The packaging of DNA into chromatin appears to play a central role in the regulation of nearly every aspect of gene transcription. In particular, an increasing number of complexes associated with the activation or repression of transcription have been shown to contain chromatin remodeling activity or enzymes that covalently modify histones or both (25
). The silent mating type loci (HML
) and telomeric DNA regions in the budding yeast Saccharomyces cerevisiae
are packaged into a specialized chromatin structure that silences transcription and, like heterochromatin in metazoans, is epigenetically inherited (29
; reviewed in reference 19
). Moreover, a distinct example of silencing occurs at the highly repetitive yeast ribosomal DNA genes (rDNA) where RNA polymerase II reporter genes inserted within the repeats are inactivated (8
). Silent chromatin is also associated with DNA regions that are involved in chromosome maintenance and transmission, such as telomeres in budding yeast and centromeres in many other eukaryotes. In these regions, silencing appears to play a structural role that is independent of transcriptional repression (1
Silencing at each of the above loci requires Sir2, a highly conserved protein which possesses an intrinsic NAD-dependent protein and histone deacetylation activity (2
). Sir2 is an unusual deacetylase in that it couples deacetylation to the hydrolysis of a high-energy bond in NAD and transfers the acetyl group from its protein substrate to ADP-ribose to generate a novel compound, 2′,3′-O
). Mutations that abolish the in vitro activity of Sir2 result in a complete loss of silencing in vivo, suggesting that direct deacetylation and/or another aspect of this activity is required for silencing (24
Genetic and biochemical evidence suggests that Sir2 is a component of two distinct complexes that carry out its telomeric/mating type and rDNA-silencing activities, respectively (16
). Sir2 binds to Sir4 affinity columns and coimmunoprecipitates with Sir4 from yeast extracts (40
). Although Sir2 or Sir4 immunoprecipitates contain only trace amounts of Sir3 (41
), Sir3 and Sir4 interact in two-hybrid assays (42
), and truncations of the Sir4 protein can associate efficiently with Sir3 (41
), suggesting that the three proteins interact physically at some stage during the assembly of telomeric/HM silent chromatin (22
). At the rDNA repeats, Sir2 assembles with a different set of proteins into a second complex called RENT (regulator of nucleolar silencing and telophase exit) (52
). In addition to Sir2, RENT contains at least two other proteins, Net1 and Cdc14 (52
), and Net1 is required for the localization of Sir2 to rDNA and for rDNA silencing (57
). Despite their central role in gene silencing, neither Sir2-containing complex has been purified to homogeneity. In particular, the molecular composition of the complex containing the Sir2 and Sir4 proteins or the putative Sir3 complex has not been defined.
The amino termini of histones H3 and H4 play an essential role in silencing at the HM
loci and telomeres (2
). At these loci, the H3 and H4 N termini are fully hypoacetylated, and a number of studies suggest that this hypoacetylated state is critical for silencing and provides a binding site for the Sir3 and Sir4 proteins (6
). The discovery of histone deacetylation activity in Sir2 provides a direct link between Sir2-containing silencing complexes and the hypoacetylated state of histone tails in silent chromatin domains (24
). However, the idea that Sir2 deacetylates histones in vivo has not been tested. Furthermore, while a strong genetic link exists between silencing and histone hypoacetylation at HM
loci and telomeres, the role of histone tails in rDNA silencing is unknown. Thus, it is unclear whether Sir2 activity at rDNA is required to deacetylate histones or other proteins.
Little is known about how the Sir proteins assemble onto chromatin and what role, if any, each Sir protein might have in the initial nucleation, stable association, and spreading of silent domains. Immunoprecipitation of chromatin from in vivo cross-linked cells has shown that Sir2 and its associated proteins are structural components of silent chromatin domains (17
). Moreover, the association of each Sir2, Sir3, and Sir4 with extended silent chromatin regions at the HM
loci and telomeres is disrupted in cells that carry a single deletion of either gene, suggesting that the three proteins are recruited to chromatin in a cooperative fashion or as components of a single complex (55
). However, these studies do not distinguish between interactions that may be involved in nucleation of silent chromatin domains from those that may be required for the spreading of silencing proteins away from nucleation sites.
To gain better insight into the nature of the yeast silencing complexes, we purified each of the Sir proteins to near homogeneity using a tandem-affinity purification (TAP) approach. Despite their large apparent sizes, the composition of the purified complexes is surprisingly simple. A large complex of approximately 700 kDa, purified using affinity tags on Sir4, is composed of Sir2 and Sir4. A smaller complex of approximately 450 kDa, purified using tagged Sir3, is composed primarily of Sir3. Having defined the composition of these complexes, we determined the requirement for individual silencing proteins and the NAD-dependent deacetylase activity of Sir2 for assembly of each of the above proteins on chromatin. Our results show that the enzymatic activity of Sir2 is not absolutely required for the association of the Sir proteins with DNA sites that initiate silencing at the HM loci and telomeres or for the binding of Sir2 itself to rDNA. However, at the HM loci and telomeres, Sir2 and it enzymatic activity are required for the efficient association of the Sir proteins with DNA regions that are distal from nucleation sites.
Furthermore, by testing the requirement for each Sir protein in the assembly of silencing complexes on chromatin, we found that the Sir2 and Sir4 proteins could partially associate with silencers and DNA regions immediately adjacent to telomeric repeats, independently of Sir3. These results define independent steps in assembly of the Sir proteins at sites that nucleate the initiation of silent chromatin and suggest that Sir4 is the most upstream protein in the assembly pathway. Finally, we show that histone H4 is hypoacetylated in silent chromatin domains in a Sir2 activity-dependent manner and that, similar to what has been described previously for the HM loci and telomeres, the N termini of histones H3 and H4 play a critical role in rDNA silencing.