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The MET3 promoter (MET3pr) inserted into the silenced chromosome in budding yeast can overcome Sir2-dependent silencing upon induction and activate transcription in every single cell among a population. Despite the fact that MET3pr is turned on in all the cells, its activity still shows very high cell-to-cell variability. To understand the nature of such “gene expression noise,” we followed the dynamics of the MET3pr-GFP expression inserted into ribosomal DNA (rDNA) using time-lapse microscopy. We found that the noisy “on” state is comprised of multiple substable states with discrete expression levels. These intermediate states stochastically transition between each other, with “up” transitions among different activated states occurring exclusively near the mitotic exit and “down” transitions occurring throughout the rest of the cell cycle. Such cell cycle dependence likely reflects the dynamic activity of the rDNA-specific RENT complex, as MET3pr-GFP expression in a telomeric locus does not have the same cell cycle dependence. The MET3pr-GFP expression in rDNA is highly correlated in mother and daughter cells after cell division, indicating that the silenced state in the mother cell is inherited in daughter cells. These states are disrupted by a brief repression and reset upon a second activation. Potential mechanisms behind these observations are further discussed.

The NAD-dependent histone deacetylase sirtuin 2 (SIRT2) plays critical roles in mitosis and cell cycle progression and recently was shown to suppress tumor growth and to be downregulated in several types of cancers. However, the underlying mechanism of SIRT2 downregulation remains unknown. In this study, using bioinformatics, gene expression profiling, protein overexpression approaches, and cell migration assays, we showed that E3 ubiquitin ligase 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase degradation 1 (HRD1) interacts with SIRT2 and promotes its ubiquitination and degradation., The NAD-dependent histone deacetylase sirtuin 2 (SIRT2) plays critical roles in mitosis and cell cycle progression and recently was shown to suppress tumor growth and to be downregulated in several types of cancers. However, the underlying mechanism of SIRT2 downregulation remains unknown. In this study, using bioinformatics, gene expression profiling, protein overexpression approaches, and cell migration assays, we showed that E3 ubiquitin ligase 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase degradation 1 (HRD1) interacts with SIRT2 and promotes its ubiquitination and degradation. Furthermore, we found that HRD1 deficiency induces SIRT2 upregulation and inhibits the growth and tumor formation of lung cancer cells both in vitro and in vivo. Of note, we observed that SIRT2 expression is downregulated in human lung cancer and also negatively correlates with HRD1 expression in these cancers. Additionally, we found that patients with lung adenocarcinoma having lower HRD1 or higher SIRT2 expression levels tend to survive longer. On the basis of these results, we propose a mechanism of lung tumorigenesis that involves HRD1-mediated downregulation of SIRT2 and suggest that interventions targeting HRD1 activity could be a potential therapeutic strategy to treat patients with lung cancer.

Cohesin associates with distinct sites on chromosomes to mediate sister chromatid cohesion. Single cohesin complexes are thought to bind by encircling both sister chromatids in a topological embrace. Transcriptionally repressed chromosomal domains in the yeast Saccharomyces cerevisiae represent specialized sites of cohesion where cohesin binds silent chromatin in a Sir2-dependent fashion. In this study, we investigated the molecular basis for Sir2-mediated cohesion. We identified a cluster of charged surface residues of Sir2, collectively termed the EKDK motif, that are required for cohesin function. In addition, we demonstrated that Esc8, a Sir2-interacting factor, is also required for silent chromatin cohesion. Esc8 was previously shown to associate with Isw1, the enzymatic core of ISW1 chromatin remodelers, to form a variant of the ISW1a chromatin remodeling complex. When ESC8 was deleted or the EKDK motif was mutated, cohesin binding at silenced chromatin domains persisted but cohesion of the domains was abolished. The data are not consistent with cohesin embracing both sister chromatids within silent chromatin domains. Transcriptional silencing remains largely intact in strains lacking ESC8 or bearing EKDK mutations, indicating that silencing and cohesion are separable functions of Sir2 and silent chromatin.

The G2-to-M transition (or prophase) checkpoint of the cell cycle is a critical regulator of mitotic entry. SIRT2, a tumor suppressor gene, contributes to the control of this checkpoint by blocking mitotic entry under cellular stress. However, the mechanism underlying both SIRT2 activation and regulation of the G2-to-M transition remains largely unknown. Here, we report the formation of a multiprotein complex at the G2-to-M transition in vitro and in vivo. Group IVA cytosolic phospholipase A2 (cPLA2α) acts as a bridge in this complex to promote binding of SIRT2 to cyclin A-Cdk2. Cyclin A-Cdk2 then phosphorylates SIRT2 at Ser331. This phosphorylation reduces SIRT2 catalytic activity and its binding affinity to centrosomes and mitotic spindles, promoting G2-to-M transition. We show that the inhibitory effect of cPLA2α on SIRT2 activity impacts various cellular processes, including cellular levels of histone H4 acetylated at K16 (Ac-H4K16) and Ac-α-tubulin. This regulatory effect of cPLA2α on SIRT2 defines a novel function of cPLA2α independent of its phospholipase activity and may have implications for the impact of SIRT2-related effects on tumorigenesis and age-related diseases.

