Sir2 and Fun30 regulate ribosomal DNA replication timing via Mcm helicase positioning and nucleosome occupancy.

The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well-known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA arrays (rDNA). We have previously reported that in the absence of SIR2, a derepressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the non-displaced MCMs. Furthermore, we found that both, activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30. Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler.

BY4741 cannot enter quiescence from rich medium.

In rich medium, W303 Saccharomyces cerevisiae begins to accumulate in G1 an hour before it exhausts the available glucose. It undergoes one more asymmetrical cell division, then stops dividing in G1. In contrast, BY4741, stops dividing four hours before glucose exhaustion, at one-fourth the cell density achieved by W303. There is no asymmetrical cell division and only 50% of the cells arrest in G1. We conclude that BY4741 growth is not limited by glucose and they do not go through the stereotypical events carried out by other strains as they enter quiescence from rich medium. In W303, the timing of glucose limitation and the transition to quiescence is correlated with the rate of biomass accumulation and cell doubling time.

A common SSD1 truncation is toxic to cells entering quiescence and promotes sporulation.

Ssd1p is an RNA binding protein in Saccharomyces cerevisiae that plays an important role in cell division, cell fate decisions, stress response and virulence. Lab strain W303 encodes the terminal truncation ssd1-2, which is typically interpreted to be a loss of function allele. We have shown that ssd1-2 is toxic to mpt5-Δ mutants and to diploids entering stationary phase and quiescence. The ssd1-Δ null shows no toxicity, indicating that ssd1-2 is disrupting an essential function that does not solely require Ssd1p. ssd1-2 cells are also more sensitive to stress than ssd1-Δ . These phenotypes are recessive to SSD1-1 . In contrast, ssd1-2 plays a dominant role in promoting sporulation.

Whi7/Srl3 polymorphisms reveal its role in cell size and quiescence.

Whi5 and Srl3/Whi7 are related proteins that resulted from the whole genome duplication of S. cerevisiae (Wolfe and Shields 1997). Whi5 plays an Rb-like function in binding and inhibiting the late G1 transcription that promotes progression from G1 to S (Costanzo et al. 2004; de Bruin et al. 2004). Whi7 can also associate with G1 transcription complexes and promotes G1 arrest when overproduced (Gomar-Alba et al. 2017), but its transcription is primarily induced by stress (Ragni et al. 2011; Mendez et al. 2020). We have used polymorphisms in two laboratory yeast strains to uncover novel functions of Whi7 in log and quiescent cells. These include small cell size during log phase and defects in entry, maintenance and recovery from quiescence.

The budding yeast transition to quiescence.

A subset of Saccharomyces cerevisiae cells in a stationary phase culture achieve a unique quiescent state characterized by increased cell density, stress tolerance, and longevity. Trehalose accumulation is necessary but not sufficient for conferring this state, and it is not recapitulated by abrupt starvation. The fraction of cells that achieve this state varies widely in haploids and diploids and can approach 100%, indicating that both mother and daughter cells can enter quiescence. The transition begins when about half the glucose has been taken up from the medium. The high affinity glucose transporters are turned on, glycogen storage begins, the Rim15 kinase enters the nucleus and the accumulation of cells in G1 is initiated. After the diauxic shift (DS), when glucose is exhausted from the medium, growth promoting genes are repressed by the recruitment of the histone deacetylase Rpd3 by quiescence-specific repressors. The final division that takes place post-DS is highly asymmetrical and G1 arrest is complete after 48 h. The timing of these events can vary considerably, but they are tightly correlated with total biomass of the culture, suggesting that the transition to quiescence is tightly linked to changes in external glucose levels. After 7 days in culture, there are massive morphological changes at the protein and organelle level. There are global changes in histone modification. An extensive array of condensin-dependent, long-range chromatin interactions lead to genome-wide chromatin compaction that is conserved in yeast and human cells. These interactions are required for the global transcriptional repression that occurs in quiescent yeast.

Ssd1 and the cell wall integrity pathway promote entry, maintenance, and recovery from quiescence in budding yeast.

