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Condensins are highly conserved protein complexes that regulate chromosome organization, affecting transcription and preparing chromosomes for mitosis.1 Experiments conducted in budding yeast cells...

The establishment of transcriptional silencing in yeast requires cell-cycle progression, but the nature of this requirement is unknown. Sir2 is a protein deacetylase that is required for gene silencing in yeast. We have used temperature-sensitive alleles of the SIR2 gene to assess Sir2's contribution to silencing as a function of the cell cycle. When examined in vivo, these conditional alleles fall into two classes: one class exhibits a loss of silencing when raised to the nonpermissive temperature regardless of cell-cycle position, while the second class exhibits a mitosis-specific silencing defect. Alleles of the first class have a primary defect in protein deacetylase activity, while the alleles of the second class are specifically defective in Sir2–Sir4 interactions at nonpermissive temperatures. Using a SIR2 temperature-sensitive allele, we show that silencing can be established at the HML locus during progression through the G2/M–G1 interval. These results suggest that yeast heterochromatin undergoes structural transitions as a function of the cell cycle and support the existence of a critical assembly step for silent chromatin in mitosis.

In yeast the Sir proteins and Rap1p are key regulators of transcriptional silencing at telomeres and the silent mating-type loci. Rap1 and Sir4 also possess anchoring activity; the rotation of plasmids bound by Sir4 or Rap1 is constrained in vivo, and Rap1 or Sir4 binding can also correct the segregation bias of plasmids lacking centromeres. To investigate the mechanistic link between DNA anchoring and regulation of transcription, we examined the ability of a third defined anchor in yeast, the 2μ circle REP3 segregation element, to mediate transcriptional silencing. We find that placement of the REP3 sequence adjacent to the HML locus in a strain deleted for natural silencer sequences confers transcriptional repression on HML. This repression requires the Sir proteins and is decreased in strains lacking the REP3-binding factors Rep1 and Rep2. The yeast cohesin complex associates with REP3; we show that REP3 silencing is also decreased in strains bearing a mutated allele of the MCD1/SCC1 cohesin gene. Conventional silencing is increased in some strains lacking the 2μ circle and decreased in strains overexpressing the Rep1 and Rep2 proteins, suggesting that the Rep proteins antagonize conventional silencing.

Transcriptional silencers in the yeast Saccharomyces induce position-specific, sequence-independent repression by promoting formation of a heterochromatin-like structure across sequences adjacent to them. We have examined the role of silencers in maintenance and inheritance of repression at the silent mating-type cassettes in yeast by monitoring the expression state of one of these cassettes following in vivo deletion of the adjacent silencer. Our experiments indicate that although silencer sequences are dispensable for the maintenance of repression in the absence of cell-cycle progression, silencers are required for the stable inheritance of a repressed state. That is, silenced loci from which the silencer is deleted most often become derepressed within one generation of losing the silencer. Thus, the heritability of a repressed state is not intrinsic to a silenced locus or to the chromatin encompassing it; rather, heritability of repression appears to be a property of the silencer itself.

Sir2 is an NAD+-dependent histone deacetylase required to mediate transcriptional silencing and suppress rDNA recombination in budding yeast. We previously identified Tdh3, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as a high expression suppressor of the lethality caused by Sir2 overexpression in yeast cells. Here we show that Tdh3 interacts with Sir2, localizes to silent chromatin in a Sir2-dependent manner, and promotes normal silencing at the telomere and rDNA. Characterization of specific TDH3 alleles suggests that Tdh3's influence on silencing requires nuclear localization but does not correlate with its catalytic activity. Interestingly, a genetic assay suggests that Tdh3, an NAD+-binding protein, influences nuclear NAD+ levels; we speculate that Tdh3 links nuclear Sir2 with NAD+ from the cytoplasm., Author Summary Cells respond to changing signals or environmental conditions by altering the expression of their genes. For instance, our cells respond to the presence of glucose or insulin in the bloodstream by regulating the expression of genes involved in basic cell metabolism. The sirtuin family of proteins has been proposed to serve as a link between a cell's metabolic state and gene expression, although the molecular mechanisms that connect metabolic status with Sir2 activity remain unclear. The expression of genes is controlled in part by the structural organization of the local chromatin region within which they reside. The yeast sirtuin protein, Sir2, mediates repression (“silencing”) of sets of genes by modulating the structural organization of specific chromatin regions. In this study we describe a novel link between a key metabolic enzyme and Sir2 function. We show that a yeast GAPDH protein, which plays a central role in glucose metabolism, also associates with Sir2 in the nucleus and promotes Sir2-dependent gene silencing. Sirtuin activity requires a small molecule, NAD+, whose availability may fluctuate depending on the metabolic state of the cell. Based on our data, we suggest that Tdh3 may promote silencing by maintaining sufficient levels of NAD+ available to Sir2 within the nucleus.