The epigenetic writer Sir2 maintains the heterochromatin state of chromosome in three chromosomal regions, namely, the silent mating type loci, telomeres, and the ribosomal DNA (rDNA). In this study, we demonstrated the mechanism by which Sir2 is regulated under heat stress. Our study reveals that a transient heat shock causes a drastic reduction in the SIR2 transcript which results in sustained failure to initiate silencing for as long as 90 generations. Hsp82 overexpression, which is the usual outcome of heat shock treatment, leads to a similar downregulation of SIR2 transcription. Using a series of genetic experiments, we have established that heat shock or Hsp82 overexpression causes upregulation of CUP9 that, in turn, represses SIR2 transcription by binding to its upstream activator sequence. We have mapped the cis regulatory element of SIR2. Our study shows that the deletion of cup9 causes reversal of the Hsp82 overexpression phenotype and upregulation of SIR2 expression in heat-induced Hsp82-overexpressing cells. On the other hand, we found that Cup9 overexpression represses SIR2 transcription and leads to a failure in the establishment of heterochromatin. The results of our study highlight the mechanism by which environmental factors amend the epigenetic configuration of chromatin.

Meiosis divides the chromosome number of the cell in half by having two rounds of chromosome segregation follow a single round of chromosome duplication. The first meiotic division is unique in that homologous pairs of sister chromatids segregate to opposite poles. Recent work in budding and fission yeast has shown that the cell cycle kinase, Cdc7-Dbf4, is required for many meiosis-specific chromosomal functions necessary for proper disjunction at meiosis I. This work reveals another role for Cdc7 in meiosis as a gene-specific regulator of the global transcription factor, Ndt80, which is required for exit from pachytene and entry into the meiotic divisions in budding yeast. Cdc7-Dbf4 promotes NDT80 transcription by relieving repression mediated by a complex of Sum1, Rfm1, and a histone deacetylase, Hst1. Sum1 exhibits meiosis-specific Cdc7-dependent phosphorylation, and mass spectrometry analysis reveals a dynamic and complex pattern of phosphorylation events, including four constitutive cyclin-dependent kinase (Cdk1) sites and 11 meiosis-specific Cdc7-Dbf4-dependent sites. Analysis of various phosphorylation site mutants suggests that Cdc7 functions with both Cdk1 and the meiosis-specific kinase Ime2 to control this critical transition point during meiosis.

Genes encoding thiamine biosynthesis enzymes in microorganisms are tightly regulated such that low environmental thiamine concentrations activate transcription and high concentrations are repressive. We have determined that multiple thiamine (THI) genes in Saccharomyces cerevisiae are also regulated by the intracellular NAD(+) concentration via the NAD(+)-dependent histone deacetylase (HDAC) Hst1 and, to a lesser extent, Sir2. Both of these HDACs associate with a distal region of the affected THI gene promoters that does not overlap with a previously defined enhancer region bound by the thiamine-responsive Thi2/Thi3/Pdc2 transcriptional activators. The specificity of histone H3 and/or H4 deacetylation carried out by Hst1 and Sir2 at the distal promoter region depends on the THI gene being tested. Hst1/Sir2-mediated repression of the THI genes occurs at the level of basal expression, thus representing the first set of transcription factors shown to actively repress this gene class. Importantly, lowering the NAD(+) concentration and inhibiting the Hst1/Sum1 HDAC complex elevated the intracellular thiamine concentration due to increased thiamine biosynthesis and transport, implicating NAD(+) in the control of thiamine homeostasis.

The cellular role of the Ada2 coactivator is currently understood in the context of the SAGA histone acetyltransferase (HAT) complex, where Ada2 increases the HAT activity of Gcn5 and interacts with transcriptional activators. Here we report a new function for Ada2 in promoting transcriptional silencing at telomeres and ribosomal DNA. This silencing function is the first characterized role for Ada2 distinct from its involvement with Gcn5. Ada2 binds telomeric chromatin and the silencing protein Sir2 in vivo. Loss of ADA2 causes the spreading of Sir2 and Sir3 into subtelomeric regions and decreased histone H4 K16 acetylation. This previously uncharacterized boundary activity of Ada2 is functionally similar to, but mechanistically distinct from, that of the MYST family HAT Sas2. Mounting evidence in the literature indicates that boundary activities create chromosomal domains important for regulating gene expression in response to environmental changes. Consistent with this, we show that upon nutritional changes, Ada2 occupancy increases at a subtelomeric region proximal to a SAGA-inducible gene and causes derepression of a silenced telomeric reporter gene. Thus, Ada2, likely in the context of SAGA, is positioned at chromosomal termini to participate in both transcriptional repression and activation in response to nutrient signaling.

Sir2 and Hst1 are NAD(+)-dependent histone deacetylases of budding yeast that are related by strong sequence similarity. Nevertheless, the two proteins promote two mechanistically distinct forms of gene repression. Hst1 interacts with Rfm1 and Sum1 to repress the transcription of specific middle-sporulation genes. Sir2 interacts with Sir3 and Sir4 to silence genes contained within the silent-mating-type loci and telomere chromosomal regions. To identify the determinants of gene-specific versus regional repression, we created a series of Hst1::Sir2 hybrids. Our analysis yielded two dual-specificity chimeras that were able to perform both regional and gene-specific repression. Regional silencing by the chimeras required Sir3 and Sir4, whereas gene-specific repression required Rfm1 and Sum1. Our findings demonstrate that the nonconserved N-terminal region and two amino acids within the enzymatic core domain account for cofactor specificity and proper targeting of these proteins. These results suggest that the differences in the silencing and repression functions of Sir2 and Hst1 may not be due to differences in enzymatic activities of the proteins but rather may be the result of distinct cofactor specificities.

