and HIR2 have been shown to interact with Asf1 in vitro and in vivo (Sharp, et al., 2001);(Sutton, et al., 2001), implying that they function together in a silencing pathway that is also PCNA dependent and partially overlaps with the CAF-I silencing pathway (Krawitz, et al., 2002).

The HIR genes HIR1, HIR2, HIR3 and HPC2 encode proteins that tightly regulate histone gene transcription (Osley and Lycan, 1987);(Xu, et al., 1992). They code for repressors proteins that bind to the histone promotors in early G1-, late S- and in G2/M-phase and therefore prevent the histone genes from being transcribed (Osley, et al., 1986). Additionally, Hir proteins contribute to Asf1-mediated nucleosome assembly (Sharp, et al., 2001). The HIR-genes are exclusively expressed during G1/S transition in yeast, whereas in other phases of the cell cycle they become repressed by a mechanism that is thought to involve a specialized chromatin structure (Dimova, et al., 1999).

Mutations in HIR genes have only minor effects on silencing at the telomeres and the HM loci (Kaufman, et al., 1998), but when combined with mutations in CAF-I subunits, yeast cells display synergistic reduction of position-dependent gene silencing both at the HM loci and the telomeres, increased sensitivity to DNA damaging agents and slow growth (Kaufman, et al., 1998);(Qian, et al., 1998);(Sharp, et al., 2001). A new role for histone interacting and –deposition proteins has been described at the centromere, where CAF-I and Hir proteins function in maintaining the centromeric chromatin structure (Sharp, et al., 2002). cac1Δhir1Δ cells have increased chromosome missegregation, genetic synergies with mutations in kinetochore protein genes and an alteration in centromeric chromatin structure. CAF-I and Hir proteins are not absolutely required for deposition of the histone H3 variant Cse4 at the centromere, but their absence leads to Cse4 deposition outside of centromeric chromatin.

Nucleosome assembly protein 1 (Nap1)

After deposition of histone H3 and H4, histone H2A and H2B are integrated into chromatin to form the core nucleosome. The nucleocytoplasmatic shuttle protein Nap1 (nucleosome assembly protein 1) binds to histone H2A and H2B and helps to assemble chromatin from newly synthesized DNA (Ito, et al., 1996). Since Nap1 has been localized in several cell compartments, the current model posits that Nap1 functions as a histone chaperone that binds H2A and H2B already in the cytoplasm. Due to interaction with Kar114p, the primary karyopherin/importin responsible for the nuclear import of H2A and H2B, it helps to import them into the nucleus, where it incorporates the histones into newly replicated DNA (Mosammaparast, et al., 2002).

Histones consist of a highly conserved globular histone-fold domain and an N-terminal domain that remains outside of the nucleosome (Luger, et al., 1997). The N-termini as well as the core region are targets for posttranslational modifications, e.g. acetylation, phosphorylation, methylation and ubiquitination, which result in changes of gene activity (Spencer and Davie, 1999).

Methylation or acetylation occurs on the ε-amino group of positively charged lysines (Fig. 2). In the case of acetylation, the charge of the amino group is then changed to neutral, whereas methylation does not change the positive charge. Additionally, histones can be methylated on arginine groups.

Fig. 2 : Possible modifications of amino acids at the example of the N-terminus of histone H3 from S. cerevisiae. A=acetylation, M=methylation, P=phosphorylation

Several histone methyltransferases (HMTs) have been characterized. One example are the SUV39 family proteins that exist in D. melanogaster, humans and S. pombe, and selectively methylate histone H3 lysine 9 (Rea, et al., 2000). This methylation has been shown to be a prerequisite for binding of the heterochromatin binding protein 1 (HP1), which connects H3 K9 methylation to silencing and heterochromatin formation (Bannister, et al., 2001);(Lachner, et al., 2001). Additionally, methylation on H3 K9 prevents phosphorylation of H3 S10, which is known as a modification involved in gene activation (Lo, et al., 2001);(Lo, et al., 2000). Methylation can also lead to transcriptional activation, e.g. methylation of H3 K4 in S. cerevisiae (Noma, et al., 2001). It is becoming clear that histone modifications are able to influence each other and work together in the form of a “histone code”, thereby creating a specific chromatin structure that is connected with gene activity.

Histone acetylation is carried out by several known histone acetyltransferase (HATs), and as acetylation is a reversible process, there also exist histone deacetylases (HDACs) as antagonists in the cell. In general, histones tend to be hyperacetylated in actively transcribed regions, whereas histones are hypoacetylated in transcriptionally repressed regions. The simplest model of gene repression involves a histone deacetylase that removes acetyl groups from the N-termini of histones in a defined area, thereby building up a repressive chromatin structure. As a consequence, transcriptional activators or other transcription factors are unable to bind to their promotor elements, which leads to transcriptional inhibition.