Wild Saccharomyces cerevisiae strains are typically diploid. When faced with glucose and nitrogen limitation they can undergo meiosis and sporulate. Diploids can also enter a protective, nondividing cellular state or quiescence. The ability to enter quiescence is highly reproducible but shows broad natural variation. Some wild diploids can only enter cellular quiescence, which indicates that there are conditions in which sporulation is lost or selected against. Others only sporulate, but if sporulation is disabled by heterozygosity at the IME1 locus, those diploids can enter quiescence. W303 haploids can enter quiescence, but their diploid counterparts cannot. This is the result of diploidy, not mating type regulation. Introduction of SSD1 to W303 diploids switches fate, in that it rescues cellular quiescence and disrupts the ability to sporulate. Ssd1 and another RNA-binding protein, Mpt5 (Puf5), have parallel roles in quiescence in haploids. The ability of these mutants to enter quiescence, and their long-term survival in the quiescent state, can be rescued by exogenously added trehalose. The cell wall integrity pathway also promotes entry, maintenance, and recovery from quiescence through the Rlm1 transcription factor.

A common strategy for initiating the transition from proliferation to quiescence.

Development, tissue renewal and long term survival of multi-cellular organisms is dependent upon the persistence of stem cells that are quiescent, but retain the capacity to re-enter the cell cycle to self-renew, or to produce progeny that can differentiate and re-populate the tissue. Deregulated release of these cells from the quiescent state, or preventing them from entering quiescence, results in uncontrolled proliferation and cancer. Conversely, loss of quiescent cells, or their failure to re-enter cell division, disrupts organ development and prevents tissue regeneration and repair. Understanding the quiescent state and how cells control the transitions in and out of this state is of fundamental importance. Investigations into the mechanics of G1 arrest during the transition to quiescence continue to identify striking parallels between the strategies used by yeast and mammals to regulate this transition. When cells commit to a stable but reversible arrest, the G1/S genes responsible for promoting S phase must be inhibited. This process, from yeast to humans, involves the formation of quiescence-specific complexes on their promoters. In higher cells, these so-called DREAM complexes of E2F4/DP/RBL/MuvB recruit the highly conserved histone deacetylase HDAC1, which leads to local histone deacetylation and repression of S phase-promoting transcripts. Quiescent yeast cells also show pervasive histone deacetylation by the HDAC1 counterpart Rpd3. In addition, these cells contain quiescence-specific regulators of G1/S genes: Msa1 and Msa2, which can be considered components of the yeast equivalent of the DREAM complex. Despite a lack of physical similarities, the goals and the strategies used to achieve a reversible transition to quiescence are highly conserved. This motivates a detailed study of this process in the simple model organism: budding yeast.

Msa1 and Msa2 Modulate G1-Specific Transcription to Promote G1 Arrest and the Transition to Quiescence in Budding Yeast.

Yeast that naturally exhaust their glucose source can enter a quiescent state that is characterized by reduced cell size, and high cell density, stress tolerance and longevity. The transition to quiescence involves highly asymmetric cell divisions, dramatic reprogramming of transcription and global changes in chromatin structure and chromosome topology. Cells enter quiescence from G1 and we find that there is a positive correlation between the length of G1 and the yield of quiescent cells. The Swi4 and Swi6 transcription factors, which form the SBF transcription complex and promote the G1 to S transition in cycling cells, are also critical for the transition to quiescence. Swi6 forms a second complex with Mbp1 (MBF), which is not required for quiescence. These are the functional analogues of the E2F complexes of higher eukaryotes. Loss of the RB analogue, Whi5, and the related protein Srl3/Whi7, delays G1 arrest, but it also delays recovery from quiescence. Two MBF- and SBF-Associated proteins have been identified that have little effect on SBF or MBF activity in cycling cells. We show that these two related proteins, Msa1 and Msa2, are specifically required for the transition to quiescence. Like the E2F complexes that are quiescence-specific, Msa1 and Msa2 are required to repress the transcription of many SBF target genes, including SWI4, the CLN2 cyclin and histones, specifically after glucose is exhausted from the media. They also activate transcription of many MBF target genes. msa1msa2 cells fail to G1 arrest and rapidly lose viability upon glucose exhaustion. msa1msa2 mutants that survive this transition are very large, but they attain the same thermo-tolerance and longevity of wild type quiescent cells. This indicates that Msa1 and Msa2 are required for successful transition to quiescence, but not for the maintenance of that state.