The Saccharomyces cerevisiae Swi/Snf complex is an ATP-dependent chromatin remodeling complex that can activate or repress transcription (see references 3, 34, and 44 for recent reviews). Swi/Snf contains 11 different subunits, including Snf2, a highly conserved ATPase. The Snf2 subunit is the catalytic core of Swi/Snf; single amino acid changes in the DNA-dependent ATPase domain eliminate both the ATPase activity and the chromatin remodeling activity of Swi/Snf (13, 53). While Swi/Snf binds to both DNA and nucleosomes, it does not do so in a site-specific manner (12, 52). Rather, sequence-specific transcriptional activators and repressors have been shown to target Swi/Snf to specific promoters (for examples, see references 16, 46-48, 51, and 72; see reference 23 for a review). Gene expression microarray analysis has shown that the mRNA levels of a small subset of S. cerevisiae genes are significantly affected by the loss of Swi/Snf activity (25, 65). This apparent specificity of Swi/Snf control is likely caused by several factors, including its recruitment by particular transcriptional regulators. In addition, there is strong evidence that Swi/Snf is redundant with other transcription complexes in vivo and may therefore play a wider role than is indicated by microarray analysis (4, 50, 54, 64). A third factor is that Swi/Snf may be required only at promoters with a particular chromatin structure. Indeed, one study has suggested that different chromatin structures can determine the dependency upon Swi/Snf (9). Since chromosomal context can influence chromatin structure, we wanted to test whether genomic position might affect the Swi/Snf dependence of a gene. To address this issue, we randomly integrated the SUC2 gene, which is strongly Swi/Snf dependent (69), into the yeast genome to identify locations where SUC2 expression becomes independent of Swi/Snf. Surprisingly, we discovered that when SUC2 is integrated into the ribosomal DNA (rDNA) locus (RDN1, hereafter referred to as rDNA), its dependence on Swi/Snf is reversed. That is, when SUC2 is located in the rDNA, SUC2 transcription is repressed rather than activated by Swi/Snf. The S. cerevisiae rDNA consists of a tandem array of 9.1-kb units repeated 100 to 200 times on chromosome XII (49) (Fig. ​(Fig.1).1). The rDNA is located in the nucleolus in an arrangement reminiscent of the heterochromatin of higher eukaryotes (reviewed in references 45 and 57). Each rDNA repeat unit includes the 5S rRNA gene, transcribed by RNA polymerase III, and a 35S precursor rRNA gene, transcribed by RNA polymerase I. About half of the tandemly repeated rRNA genes are transcriptionally active; the active rRNA gene copies are randomly distributed along the ribosomal rRNA gene locus (14). Unlike the results seen with rRNA genes, the expression of several different Pol II-transcribed genes, when integrated into various regions of the rDNA, is repressed (7, 18, 60). rDNA silencing also represses recombination, believed to play an important role in preventing rDNA loss (21). FIG. 1. Diagram of the SUC2 insertion in the rDNA. The top line represents the rDNA array on the right arm of chromosome XII. A representative rDNA repeat, indicating the position of the integration of the SUC2 URA3 cassette (see text), is shown below. The thick ... Many trans-acting proteins required for rDNA silencing of Pol II transcription have been identified (6-8, 60, 62, 63, 66). These include Sir2, a member of a highly conserved family of NAD-dependent protein and histone deacetylases (30, 40, 61), and Set1, a histone methyltransferase (6, 8). The experiments presented in this paper identify another factor required for rDNA silencing, the Swi/Snf complex. Our results strongly suggest that Swi/Snf-mediated silencing occurs by a mechanism independent of Sir2 and Set1. Additional experiments show that Swi/Snf is also required for silencing at telomeres but not at silent-mating-type cassettes.

Studies of yeast have shown that the SIR2 gene family is involved in chromatin structure, transcriptional silencing, DNA repair, and control of cellular life span. Our functional studies of human SIRT2, a homolog of the product of the yeast SIR2 gene, indicate that it plays a role in mitosis. The SIRT2 protein is a NADdependent deacetylase (NDAC), the abundance of which increases dramatically during mitosis and is multiply phosphorylated at the G2/M transition of the cell cycle. Cells stably overexpressing the wild-type SIRT2 but not missense mutants lacking NDAC activity show a marked prolongation of the mitotic phase of the cell cycle. Overexpression of the protein phosphatase CDC14B, but not its close homolog CDC14A, results in dephosphorylation of SIRT2 with a subsequent decrease in the abundance of SIRT2 protein. A CDC14B mutant defective in catalyzing dephosphorylation fails to change the phosphorylation status or abundance of SIRT2 protein. Addition of 26S proteasome inhibitors to human cells increases the abundance of SIRT2 protein, indicating that SIRT2 is targeted for degradation by the 26S proteasome. Our data suggest that human SIRT2 is part of a phosphorylation cascade in which SIRT2 is phosphorylated late in G2, during M, and into the period of cytokinesis. CDC14B may provoke exit from mitosis coincident with the loss of SIRT2 via ubiquitination and subsequent degradation by the 26S proteasome. As the founding member of a vast gene family with members present in archaebacteria, eubacteria, and eukaryotes, SIR2 was first described in the budding yeast as a gene mediating the transcriptional silencing of the silent mating type (MAT) loci HML and HMR (14, 19). Additional functions for SIR2 in budding have been described, including the silencing of subtelomeric genes (telomere position effect [TPE]) and the regulation of transcription and recombination in the multiple tandem copies of ribosomal DNA (rDNA) (for a review, see reference 12). Guarente, Sinclair, and coworkers have shown that the SIR2 gene may suppress aging in budding yeast, through a mechanism involving the suppression of extrachromosomal rDNA circles (ERCs) derived from errant intralocus recombination and suggested that SIR2-related genes in other organisms may be involved in the aging process as well, even in multicellular eukaryotes (13). The mechanism by which SIRT (an acronym for SIR2 related) genes retard aging in metazoans may involve caloric restriction (CR) instead of the ERCs found in yeast (22). Support for this hypothesis has recently come from the key finding that providing the nematode Caenorhabditis elegans with two copies of one of its SIR2-related genes (the gene normally found on chromosome IV) can extend the worm’s life span by 50% (36). This extension of life span function is seen only for one of the three SIR2-related genes, Sir-2.1, encoding a large nuclear protein orthologous to that coded for by the SIR2-related gene known as SIRT1 in humans and SIR2 in mice. Neither of the other two SIRT genes in the

