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The Mediator complex is required for the regulated transcription of nearly all RNA polymerase II-dependent genes. Here we demonstrate a new role for Mediator which appears to be separate from its function as a transcriptional coactivator. Mediator associates directly with heterochromatin at telomeres and influences the exact boundary between active and inactive chromatin. Loss of the Mediator Med5 subunit or mutations in Med7 cause a depletion of the complex from regions located near subtelomeric X elements, which leads to a change in the balance between the Sir2 and Sas2 proteins. These changes in turn result in increased levels of H4K16 acetylation near telomeres and in desilencing of subtelomeric genes. Increases in H4K16 acetylation have been observed at telomeres in aging cells. In agreement with this observation, we found that the loss of MED5 leads to shortening of the Saccharomyces cerevisiae (budding yeast) replicative life span.In Saccharomyces cerevisiae (budding yeast), telomeric DNA consists of imperfect tandem repeats of the consensus sequence (TG 1-3 ) n , with a combined length of about 300 nucleotides (21). Next to the telomere is the subtelomeric region, which often contains two types of repeats, the YЈ and X elements. The YЈ elements are between 4 and 12 kb long and are located next to the telomeres at many chromosome ends (26). The sizes of X elements vary, but they always contain a "core X" repeat region that is found at nearly all telomeres. Depending on how the YЈ and X elements are distributed, S. cerevisiae chromosome ends can be divided into X and X-YЈ types (22,23).Genes situated close to telomeres undergo reversible silencing, a phenomenon that has been termed the telomere position effect (TPE) (28). This effect was first observed when a reporter gene was inserted next to a telomeric TG 1-3 tract of an artificial telomere. TPE can also be observed at native yeast telomeres, but the phenomenon appears to be a bit more complex at these locations, since TPE varies between telomeres and in different strain backgrounds (29,40,41). The molecular basis of TPE is believed to be the Rap1, Ku, and Sir protein-mediated spreading of heterochromatin-like structures from the telomeric DNA inwards, which represses genes located in the subtelomeric region (42). According to this model, the Rap1/Ku/Sir structures are formed at telomeres and propagate toward the subtelomeres via interactions between the Sir proteins and histone tails. Sir2 is an active histone deacetylase that removes the acetyl group on lysine 16 of histone H4 (H4K16), which allows Sir3 and Sir4 to bind the nonacetylated histone tails (11, 35).As mentioned, TPE varies between individual chromosome ends, and the exact repeat structure of the subtelomeric region may in fact influence the spread of heterochromatin. YЈ elements counteract the spread of Sir proteins, and the YЈ regions display high levels of H4K16 acetylation. Furthermore, YЈ elements are highly enriched in nucleosomes and are transcriptionally active. In contrast, even X ...

The Med2, Med3 and Med15 proteins form a heterotrimeric subdomain in the budding yeast Mediator complex. This Med15 module is an important target for many gene specific transcription activators. A previous proteome wide screen in yeast identified Med3 as a protein with priogenic potential. In the present work, we have extended this observation and demonstrate that both Med3 and Med15 form amyloid-like protein aggregates under H2O2 stress conditions. Amyloid formation can also be stimulated by overexpression of Med3 or of a glutamine-rich domain present in Med15, which in turn leads to loss of the entire Med15 module from Mediator and a change in stress response. In combination with genome wide transcription analysis, our data demonstrate that amyloid formation can change the subunit composition of Mediator and thereby influence transcriptional output in budding yeast.

dAt Schizosaccharomyces pombe centromeres, heterochromatin formation is required for de novo incorporation of the histone H3 variant CENP-A Cnp1 , which in turn directs kinetochore assembly and ultimately chromosome segregation during mitosis. Noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (Pol II) directs heterochromatin formation through not only the RNA interference (RNAi) machinery but also RNAi-independent RNA processing factors. Control of centromeric ncRNA transcription is therefore a key factor for proper centromere function. We here demonstrate that Mediator directs ncRNA transcription and regulates centromeric heterochromatin formation in fission yeast. Mediator colocalizes with Pol II at centromeres, and loss of the Mediator subunit Med20 causes a dramatic increase in pericentromeric transcription and desilencing of the core centromere. As a consequence, heterochromatin formation is impaired via both the RNAi-dependent and -independent pathways, resulting in loss of CENP-A Cnp1 from the core centromere, a defect in kinetochore function, and a severe chromosome segregation defect. Interestingly, the increased centromeric transcription observed in med20⌬ cells appears to directly block CENP-A Cnp1 incorporation since inhibition of Pol II transcription can suppress the observed phenotypes. Our data thus identify Mediator as a crucial regulator of ncRNA transcription at fission yeast centromeres and add another crucial layer of regulation to centromere function.

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