In this work, we demonstrate that Mediator associates with telomeric regions and influences the exact boundary between active and inactive chromatin. We suggest that Mediator influences the delicate balance that exists between Sir2 and Sas2. The opposing effects of these enzymes are believed to generate a gradient of H4K16 acetylation, which in turn marks the boundary between active and silenced chromatin near telomeres. Our data suggest that Mediator may interact directly with the subtelomeric region and physically regulate this balance. Mutations that lead to a loss of Mediator from this region cause a decrease of subtelomeric silencing and a shift in the H4K16 acetylation gradient.
Our findings are distinctly different from those presented in a recent report that identified the SAGA subunit Ada2 as a possible regulator of the Sir2-Sas2 balance at telomeres (13
). Ada2 was shown to bind telomeric chromatin and the silencing protein Sir2 in vivo
, and loss of ADA2
caused a spread of Sir2 and Sir3 into subtelomeric regions and decreased histone H4K16 acetylation. Interestingly, a series of publications demonstrated a close link between the SAGA complex and Mediator (17
). The two protein complexes appear to be required for stepwise activation of transcription at many promoters. In addition, recruitment of Mediator and the SAGA complexes by Gcn4 has been shown to be interdependent (30
). Furthermore, the SAGA complex makes physical contacts with Mediator, and mutations/deletions of many genes encoding SAGA components (including ADA2
) synthetically interact with mutations/deletions of Mediator-encoding genes (6
In our studies, we observed a dramatic increase of H4K16 acetylation at the conserved X core element and also noticed a slight increase of histone H4 levels. The function of this element is not well understood, but in wt cells it is depleted of nucleosomes and instead bound by typical telomeric proteins, e.g., Sir2 and Rap1 (45
). Based on our findings, we suggest that Mediator helps to set up the boundary between X elements bound by Sir proteins and surrounding regions bound by Sas2. In support of this notion, we found that deletion of MED5
or temperature-sensitive mutations in MED7
could disturb the balance between Sir2 and Sas2, consequently allowing nucleosomes acetylated at H4K16 to “leak” into nearby X elements.
How Mediator is recruited to X element border regions is not yet clear, but one factor could be the modification status of the histones, since Mediator can interact with histone H4 N-terminal tail peptides and H4K16 acetylation has a strong negative effect on this interaction (X. Zhu et al., submitted for publication). Even if Mediator and Sir3 can both bind to deacetylated H4 N-terminal tails and nucleosomes in vitro
, they cannot do so simultaneously. Instead, our experiments suggest that they are mutually exclusive, supporting the idea that Mediator may function as a border element. But Mediator blocks not only Sir3 binding but also spreading of Sas2, which may explain why the loss of Mediator does not lead to increased Sir3 occupancy but instead to a spreading of Sas2 and H4K16 acetylation into X elements. In support of this idea, overexpression of the Sas2 protein causes lower occupancy of the Sir2 protein and higher H4K16 acetylation levels in telomeric regions (32
). Interestingly, the increase of H4K16 acetylation seen in aging cells is also especially pronounced within X elements (7
Med5 is structurally located at the interface of the tail and middle modules of Mediator, and a med5
Δ mutant strain displays increased respiration and mitochondrial activity (4
). This result could potentially be coupled to our observation of a shortening life span in med5
Δ deletion cells, since impaired respiratory chain function has been linked to premature aging in many organisms. However, we determined the life span of another Mediator tail component mutant strain, the med16
Δ strain, that also displayed a similar increase in respiratory activity. We failed to observe changes in replicative life span in the med16
Δ strain, arguing against changed mitochondrial activity being the reason for aging in the med5
Δ mutant cells (data not shown). The Med5 protein has also been identified as an active histone acetyltransferase, but its substrate specificity remains unknown (12
). We do, however, find it unlikely that Med5 directly acetylates H4K16, since the observed increase in H4K16 acetylation levels was associated with a decrease of Mediator in the subtelomeric region. In addition, we cannot rule out the possibility that Med5 acts in concert with other components of the Mediator complex. We previously reported that Mediator interacts directly with Med16 and forms a specific Mediator subcomplex in the tail module of the Mediator complex. Previous reports have demonstrated that loss of MED16
is associated with global alterations in chromatin accessibility (1
), and as demonstrated here, deletion of MED16
also affects subtelomeric silencing. It is therefore possible that Med5 and Med16 together form a Mediator submodule that affects chromatin structure at specific genomic locations. Med16 (in contrast to Med5) is conserved in evolution, and Mediator may therefore affect chromatin structure in higher eukaryotes as well.
A large number of studies have identified the Mediator complex as the component of the transcription preinitiation complex required to stimulate Pol II-dependent transcription. Genome-wide occupancy studies have slightly modified this view, however, and demonstrated distinctly different patterns for Mediator and Pol II (1
). Mediator is present at many regions throughout the subtelomeric regions, whereas Pol II is depleted from these locations. Furthermore, Mediator is also enriched at other heterochromatin regions, such as the centromere (10
). Our observations suggest that Mediator could contribute to the formation of a border between active and inactive chromatin regions. In support of this notion, loss of Med5 leads to a pronounced change in histone occupancy and H4K16 acetylation levels in X elements but does not change Mediator occupancy within the X elements. Instead, loss of Med5 affects Mediator occupancy at adjacent regions bordering the X elements. It is tempting to speculate that Mediator may have a similar function at some gene regulatory regions and that a role for Mediator in defining the border between active and inactive chromatin may be a recurring theme at many genomic locations.