Transcriptional repression is often correlated with the alteration of chromatin structure through modifications of the nucleosomes in the promoter region, such as by deacetylation of the N-terminal histone tails. This is presumed to make the promoter region inaccessible to other regulatory factors and the general transcription machinery. To accomplish this, histone deacetylases are recruited to specific promoters via DNA-binding proteins and tethering factors. We have previously reported the requirement for the NAD(+)-dependent histone deacetylase Hst1 and the DNA-binding protein Sum1 for vegetative repression of many middle sporulation genes in Saccharomyces cerevisiae. Here we report the identification of a novel tethering factor, Rfm1, that is required for Hst1-mediated repression. Rfm1 interacts with both Sum1 and Hst1 and is required for the Sum1-Hst1 interaction. DNA microarray and Northern blot analyses showed that Rfm1 is required for repression of the same subset of Sum1-repressed genes that require Hst1. These results suggest that Rfm1 is a specificity factor that targets the Hst1 deacetylase to a subset of Sum1-regulated genes.

Calorie restriction (CR) extends life span in many different organisms, including mammals. We describe here a novel pathway that extends the life span of Saccharomyces cerevisiae mother cells but does not involve a reduction in caloric content of the media, i.e., there is growth of yeast cells in the presence of a high concentration of external osmolytes. Like CR, this longevity-promoting response to high osmolarity requires SIR2, suggesting a common mechanism of life span regulation. Genetic and microarray analysis indicates that high osmolarity extends the life span by activating Hog1p, leading to an increase in the biosynthesis of glycerol from glycolytic intermediates. This metabolic shift likely increases NAD levels, thereby activating Sir2p and promoting longevity.

Transcriptional silencing at the budding yeast silent mating type (HM) loci and telomeric DNA regions requires Sir2, a conserved NAD-dependent histone deacetylase, Sir3, Sir4, histones H3 and H4, and several DNA-binding proteins. Silencing at the yeast ribosomal DNA (rDNA) repeats requires a complex containing Sir2, Net1, and Cdc14. Here we show that the native Sir2/Sir4 complex is composed solely of Sir2 and Sir4 and that native Sir3 is not associated with other proteins. We further show that the initial binding of the Sir2/Sir4 complex to DNA sites that nucleate silencing, accompanied by partial Sir2-dependent histone deacetylation, occurs independently of Sir3 and is likely to be the first step in assembly of silent chromatin at the HM loci and telomeres. The enzymatic activity of Sir2 is not required for this initial binding, but is required for the association of silencing proteins with regions distal from nucleation sites. At the rDNA repeats, we show that histone H3 and H4 tails are required for silencing and rDNA-associated H4 is hypoacetylated in a Sir2-dependent manner. However, the binding of Sir2 to rDNA is independent of its histone deacetylase activity. Together, these results support a stepwise model for the assembly of silent chromatin domains in Saccharomyces cerevisiae.

Surprisingly, the contribution of defects in DNA replication to the determination of yeast life span has never been directly investigated. We show that a replicative yeast helicase/nuclease, encoded by DNA2 and a member of the same helicase subfamily as the RecQ helicases, is required for normal life span. All of the phenotypes of old wild-type cells, for example, extended cell cycle time, age-related transcriptional silencing defects, and nucleolar reorganization, occur after fewer generations in dna2 mutants than in the wild type. In addition, the life span of dna2 mutants is extended by expression of an additional copy of SIR2 or by deletion of FOB1, which also increase wild-type life span. The ribosomal DNA locus and the nucleolus seem to be particularly sensitive to defects in dna2 mutants, although in dna2 mutants extrachromosomal ribosomal circles do not accumulate during the aging of a mother cell. Several other replication mutations, such as rad27{Delta}, encoding the FEN-1 nuclease involved in several aspects of genomic stability, also show premature aging. We propose that replication fork failure due to spontaneous, endogenous DNA damage and attendant genomic instability may contribute to replicative senescence. This may imply that the genomic instability, segmental premature aging symptoms, and cancer predisposition associated with the human RecQ helicase diseases, such as Werner, Bloom, and Rothmund-Thomson syndromes, are also related to replicative stress.

Multiple genetic pathways have been shown to regulate life span and aging in the yeast Saccharomyces cerevisiae. Here we show that loss of a component of the RNA polymerase II complex, Hpr1p, results in a decreased life span. Although hpr1Delta mutants have an increased rate of recombination within the ribosomal DNA (rDNA) array, this is not accompanied by an increase in extrachromosomal rDNA circles (ERCs). Analyses of mutants that affect replication of the rDNA array and suppressors that reverse the phenotypes of the hpr1Delta mutant show that the reduced life span is associated with increased genomic instability but not with increased ERC formation. The hpr1Delta mutant acts in a pathway distinct from previously described mutants that reduce life span.