Histone deacetylases (HDACs)

Histone deacetylation has been linked to the establishment of transcriptional inactive heterochromatin. At least ten HDACs exist in S. cerevisiae: Hda1, Hos1-3, Rpd3, Hst1-4 and Sir2. Hos 1/Hos3 and Hos 2 mainly affect ribosomal DNA and ribosomal protein genes, whereas Rpd3 and Hda1, which preferentially deacetylate distinct promotors, function in histone deacetylase complexes and their absence results in hyperacetylation of histone H3 and H4 (Kuo and Allis, 1998);(Robyr, et al., 2002).

The first hint that Sir2 functions as a histone deacetylase was the observation that SIR2 overexpression leads to hypoacetylation of histones in yeast (Braunstein, et al., 1993). Sir2 has now been characterized as a phylogenetically conserved NAD⁺-dependent HDAC that efficiently deacetylates histone H4 lysine 16 and histone H3 lysine 9 (Imai, et al., 2000);(Landry, et al., 2000);(Smith, et al., 2000). One role of Sir2 is the establishment of silencing at the rDNA locus (Bryk, et al., 1997);(Smith and Boeke, 1997), as well as at silent mating-type loci HML and HMR and at the telomeres in a complex with other Sir proteins (Sir3 and Sir4, at the HM loci additionally Sir1) (Moretti, et al., 1994);(Rine and Herskowitz, 1987). Recently, a role in ageing has been described for Sir2 in C. elegans and in other organisms, including yeast (Guarente, 2000);(Guarente, 2001);(Imai, et al., 2000). Overexpression of Sir2 leads to an increase in life span (Kaeberlein, et al., 1999), which is determined in yeast by how many times a mother yeast cell is able to divide, whereas SIR2 deletion or NAD⁺-deprivation leads to a increased recombination rate and prevents longevity (Lin, et al., 2000).

Histone acetyltransferases (HATs)

In contrast to HDACs, histone acetyltransferases have the opposite effect on silencing, since the acetylation of lysine residues generally leads to the formation of euchromatin and transcriptional activation. The various existing HATs are grouped into five families based on sequence similarities. These families also include the MYST family with its representatives in yeast (Sas2, Sas3, Esa1), D. melanogaster (MOF, Chameau) and human (MOZ, MORF, Tip60, HBO1). Members of the evolutionary conserved MYST family share the MYST homology domain with a binding site for acetyl-CoA (HAT domain) and an atypical C2HC zink-finger motif.

SAS2 was originally identified in a screen for suppressors of silencing defects at HMR (Ehrenhofer-Murray, et al., 1997). In contrast to its role at the HMR locus, a deletion of SAS2 (sas2Δ) decreases silencing at HML in sir1Δ strains and derepresses silencing at the telomeres (Ehrenhofer-Murray, et al., 1997);(Reifsnyder, et al., 1996). The conserved acetyl-CoA binding domain as well as the HAT domain is essential for HML and telomeric silencing, because mutations in these domains lead to the same silencing phenotypes as a SAS2 deletion (Meijsing and Ehrenhofer-Murray, 2001);(Osada, et al., 2001). Additionally, (Meijsing and Ehrenhofer-Murray, 2001) have shown that a mutation in histone H4 lysine 16 to arginine phenocopies the effect of a sas2Δ, implying that H4 K16 is one target for Sas2 acetylation.

Sas2 exists in a complex called SAS-I together with two other proteins involved in silencing, Sas4 and Sas5 (Meijsing and Ehrenhofer-Murray, 2001);(Osada, et al., 2001) and acetylates next to histone H4 lysine 16 also H3 lysine 14 in vitro (Sutton, et al., 2003). The association with Sas4 is essential for Sas2’s acetyltransferase activity, whereas binding of Sas5 is required for improving the activity of the complex (Sutton, et al., 2003). Furthermore, Sas2 has been shown to interact with the chromatin assembly factors CAF-I and Asf1 (Meijsing and Ehrenhofer-Murray, 2001);(Osada, et al., 2001), proposing a model where Sas2 is recruited to the replication fork via Cac1 and/or Asf1, where it can acetylate the incorporated histones. Thereby, Sas2 might link DNA replication and chromatin assembly to histone modification. It is unknown whether Sas2 also acetylates histone variants that exist in the cell.

Three silenced regions are known in the yeast S. cerevisiae: the silent mating-type loci HML and HMR on chromosome III, the rDNA locus on chromosome XII and the telomeres. Both silencing at the HM loci and at the telomeres requires a common set of trans-acting factors, which include the Sir (silent information regulator)-proteins, the N-termini of histone H3 and H4, Abf1 and the repressor-activator protein Rap1. The structure of the different silenced regions and their common and distinct features are discussed below.

