Yeast Tdh3 (glyceraldehyde 3-phosphate dehydrogenase) is a Sir2-interacting factor that regulates transcriptional silencing and rDNA recombination.

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.

Histone variant H2A.Z and linker histone H1 influence chromosome condensation in Saccharomyces cerevisiae.

Chromosome condensation is essential for the fidelity of chromosome segregation during mitosis and meiosis. Condensation is associated both with local changes in nucleosome structure and larger-scale alterations in chromosome topology mediated by the condensin complex. We examined the influence of linker histone H1 and variant histone H2A.Z on chromosome condensation in budding yeast cells. Linker histone H1 has been implicated in local and global compaction of chromatin in multiple eukaryotes, but we observe normal condensation of the rDNA locus in yeast strains lacking H1. However, deletion of the yeast HTZ1 gene, coding for variant histone H2A.Z, causes a significant defect in rDNA condensation. Loss of H2A.Z does not change condensin association with the rDNA locus or significantly affect condensin mRNA levels. Prior studies reported that several phenotypes caused by loss of H2A.Z are suppressed by eliminating Swr1, a key component of the SWR complex that deposits H2A.Z in chromatin. We observe that an htz1Δ swr1Δ strain has near-normal rDNA condensation. Unexpectedly, we find that elimination of the linker histone H1 can also suppress the rDNA condensation defect of htz1Δ strains. Our experiments demonstrate that histone H2A.Z promotes chromosome condensation, in part by counteracting activities of histone H1 and the SWR complex.

Histone variant H2A.Z promotes meiotic chromosome axis organization in Saccharomyces cerevisiae.

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.

Ccq1p and the condensin proteins Cut3p and Cut14p prevent telomere entanglements in the fission yeast Schizosaccharomyces pombe.

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.

Isolation and characterization of conditional alleles of the yeast SIR2 gene.

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.

New alleles of SIR2 define cell-cycle-specific silencing functions.

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.

SIR2-induced inviability is suppressed by histone H4 overexpression.

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.

Heterochromatin spreading at yeast telomeres occurs in M phase.

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.

REP3-mediated silencing in Saccharomyces cerevisiae.

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 2micro 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 2micro circle and decreased in strains overexpressing the Rep1 and Rep2 proteins, suggesting that the Rep proteins antagonize conventional silencing.

Histone variant H2A.Z functions in sister chromatid cohesion in Saccharomyces cerevisiae.

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.

Sir3 and epigenetic inheritance of silent chromatin in Saccharomyces cerevisiae.

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.

A cis-acting tRNA gene imposes the cell cycle progression requirement for establishing silencing at the HMR locus in yeast.

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.

H2A.Z (Htz1) controls the cell-cycle-dependent establishment of transcriptional silencing at Saccharomyces cerevisiae telomeres.

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 G(1) 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.