In the yeast Saccharomyces cerevisiae, a and alpha mating-type information is stored in transcriptionally silenced cassettes called HML and HMR. Silencing of these loci, maintained by the formation of a specialized type of heterochromatin, requires trans-acting proteins and cis-acting elements. Proteins required for silencing include the Sir2 NAD(+)-dependent deacetylase, Sir3, and Sir4. Factors that bind to the cis elements at HMR and HML and that are important for silencing include the origin recognition complex (ORC). Mutations of any of these Sir proteins or combinations of cis elements result in loss of silencing. SUM1-1 was previously identified as a dominant mutation that restores silencing to HMR in the absence of either the Sir proteins or some of the cis elements. We have investigated the novel mechanism whereby Sum1-1 causes Sir-independent silencing at HMR and present the following findings: Sum1-1 requires the Sir2 homolog, Hst1, for silencing and most probably requires the NAD(+)-dependent deacetylase activity of this protein. Sum1-1 interacts strongly with ORC, and this strong interaction is dependent on HMR DNA. Furthermore, ORC is required for Sum1-1-mediated silencing at HMR. These observations lead to a model for Sum1-1 silencing of HMR in which Sum1-1 is recruited to HMR by binding to ORC. Sum1-1, in turn, recruits Hst1. Hst1 then deacetylates histones or other chromatin-associated proteins to cause chromatin condensation and transcriptional silencing.

Strains of the yeast Saccharomyces cerevisiae defective in transcription factor UAF give rise to variants able to grow by transcribing endogenous ribosomal DNA (rDNA) by RNA polymerase II (Pol II). We have demonstrated that the switch to growth using the Pol II system consists of two steps: a mutational alteration in UAF and an expansion of chromosomal rDNA repeats. The first step, a single mutation in UAF, is sufficient to allow Pol II transcription of rDNA. In contrast to UAF mutations, mutations in Pol I or other Pol I transcription factors can not independently lead to Pol II transcription of rDNA, suggesting a specific role of UAF in preventing polymerase switch. The second step, expansion of chromosomal rDNA repeats to levels severalfold higher than the wild type, is required for efficient cell growth. Mutations in genes that affect recombination within the rDNA repeats, fob1 and sir2, decrease and increase, respectively, the frequency of switching to growth using Pol II, indicating that increased rDNA copy number is a cause rather than a consequence of the switch. Finally, we show that the switch to the Pol II system is accompanied by a striking alteration in the localization and morphology of the nucleolus. The altered state that uses Pol II for rDNA transcription is semistable and heritable through mitosis and meiosis. We discuss the significance of these observations in relation to the plasticity of rDNA tandem repeats and nucleolar structures as well as evolution of the Pol I machinery.

Although a surprisingly large number of genes affect yeast telomeres, in most cases it is not known if their products act directly or indirectly. We describe a one-hybrid assay for telomere binding proteins and use it to establish that six proteins that affect telomere structure or function but which had not been shown previously to bind telomeres in vivo are indeed telomere binding proteins. A promoter-defective allele of HIS3 was placed adjacent to a chromosomal telomere. Candidate proteins fused to a transcriptional activation domain were tested for the ability to activate transcription of the telomere-linked HIS3 gene. Using this system, Rif1p, Rif2p, Sir2p, Sir3p, Sir4p, and Cdc13p were found to be in vivo telomere binding proteins. None of the proteins activated the same reporter gene when it was at an internal site on the chromosome. Moreover, Cdc13p did not activate the reporter gene when it was adjacent to an internal tract of telomeric sequence, indicating that Cdc13p binding was telomere limited in vivo. The amino-terminal 20% of Cdc13p was sufficient to target Cdc13p to a telomere, suggesting that its DNA binding domain was within this portion of the protein. Rap1p, Rif1p, Rif2p, Sir4p, and Cdc13p activated the telomeric reporter gene in a strain lacking Sir3p, which is essential for telomere position effect (TPE). Thus, the telomeric association of these proteins did not require any of the chromatin features necessary for TPE. The data support models in which the telomere acts as an initiation site for TPE by recruiting silencing proteins to the chromosome end.

Protein families are generated by successive rounds of gene duplication and subsequent diversification. However, the paths by which duplicated genes acquire distinct functions are not well characterized. We focused on a pair of duplicated deacetylases from Saccharomyces cerevisiae, Sir2 and Hst1, that subfunctionalized after duplication. As a proxy for the ancestral, nonduplicated deacetylase, we studied Sir2 from another yeast, Kluyveromyces lactis. We compared the interaction domains of these deacetylases for the Sir transcriptional silencing complex, which acts with ScSir2, and the Sum1 repressor, which acts with ScHst1, and found that these interaction domains have been retained over the course of evolution and can be disrupted by simple amino acid substitutions. Therefore, Sir2 and Hst1 subfunctionalized by acquiring complementary inactivating mutations in these interaction domains.