Silencing at the mating-type loci HML and HMR

Haploid yeast cells can display either an a- or a α-mating-type. The cell type is defined by the allele present in the MAT locus on chromosome III. Whereas cells with a MATa allele are of a-mating-type, and cells with the MATα allele are of α-mating-type. Under certain circumstances, a cell can switch its mating-type by cleaving the mating-type locus with HO endonuclease and replacing the MAT with information from HML or HMR.

Additionally to MAT, the cell contains unexpressed copies of the mating-type information genes near the telomeres on the same chromosome: HMR generally codes for a-information and HML for α-information. The repression of the HM loci is maintained by flanking silencing elements (E and I silencer) as well as by silencer binding proteins. Whereas the E-silencer is absolutely essential for silencing, a deletion of the I-silencer does not influence the repression (Brand, et al., 1985). Since the I-silencer is a protosilencer, it becomes important in compromised situations, e.g. when the E silencer is not fully functional. The flanking silencers from the HM loci cause repression of a- or α-information transcription in addition to MAT gene expression.

Fig. 3 : Structure of the silent mating-type locus HMR in S. cerevisiae with silencer binding proteins and proteins involved in the establishment and maintenance of silencing.

To exemplify silencing at the HM loci, silencing at the well-studied HMR silencer is explained subsequently and in Fig. 3. The a-information at HMR is flanked by the HMR-E and the HMR-I silencers. The ~140 bp HMR-E contains binding sites for the origin recognition complex ORC, Rap1 and Abf1, whereas the HMR-I silencer lacks the Rap1 binding site. Rap1 and Abf1 are regulatory proteins that function as transcriptional activators in other positions in the genome beside their role in silencing (Shore, 1997).

ORC, Rap1 and Abf1 serve as a platform for binding of the Sir-proteins to HMR-E. Deletion of Sir1 only causes a weak silencing defect, whereas deletion of SIR2-4 leads to complete derepression. Through the interaction with ORC, Sir1 facilitates the binding of Sir2, Sir3 and Sir4 to the silencer. Hereby, Sir3 and Sir4 interact directly with Rap 1 and the N-termini of histone H3 and H4 (Moretti, et al., 1994);(Triolo and Sternglanz, 1996). Sir2 further improves efficient spreading of the Sir-proteins throughout HMR via its NAD⁺-dependent deacetylation activity (Rusche, et al., 2002).

Silencing at the rDNA locus

Another silencing locus in S. cerevisiae is the highly repetitive rDNA locus on chromosome XII. The rDNA locus encodes the ribosomal RNAs, which are not translated into proteins, but establish the ribosome together with ribosomal proteins. One rDNA unit consists of ~9.1 kb, which is tandemly repeated in 100-200 copies per cell. Silencing at this locus is thought to regulate the access of RNA polymerase I, but prevents RNA polymerase II from transcription (Shou, et al., 2001).

At the rDNA locus, Sir2 assembles together with Net1, Cdc14, Nan1 and PolI into the so-called RENT (regulator of nucleolar silencing and telophase exit) complex. In sir2Δ cells, the rDNA silencing is decreased and the chromatin structure is less compact (Smith and Boeke, 1997), whereas the recombination rate is increased (Gottlieb and Esposito, 1989). Thus, Sir2 improves rDNA silencing via its deacetylation activity.

The structural core component of the RENT complex is Net1, which recruits the other RENT-proteins to the rDNA locus. Net1 binds within the RENT complex to PolI, the RNA polymerase I, and stimulates its enzymatic activity (Shou, et al., 2001). Whether Net1 directly binds to DNA is still unknown, but it is possible that other still unknown proteins contribute to rDNA silencing. Interestingly, when NET1 is overexpressed, it is also associated with the HMR-E silencer, whereas net1-1 acts indirectly in HMR silencing by releasing Sir2 from the nucleolus (Kasulke, et al., 2002).

Silencing at the telomeres

Telomeres can be found at the ends of all 16 chromosomes in yeast. They consist of a 300-350 bp region with irregular TG_1-3 repeats, which are disrupted by X- and Y-elements of variable size (Palladino and Gasser, 1994). The Rap1 binds on average every 18 bp in the telomeric repeats and is a structural core component of the telomeres. The Rap1 binding region is also referred to as the telosome and is characterized by its nuclease resistance.

Rap1 in turn recruits Sir2, Sir3 and Sir4 to the telomeres, where Sir3 and Sir4 bind to the N-termini of histone H3 and H4. The Sir proteins spread in a histone-dependent manner up to 2.8 kb into the core telomeric heterochromatin. (Strahl-Bolsinger, et al., 1997) have proposed a model in which telosomal Rap1 folds back onto subtelomeric regions. This allows a condensation at telomeric heterochromatin due to the interaction between Rap1 and the Sir’s, as well as among the Sir proteins themselves.

Silencing at the telomeres and subsequent condensation prevents the chromosome ends from being degraded and improves their replication (Palladino, et al., 1993). Additionally, the positioning of the telomeres to the nuclear periphery is maintained.