The packaging of DNA into transcriptionally repressed heterochromatin is an important process common to eukaryotic organisms. Just as important is the restriction of this heterochromatin to appropriate genomic loci. To understand why repressive chromatin forms in particular locations, we compared the rates of assembly of silenced chromatin at various loci in the budding yeast Saccharomyces cerevisiae.
In S. cerevisiae
, domains of silenced chromatin are found at the silent mating-type cassettes HMR
and at most telomeres. The structural components of this chromatin are Sir2p, Sir3p, and Sir4p. The first step in establishment of silenced chromatin is the recruitment of Sir proteins to the chromosome, which is mediated by DNA sequences termed silencers. At the silent mating-type cassettes, the silencers HMR-E
and the silencers HML-E
. Each of these silencers consists of binding sites for the origin recognition complex (ORC) and for either Rap1p, Abf1p, or both. Sir proteins are assembled at the silencers through physical interactions with the DNA-binding proteins and each other. A fourth Sir protein, Sir1p, also participates in the initial assembly process through interactions with ORC and Sir4p (49
). Telomeric silencers are distinct from the silencers at HM
loci in that they are composed of an array of Rap1p binding sites embedded in the telomeric (TG1-3
repeats. Despite variation in composition, most silencers function to recruit the Sir complex and thus initiate silencing. The one exception is HMR-I
, which does not recruit Sir proteins on its own and appears to play a supporting role in silencing HMR
Once recruited to a silencer, Sir2p, Sir3p, and Sir4p spread along the chromosome. A working model for spreading proposes that Sir proteins propagate in a stepwise manner facilitated by sequential deacetylation of histones (reviewed in reference 36
). Sir2p, a histone deacetylase, generates hypoacetylated histone H3 and H4 tails that are preferentially bound by Sir3p and Sir4p, which in turn recruit additional Sir2p to deacetylate the next nucleosome (5
). Thus, the Sir proteins are dependent on each other for assembly. This model predicts that Sir-silenced chromatin should propagate linearly along a chromosome. Because silencers serve as the initiators of the spreading process, they determine where silenced chromatin will form. Although Sir proteins are recruited differently by silencers at HM
loci and telomeres, spreading of the Sir complex occurs at all sites.
The ability of Sir proteins to propagate along a chromosome could potentially be toxic to the cell if silenced chromatin spreads beyond its appropriate domain or fortuitously assembles in the wrong locations. Consequently, mechanisms must exist to damp down the spreading of the Sir proteins. However, such damping mechanisms could prevent the Sir proteins from stably repressing promoters distant from a silencer, as required to maintain cell type identity. How the assembly of silenced chromatin is opposed throughout most of the genome and promoted in particular locations is incompletely understood. Two general models have been proposed: competition with euchromatin and discrete DNA sequences that generate barriers between active and silenced chromatin. A naturally occurring barrier is a tRNAThr
gene located on the telomere-proximal side of HMR
). However, barrier elements have not been identified at most junctions between silenced and active chromatin in S. cerevisiae
, and competition is proposed to limit spreading at these sites. In this model, active chromatin is characterized by a set of histone modifications that reduce the affinity of the Sir proteins for nucleosomes. Thus, an equilibrium between active and silenced chromatin is reached, and silenced chromatin spreads only a short distance from a silencer. Evidence for this model comes from observations that in the absence of proteins that characterize active chromatin, Sir proteins spread farther at telomeres. These proteins include, but are not necessarily limited to, the histone acetyltransferase Sas2p, the bromodomain protein Bdf1p, the histone variant H2A.Z, and the histone methyltransferases Dot1p and Set1p (20
). Although these “antisilencers” clearly play a role in restricting the spread of Sir proteins, their absence results in only modest extensions of silenced chromatin rather than global redistribution of Sir proteins.
The impact of antisilencers on silenced chromatin often varies at different genomic loci. Sas2p, for example, is arguably the most significant antagonist of telomeric silencing through its acetylation of histone H4 at the K16 residue (H4K16). In addition to competing with Sir2p for the state of H4K16, Sas2p also fortifies the function of other antisilencers by facilitating the deposition of H2A.Z and boosting the ability of Dot1p to compete with Sir3p for access to histones (1
). Despite exhibiting a clear antisilencing phenotype at telomeres, SAS2
has a much less severe impact at HMR
). Furthermore, SAS2
differentially influences silencing at the two mating-type loci, HMR
). Such discrepancies have been observed for other antisilencers (39
) and suggest that silenced chromatin may not assemble equivalently at all locations.
In this study, we characterized the rates of Sir complex assembly at several genomic locations. We discovered that spreading rates vary at different genomic loci and that much of this variation can be attributed to the silencer. Sir proteins assembled rapidly at HMR over a region of about 3 kb, and the association of Sir proteins occurred virtually simultaneously throughout the locus. In contrast, assembly at a telomere (VI-R) was significantly slower and proceeded in a linear fashion, such that the Sir proteins associated with regions closer to the telomere earlier than regions farther from the telomere. Remarkably, despite the differences in the rates of spreading, the Sir proteins were recruited to the silencers (HMR-E or the telomeric repeat) at equivalent rates and at similar levels. Furthermore, insertion of the HMR-E silencer into the telomere resulted in more-rapid spreading of Sir proteins, indicating that the slower spreading observed at the telomere was not simply due to the telomeric chromatin being restrictive to spreading. From these observations, we conclude that the HMR-E silencer does not simply recruit Sir proteins to the chromosome. It also has the capacity, which the telomeric repeat does not have, to promote the assembly of silenced chromatin over a distance of several kilobases. We propose that silencers permit a specialized chromatin structure to exist that would otherwise be unstable and that the silencer's ability to promote spreading is an important parameter for determining the size and stability of silenced chromatin domains. Furthermore, we hypothesize that the HMR-E silencer promotes the assembly of Sir proteins over a distance by creating a situation in which spreading is not strictly linear, as predicted by the stepwise model of sequential deacetylation.