Meiotic development is the pathway that produces haploid gametes from diploid precursors. Following the induction of the pathway, cells duplicate the genome and then enter an elongated prophase when homolog pairing, synapsis, and genetic recombination take place. Exit from prophase is followed by two sequential rounds of chromosome segregation. In many organisms, exit from prophase is a key control point where meiotic progression is regulated. Meiotic development in the yeast Saccharomyces cerevisiae (sporulation) is tightly regulated by a transcriptional program. The transcriptional program of sporulation is characterized by the sequential expression of temporally distinct sets of genes that are induced as different steps in the program take place (5, 30). Although there are at least 12 temporally distinct subclasses of sporulation-specific genes, they can be broadly grouped into early, middle, and late classes (reviewed in reference 39). Early promoters are activated by the Ime1 transcription factor, which is expressed and activated in diploids in response to starvation signals (reviewed in references 13 and 39). Early genes are expressed throughout meiotic S phase and prophase (5, 30). Toward the end of prophase, at the pachytene stage, homologs are fully connected by synaptonemal complexes (SCs), joint molecules have been formed but crossover recombinants have not been resolved (1, 24), and the spindle pole body (centrosome) has duplicated in preparation for the first meiotic division (3, 43). Exit from pachytene, entry into the meiotic divisions, and spore formation require the induction of middle genes. Middle promoters are activated by the Ndt80 transcription factor, which specifically binds to DNA elements termed middle sporulation elements (MSEs) (6, 8). ndt80Δ mutants block meiotic development in pachytene (43). ndt80Δ-blocked cells can be held in pachytene for extended periods of time and efficiently resume mitotic growth if nutrients are replenished (43). In contrast, cells that have induced middle promoters and exited pachytene are largely unable to resume mitotic growth (33). These observations suggest that Ndt80 plays a role in the phenomenon of commitment to meiosis. Ndt80 also appears to be regulated by the recombination checkpoint (also known as the pachytene checkpoint) (6, 8, 26, 38) that blocks meiotic development in response to recombination intermediates such as double-strand breaks and defects in SC formation (see references 9 and 31 for reviews of the pachytene checkpoint). Ndt80 promotes the disassembly of SCs and crossover formation by activating the expression of the Cdc5 polo-like kinase (36). Ndt80 also promotes the nuclear divisions and spore morphogenesis by activating the expression of M-phase cyclins, cell cycle regulatory molecules, and gene products required for spore formation (5). NDT80 is itself a tightly regulated meiosis-specific gene that is expressed shortly before most other middle genes, and it has been classified as an early/middle (25) or subclass 3a (30) gene. Its expression is controlled through Ndt80-MSE interactions in its own promoter in a positive autoregulatory loop (6). Despite the critical role that NDT80 induction plays in meiotic regulation, the molecular events that trigger the NDT80 autoregulatory loop have not yet been elucidated. Sum1 is a DNA-binding protein that represses a subset of middle promoters in vegetative cells (42). Sum1 interacts with the core MSE and with adjacent bases (28). Ndt80-inducible MSEs therefore vary in their affinities for Sum1. The Sum1 and Ndt80 DNA-binding domains have been shown previously to compete for occupancy of MSE DNA in vitro. About 75 of the 150 middle promoters are Sum1 repressible, and these promoters are enriched with MSEs that are predicted to bind Sum1 (28, 40). The NDT80 promoter is controlled by a Sum1-repressible MSE, suggesting that the removal of Sum1 repression can regulate meiotic progression (25, 42). Sum1 represses transcription through the Hst1 NAD+-dependent histone deacetylase (Sir2 paralog) that is bound to Sum1 via the Rfm1 bridging protein (20). However, only a subset of Sum1-repressible promoters are derepressed in hst1Δ and rfm1Δ mutants, suggesting that Sum1 can repress transcription by an Hst1-Rfm1-independent mechanism. Consistent with the hypothesis that Sum1 regulates prophase exit, sum1Δ mutants bypass the pachytene checkpoint and carry out the nuclear divisions in the presence of recombination intermediates (broken chromosomes) to produce nonviable haploid products (15, 26). In addition to fulfilling a role in middle meiotic gene regulation, Sum1 interacts with a subset of origins of DNA replication and positively regulates S phase by an Hst1-dependent mechanism (12, 41). Sum1 is also required to repress α-specific promoters in a cells (44). Sum1 and Hst1 also regulate the expression of promoters controlling the salvage pathway for NAD+ (2). However, NAD+ salvage pathway genes and α-specific genes are not induced during meiosis (5, 30). It is unclear how Sum1's roles in S phase regulation, cell type specification, NAD+ biosynthesis, and meiosis are differentially regulated. Ime2 is a meiosis-specific cyclin-dependent kinase (CDK)-like kinase that regulates multiple steps in meiotic development (11). Ime2 has been shown previously to activate Ndt80 (34, 35). In addition, ime2Δ mutants show defects in middle meiotic gene expression that are partially suppressed by sum1Δ (25). These observations suggest that Ime2 promotes the transcription of middle promoters by positively regulating Ndt80 and by negatively regulating Sum1. Two models can explain how Sum1 repression is removed in meiotic cells. The first model posits that Sum1 is removed from chromatin by a mechanism that requires competition with Ndt80. The findings that the purified DNA-binding domain of Sum1 can be displaced from an MSE with purified Ndt80 in vitro (28) and that high-level ectopic Ndt80 expression in vegetative haploid cells can induce SMK1 and other Sum1-repressible middle genes in mitotic haploid cells (5) demonstrate that competition can occur. According to this competition model, Sum1 would function as a transcriptional damper to prevent adventitious induction of middle promoters by Ndt80 or other activators. The second model posits that Sum1 is actively removed from chromatin in response to meiotic signals in S-phase or prophase cells. According to this sequential model, the regulated removal of Sum1 repression would create a state that would permit the NDT80 autoregulatory loop to be induced. Distinguishing between the competition and sequential models for the removal of Sum1 repression has important implications for how meiotic progression is regulated. The SMK1 middle meiotic promoter provides an excellent model for the study of Sum1 regulation since it is controlled by a single, well-characterized MSE that interacts with Sum1 and Ndt80 and by an upstream activation sequence that binds the constitutive Abf1 transcriptional activator (28, 29, 42). Thus, Smk1 is not expressed when Sum1 is present, and it is expressed to a moderate level in an Abf1-dependent fashion when Sum1 has been removed. The derepressed SMK1 promoter is further activated by Ndt80 during meiotic development to yield peak levels of SMK1 expression (15). In this study, we show that Sum1 is removed from middle meiotic promoters in a pathway that does not require competition with Ndt80. We also show that the Ime2 CDK-like kinase phosphorylates Sum1 on T306 in vivo and that a form of Sum1 containing a nonphosphorylatable substitution at this position (Sum1-T306A) is not removed in ndt80Δ meiotic cells. Although the deletion of Hst1 does not derepress SMK1 in vegetative cells, it does bypass the Sum1 removal defect of a sum1(T306A) ndt80Δ strain. These findings suggest that Ime2 promotes a state that is permissive for middle-promoter expression by antagonizing Hst1. These data indicate that a regulated pathway promotes the removal of the Sum1 brake from chromatin prior to NDT80 induction and suggest that this pathway controls the expression of middle promoters and meiotic progression. Our findings also suggest that when this pathway is compromised, Sum1 can be removed in an Ndt80-dependent pathway.

