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Progression through meiosis is associated with significant reorganization of chromosome structure, regulated in part by changes in histones and chromatin. Prior studies observed defects in meiotic progression in yeast strains lacking the linker histone H1 or variant histone H2A.Z. To further define the contributions of these chromatin factors, we have conducted genetic and cytological analysis of cells undergoing meiosis in the absence of H1 and H2A.Z. We find that a spore viability defect observed in strains lacking H2A.Z can be partially suppressed if cells also lack histone H1, while the combined loss of both H1 and H2A.Z is associated with elevated gene conversion events. Cytological analysis of Red1 and Rec8 staining patterns indicates that a subset of cells lacking H2A.Z fail to assemble a proper chromosome axis, and the staining pattern of the synaptonemal complex protein Zip1 in htz1Δ/htz1Δ cells mimics that of cells deficient for Rec8-dependent meiotic cohesion. Our results suggest a role for H2A.Z in the establishment or maintenance of the meiotic chromosome axis, possibly by promoting the efficient chromosome cohesion.

H2A.Z is a highly conserved variant of histone H2A with well-characterized roles in transcriptional regulation. We previously reported that H2A.Z and Mcd1, a subunit of the cohesin complex, regulate the establishment of transcriptional silencing at telomeres in Saccharomyces cerevisiae and that H2A.Z broadly dissociated from chromatin during the anaphase-to-telophase transition, coincident with the dissociation of Mcd1 from chromosomes and dissolution of cohesion. In this study, we show that depletion of H2A.Z causes precocious loss of sister chromatid cohesion in yeast without loss of Mcd1 from chromosomes. H2A.Z is deposited into chromatin by the SWR1 complex and is subject to acetylation of its four N-terminal tail lysine residues by the NuA4 and SAGA histone acetyltransferase complexes. We found that cells compromised for function of the SWR1 complex were defective in cohesion, as were cells expressing a form of H2A.Z not subject to acetylation. Finally, inactivation of H2A.Z in metaphase-blocked cells led immediately to cohesion defects, suggesting a direct role for H2A.Z in the maintenance of sister chromatid cohesion.

Epigenetic mechanisms maintain the specific characteristics of differentiated cells by ensuring the inheritance of gene expression patterns through DNA replication and mitosis. We examined the mechanism of epigenetic inheritance of Sir protein-dependent transcriptional silencing in Saccharomyces cerevisiae by examining gene expression and molecular markers of silencing at the silent mating type loci under conditions of limiting Sir3 protein. We observed that silencing at HMR, as previously reported for HML, is epigenetically inherited. This inheritance is accompanied by an increased ability of previously silenced cells to retain or recruit limiting Sir3 protein to cis-acting silencer sequences. We also observed that the low H4-K16 histone acetylation and H3-K79 methylation associated with a silenced HMR locus persist in recently derepressed cells for several generations at levels of Sir3 insufficient to maintain these marks in long-term-derepressed cells. The unique ability of previously silenced cells to retain Sir3 protein, maintain silencing-specific histone modifications, and repress HMR transcription at levels of Sir3 insufficient to mediate these effects in long-term-derepressed cells suggests that a cis-acting, chromatin-based mechanism drives epigenetic inheritance at this locus.

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

Numerous studies have determined that the establishment of Sir protein-dependent transcriptional silencing in yeast requires progression through the cell cycle. In our study we examined the cell cycle requirement for the establishment of silencing at the HML and HMR loci using strains bearing conditional or inducible SIR3 alleles. Consistent with prior reports, we observed that establishing silencing at HMR required progression through the cell cycle. Unexpectedly, we found that the HML locus is far less dependent on cell cycle progression to establish silencing. Seeking cis-acting elements that could account for this difference, we found that deletion of a tRNA gene that serves as a chromatin boundary at HMR abolishes the cell cycle progression requirement at this locus, while insertion of sequences containing this tRNA gene adjacent to HML imposes dependence on cell cycle progression for the full establishment of silencing. Our results indicate that the cell cycle progression requirement is not a property intrinsic to the formation of heterochromatin in yeast, but is instead a cis-limited, locus-specific phenomenon. We show that inactivation of the Scc1 cohesin also abolishes the requirement for cell cycle progression and test models based on a possible link between the tRNA gene and cohesin association.

The establishment of transcriptional silencing in Saccharomyces cerevisiae requires progression through the cell cycle. We have previously found that transit through M-phase is necessary and sufficient to establish silencing at telomeres following induction of the Sir3 silencing factor. In this study we find that halting cell-cycle progression in either G1 or at the beginning of M-phase limits the ability of Sir3 to associate with a telomere-linked reporter gene and prevents the changes in histone modifications associated with gene repression. Deletion of genes coding for the histone variant H2A.Z (Htz1 in yeast) and histone acetyltransferase Sas2 abolish the cell-cycle progression requirement for the establishment of silencing. Cells blocked in telophase (but not at metaphase) are also able to establish silencing. We show that H2A.Z binds to the promoter of our telomere-linked reporter gene and that this binding diminishes in silenced cells. Finally, we observe a specific displacement of H2A.Z from chromatin in telophase-blocked cells, regardless of the silencing status of the reporter gene. These results suggest that the requirement for M-phase in the establishment of silencing may reflect a cell-cycle regulated relaxation of heterochromatin barriers.

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.