The Schizosaccharomyces pombe telomere-associated protein Ccq1p has previously been shown to participate in telomerase recruitment, heterochromatin formation, and suppression of checkpoint activation. Here we characterize a critical role for Ccq1p in mitotic transit. We show that mitotic cells lacking Ccq1p lose minichromosomes at high frequencies but that conditional knockdown of Ccq1p expression results in telomere bridging within one cell cycle. Elevating Ccq1p expression resolves the telomere entanglements caused by decreased Taz1p activity. Ccq1p affects telomere resolution in the absence of changes in telomere size, indicating a role for Ccq1p that is independent of telomere length regulation. Using affinity purification, we identify the condensin proteins Cut3p and Cut14p as candidate Ccq1p interactors in this activity. Condensin loss-of-function disrupts Ccq1p telomeric localization and normal intertelomere clustering, while condensin overexpression relieves the chromosome segregation defects associated with conditional Ccq1p knockdown. These data suggest that Ccq1p and condensins collaborate to mediate resolution of telomeres in mitosis and regulate intertelomeric clustering during interphase.

Transcriptional silencing at the HM loci and telomeres in yeast depends on several trans-acting factors, including Rap1p and the Sir proteins. The SUM1-1 mutation was identified by its ability to restore silencing to strains deficient in one or more of these trans-acting factors. The mechanism by which SUM1-1 bypasses the requirement for silencing proteins is not known. We identified four loci that when reduced in dosage in diploid strains increase the ability of SUM1-1 strains to suppress silencing defects. Two of the genes responsible for this effect were found to be MGA2 and SPT23. Mga2p and Spt23p were previously identified as functionally related transcription factors that influence chromatin structure. We find that deletion of MGA2 or SPT23 also increases the efficiency of silencing in haploid SUM1-1 strains. These results suggest that Mga2p and Spt23p are antagonists of silencing. Consistent with this proposal we find that deletion of MGA2 or SPT23 also suppresses the silencing defects caused by deletion of the SIR1 gene or by mutations in the HMR silencer sequences. However, we find that Mga2p and Spt23p can positively affect silencing in other contexts; deletion of either MGA2 or SPT23 decreases mating in strains bearing mutations in the HML-E silencer. Mga2p and Spt23p appear to be a novel class of factors that influence disparate pathways of transcriptional control by chromatin.

Two classes of sequences in the yeast Saccharomyces cerevisiae are subject to transcriptional silencing: the silent mating-type cassettes and telomeres. In this report we demonstrate that the silencing of these regions is strictly associated with acetylation of the epsilon-amino groups of lysines in the amino-terminal domains of three of the four core histones. Both the silent mating-type cassettes and the Y domains of telomeres are packaged in nucleosomes in vivo that are hypoacetylated relative to those packaging active genes. This difference in acetylation is eliminated by genetic inactivation of silencing: The silent cassettes from sir2, sir3, or sir4 cells show the same level of acetylation as other active genes. The correspondence of silencing and hypoacetylation of the mating-type cassettes is observed even for an allele lacking a promoter, indicating that silencing per se, rather than the absence of transcription, is correlated with hypoacetylation. Finally, overexpression of Sir2p, a protein required for transcriptional silencing in yeast, yields substantial histone deacetylation in vivo. These studies fortify the hypothesis that silencing in yeast results from heterochromatin formation and argue that the silencing proteins participate in this formation.