It is well accepted that for transcriptional silencing in budding yeast, the evolutionarily conserved lysine deacetylase Sir2, in concert with its partner proteins Sir3 and Sir4, establishes a chromatin structure that prevents RNA polymerase II (Pol II) transcription. However, the mechanism of repression remains controversial. Here, we show that the recruitment of Pol II, as well as that of the general initiation factors TBP and TFIIH, occurs unimpeded to the silent HMRa1 and HMLalpha1/HMLalpha2 mating promoters. This, together with the fact that Pol II is Ser5 phosphorylated, implies that SIR-mediated silencing is permissive to both preinitiation complex (PIC) assembly and transcription initiation. In contrast, the occupancy of factors critical to both mRNA capping and Pol II elongation, including Cet1, Abd1, Spt5, Paf1C, and TFIIS, is virtually abolished. In agreement with this, efficiency of silencing correlates not with a restriction in Pol II promoter occupancy but with a restriction in capping enzyme recruitment. These observations pinpoint the transition between polymerase initiation and elongation as the step targeted by Sir2 and indicate that transcriptional silencing is achieved through the differential accessibility of initiation and capping/elongation factors to chromatin. We compare Sir2-mediated transcriptional silencing to a second repression mechanism, mediated by Tup1. In contrast to Sir2, Tup1 prevents TBP, Pol II, and TFIIH recruitment to the HMLalpha1 promoter, thereby abrogating PIC formation.

In the budding yeast Saccharomyces cerevisiae, transcriptional silencing is defined as constitutive, chromatin-mediated repression of transcription that occurs at HM loci, ribosomal DNA (rDNA), and telomeres. The Sir2 protein, essential for S. cerevisiae transcriptional silencing, is of particular interest as the founding member of the sirtuin family of NAD-dependent protein deacetylases that is conserved throughout eukaryotes. Increased expression of SIR2 or its closest homologs increases the life span in yeast and animals, respectively (reviewed in references 31 and 44). Although these studies suggest the conservation of an aging pathway, molecular mechanisms underlying the conserved function of sirtuins in aging remain unclear. In yeast, the only reported targets of Sir2 activity are histones. By contrast, the mammalian sirtuin SIRT1 deacetylates many different targets, including p53 (reviewed in reference 55). In yeast, Sir2 binds the telomeres and HM loci in complex with Sir3 and Sir4. Deacetylation of histone H4 lysine 16 (H4K16) promotes binding of Sir3 to chromatin and spreading of transcriptional silencing (reviewed in reference 54). However, Sir3 and Sir4 have no known metazoan homologs. Thus, it seems likely that some Sir2 molecular interactions remain undiscovered. Indeed, molecular mechanisms that contribute to sir2Δ phenotypes, such as suppression of replication onset (47) and unequal inheritance of oxidative damage between mother and daughter cells (1), are undefined. To identify novel Sir2-interacting proteins that might participate in these and other uncharacterized molecular pathways, we performed a screen for Sir2 physical interactions. We identified Slx5 as a novel Sir2 interactor by two-hybrid analysis with Sir2 fused to the Gal4 DNA-binding domain (GBD). Slx5 is a RING domain protein that was first identified as essential for viability in the absence of Sgs1 (45). Sgs1 is the sole S. cerevisiae representative of the RecQ helicase family that includes human WRN and BLM. Slx5 forms a complex with another RING domain protein, Slx8 (45, 76). Mutants lacking Slx5 or Slx8 show an increase in global sumoylation (6, 28, 70). SUMO is a ubiquitin-like protein moiety that is covalently attached to lysine residues. The Slx5/8 complex may function as a SUMO E3 ligase (28). Recently, it was discovered that Slx5 contains SUMO interaction motifs and that Slx8 is a ubiquitin E3 ligase (49, 67, 73). The Slx5/8 complex is thus a member of a newly defined conserved family of factors, known as STUbLs (SUMO-targeted ubiquitin ligases), present in humans as a single protein, RNF4 (49, 62). A function shared by some members of this protein family may be ubiquitination of polysumoylated proteins. The polysumoylated and ubiquitinated proteins are then degraded by the proteasome (67), providing a previously unsuspected mechanism for down-regulation. Although Sir3 and Sir4, but not Sir2, are known to be sumoylated (11, 72), the Sir2-Slx5 two-hybrid interaction required neither protein and the GAD-Slx5 interactor lacked the N-terminal SUMO interaction motifs. Furthermore, we recapitulated the physical interaction in vitro by affinity precipitation under conditions in which protein sumoylation is not preserved. Thus, the physical interaction was not likely to be bridged by sumoylation. The proteins have a functional overlap defined by several independent assays. Yeast lacking the SLX5 gene grew poorly, and additionally deleting SIR2, SIR3, or SIR4 exacerbated this defect, indicating that Slx5 and the Sir2/3/4 complex may have parallel roles in a vital process. Deletion of the SLX5 gene caused a previously unsuspected loss of telomeric and rDNA silencing, as does sir2Δ. Furthermore, ulp2Δ mutant cells, which share the slx5Δ phenotype of increased global sumoylation, also shared the silencing defect and loss of viability in combination with the sir2Δ or sir4Δ mutation. Therefore, the role of Slx5 in silencing appears to be linked to its role in regulating sumoylation, such that excess accumulation of sumoylated proteins is toxic in the absence of silenced chromatin.