We have characterized a defect in the mitotic transmission of plasmid minichromosomes in yeast strains deleted for the more highly expressed pair of histone H3 and H4 genes. Several observations indicate that an impairment in DNA replication contributes to the decrease in minichromosome stability. First, the maintenance of ARS plasmids that lack centromeres was also defective. Second, the addition of multiple ARS elements suppressed the defect in plasmid maintenance. Third, a synergistic increase in plasmid loss rate was seen when a plasmid containing an inefficient mutated ARS was tested in a strain deleted for histone genes, implying an interaction between ARS activity and the histone gene deletion. These results support the existence of a histone-dependent step in the initiation of DNA replication. We find that the stability of native chromosomes is not affected in strains deleted for histone genes. We propose that reduced histone H3 and H4 protein decreases the efficiency of initiation at ARS elements on plasmids and chromosomes, but that the presence of multiple origins on chromosomes compensates for the reduced efficiency. We find that decreased minichromosome stability is suppressed by increases in strain ploidy. The greater stability due to ploidy increases is not due to a relative increase in the expression of histone genes. We discuss models for the effect of strain ploidy on minichromosome maintenance. Copyright © 2000 John Wiley & Sons, Ltd.

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.

Sir2 is a protein deacetylase that mediates transcriptional silencing at the HM loci, telomeres, and rDNA repeats in yeast. To identify functionally significant regions of the Sir2 protein, we have characterized two types of mutations. First, we used random mutagenesis to create temperature-sensitive alleles of the SIR2 gene. Mutations conferring conditional silencing can be isolated throughout the SIR2 gene, causing both enzymatic and protein interaction defects. We used external deletions to identify regions essential for silencing in the non-conserved regions of Sir2. Deletions of the Sir2 N-terminal 89 amino acid residues caused a subtle increase in silencing, while deletions encompassing residues 110–146 caused loss of Sir2 interactions with both Sir4 and Net1. This loss of protein interaction correlates with a loss of Sir2-mediated silencing, and is consistent with a model in which Net1 and Sir4 compete for interaction with Sir2. These results indicate that recognition of the binding partners of Sir2 is a key function of non-conserved sequences.

Heterochromatin regulation of gene expression exhibits epigenetic inheritance, in which some feature of the structure is retained and can reseed formation in new cells. To understand the cell-cycle events that influence heterochromatin assembly and maintenance in budding yeast, we have conducted two types of experiments. First we have examined the kinetics of heterochromatin spreading at telomeres. We have constructed a strain in which the efficient silencing of a telomere-linked URA3 gene depends on the inducible expression of the Sir3 silencing factor. Prior studies determined that S-phase passage was required for the establishment of silencing at the HM loci in yeast. We find that establishment of silencing in our strain occurs at a point coincident with mitosis and does not require S-phase passage. In addition, we find that passage through mitosis is sufficient to establish silencing at the HML locus in a strain bearing a conditional allele of SIR3. Finally, we have also assessed the stability of yeast heterochromatin in the absence of the cis-acting elements required for its establishment. We show that silencing is stable through S phase in the absence of silencers and therefore possesses the ability to self-propagate through DNA replication. However, silencing is lost in the absence of silencers during progression through M phase. These experiments point to crucial events in mitosis influencing the assembly and persistence of heterochromatin.

We have identified histone H4 as a high-expression suppressor of Sir2-induced inviability in yeast cells. Overexpression of histone H3 does not suppress Sir2-induced lethality, nor does overexpression of histone H4 alleles associated with silencing defects. These results suggest a direct and specific interaction between Sir2 and H4 in the silencing mechanism.

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.

The SIR gene products maintain transcriptional repression at the silent mating type loci and telomeres in Saccharomyces cerevisiae, although no enzymatic or structural activity has been assigned to any of the Sir proteins nor has the role of any of these proteins in transcriptional silencing been clearly defined. We have investigated the functions and interactions of the Sir2, Sir3, and Sir4 proteins by overexpressing them in yeast cells. We find that Sir2p and Sir3p are toxic when overexpressed, while high Sir4p levels have no toxic effect. Epistasis experiments indicate that Sir2p-induced toxicity is diminished in strains lacking the SIR3 gene, while both Sir2p and Sir4p are required for Sir3p to manifest its full toxic effect. In addition, the effects of Sir2 or Sir3 overexpression are exacerbated by specific mutations in the N-terminus of the histone H4 gene. These results are consistent with a model in which Sir2p, Sir3p and Sir4p function as a complex and interact with histones to modify chromatin structure. We find no evidence that toxicity from high levels of the Sir proteins results from widespread repression of transcription. Instead, we find that high levels of Sir2p and/or Sir3p cause a profound decrease in chromosome stability. These results can be appreciated in the context of the effects of Sir2p in histone acetylation and of chromatin structure on chromosome stability.

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.

Yeast autonomously replicating sequence (ARS) elements are composed of a conserved 11-base-pair (bp) core consensus sequence and a less well defined 3'-flanking region. We have investigated the relationship between the H4 ARS core consensus sequence and its 3'-flanking domain. The minimal sequences necessary and sufficient for function were determined by combining external 3' and 5' deletions to produce a nested set of ARS fragments. Sequences 5' of the core consensus were dispensable for function, but at least 66 bp of 3'-flanking domain DNA was required for full ARS function. The importance of the relative orientation of the core consensus element with respect to the 3'-flanking domain was tested by precisely inverting 14 bp of DNA including the core consensus sequence by oligonucleotide mutagenesis. This core inversion mutant was defective for all ARS function, showing that a fixed relative orientation of the core consensus and 3'-flanking domain is required for function. The 3'-flanking domain of the minimal functional H4 ARS fragment contains three sequences with a 9-of-11-bp match to the core consensus. The role of these near-match sequences was tested by directed mutagenesis. When all near-match sequences with an 8-of-11-bp match or better were simultaneously disrupted by point mutations, the resulting ARS construct retained full replication function. Therefore, multiple copies of a sequence closely related to the core consensus element are not required for H4 ARS function.