Unrepaired DNA double-strand breaks (DSBs) typically result in G(2) arrest. Cell cycle progression can resume following repair of the DSBs or through adaptation to the checkpoint, even if the damage remains unrepaired. We developed a screen for factors in the yeast Saccharomyces cerevisiae that affect checkpoint control and/or viability in response to a single, unrepairable DSB that is induced by HO endonuclease in a dispensable yeast artificial chromosome containing human DNA. SIR2, -3, or -4 mutants exhibit a prolonged, RAD9-dependent G(2) arrest in response to the unrepairable DSB followed by a slow adaptation to the persistent break, leading to division and rearrest in the next G(2). There are a small number of additional cycles before permanent arrest as microcolonies. Thus, SIR genes, which repress silent mating type gene expression, are required for the adaptation and the prevention of indirect lethality resulting from an unrepairable DSB in nonessential DNA. Rapid adaptation to the G(2) checkpoint and high viability were restored in sir(-) strains containing additional deletions of the silent mating type loci HML and HMR, suggesting that genes under mating type control can reduce the toleration of a single DSB. However, coexpression of MATa1 and MATalpha2 in Sir(+) haploid cells did not lead to lethality from the HO-induced DSB, suggesting that toleration of an unrepaired DSB requires more than one Sir(+) function.

The telomeres of most organisms consist of short repeated sequences that can be elongated by telomerase, a reverse transcriptase complex that contains its own RNA template for the synthesis of telomere repeats. In Saccharomyces cerevisiae, the RAP1 gene encodes the major telomere binding protein Rap1p. Here we use a quantitative telomere formation assay to demonstrate that Rap1p C termini can enhance telomere formation more than 30-fold when they are located at internal sites. This stimulation is distinct from protection from degradation. Enhancement of formation required the gene for telomerase RNA but not Sir1p, Sir2p, Sir3p, Sir4p, Tel1p, or the Rif1p binding site in the Rap1p C terminus. Our data suggest that Rap1p C termini enhance telomere formation by attracting or increasing the activity of telomerase near telomeres. Earlier work suggests that Rap1p molecules at the chromosome terminus inhibit the elongation of long telomeres by blocking the access of telomerase. Our results suggest a model where a balance between internal Rap1p increasing telomerase activity and Rap1p at the termini of long telomeres controlling telomerase access maintains telomeres at a constant length.

Heterochromatin in metazoans induces transcriptional silencing, as exemplified by position effect variegation in Drosophila melanogaster and X-chromosome inactivation in mammals. Heterochromatic DNA is packaged in nucleosomes that are distinct in their acetylation pattern from those present in euchromatin, although the role these differences play in the structure of heterochromatin or in the effects of heterochromatin on transcriptional activity is unclear. Here we report that, as observed in the facultative heterochromatin of the inactive X chromosome in female mammalian cells, histones H3 and H4 in chromatin spanning the transcriptionally silenced mating-type cassettes of the yeast Saccharomyces cerevisiae are hypoacetylated relative to histones H3 and H4 of transcriptionally active regions of the genome. By immunoprecipitation of chromatin fragments with antibodies specific for H4 acetylated at particular lysine residues, we found that only three of the four lysine residues in the amino-terminal domain of histone H4 spanning the silent cassettes are hypoacetylated. Lysine 12 shows significant acetylation levels. This is identical to the pattern of histone H4 acetylation observed in centric heterochromatin of D. melanogaster. These two observations provide additional evidence that the silent cassettes are encompassed in the yeast equivalent of metazoan heterochromatin. Further, mutational analysis of the amino-terminal domain of histone H4 in S. cerevisiae demonstrated that this observed pattern of histone H4 acetylation is required for transcriptional silencing. This result, in conjunction with prior mutational analyses of yeast histones H3 and H4, indicates that the particular pattern of nucleosome acetylation found in heterochromatin is required for its effects on transcription and is not simply a side effect of heterochromatin formation.