A Genetic Screen for Saccharomyces cerevisiae Mutants That Fail to Enter Quiescence.

Budding yeast begin the transition to quiescence by prolonging G1 and accumulating limited nutrients. They undergo asymmetric cell divisions, slow cellular expansion, acquire significant stress tolerance and construct elaborate cell walls. These morphologic changes give rise to quiescent (Q) cells, which can be distinguished from three other cell types in a stationary phase culture by flow cytometry. We have used flow cytometry to screen for genes that are required to obtain the quiescent cell fraction. We find that cell wall integrity is critical and these genes may help define quiescence-specific features of the cell wall. Genes required to evade the host innate immune response are common. These may be new targets for antifungal drugs. Acquired thermotolerance is also a common property, and we show that the stress-response transcription factors Msn2 and Msn4 promote quiescence. Many other pathways also contribute, including a subset of genes involved in autophagy, ubiquitin-mediated proteolysis, DNA replication, bud site selection, and cytokinesis.

Xbp1 directs global repression of budding yeast transcription during the transition to quiescence and is important for the longevity and reversibility of the quiescent state.

Pure populations of quiescent yeast can be obtained from stationary phase cultures that have ceased proliferation after exhausting glucose and other carbon sources from their environment. They are uniformly arrested in the G1 phase of the cell cycle, and display very high thermo-tolerance and longevity. We find that G1 arrest is initiated before all the glucose has been scavenged from the media. Maintaining G1 arrest requires transcriptional repression of the G1 cyclin, CLN3, by Xbp1. Xbp1 is induced as glucose is depleted and it is among the most abundant transcripts in quiescent cells. Xbp1 binds and represses CLN3 transcription and in the absence of Xbp1, or with extra copies of CLN3, cells undergo ectopic divisions and produce very small cells. The Rad53-mediated replication stress checkpoint reinforces the arrest and becomes essential when Cln3 is overproduced. The XBP1 transcript also undergoes metabolic oscillations under glucose limitation and we identified many additional transcripts that oscillate out of phase with XBP1 and have Xbp1 binding sites in their promoters. Further global analysis revealed that Xbp1 represses 15% of all yeast genes as they enter the quiescent state and over 500 of these transcripts contain Xbp1 binding sites in their promoters. Xbp1-repressed transcripts are highly enriched for genes involved in the regulation of cell growth, cell division and metabolism. Failure to repress some or all of these targets leads xbp1 cells to enter a permanent arrest or senescence with a shortened lifespan.

Key events during the transition from rapid growth to quiescence in budding yeast require posttranscriptional regulators.

Yeast that naturally exhaust the glucose from their environment differentiate into three distinct cell types distinguishable by flow cytometry. Among these is a quiescent (Q) population, which is so named because of its uniform but readily reversed G1 arrest, its fortified cell walls, heat tolerance, and longevity. Daughter cells predominate in Q-cell populations and are the longest lived. The events that differentiate Q cells from nonquiescent (nonQ) cells are initiated within hours of the diauxic shift, when cells have scavenged all the glucose from the media. These include highly asymmetric cell divisions, which give rise to very small daughter cells. These daughters modify their cell walls by Sed1- and Ecm33-dependent and dithiothreitol-sensitive mechanisms that enhance Q-cell thermotolerance. Ssd1 speeds Q-cell wall assembly and enables mother cells to enter this state. Ssd1 and the related mRNA-binding protein Mpt5 play critical overlapping roles in Q-cell formation and longevity. These proteins deliver mRNAs to P-bodies, and at least one P-body component, Lsm1, also plays a unique role in Q-cell longevity. Cells lacking Lsm1 and Ssd1 or Mpt5 lose viability under these conditions and fail to enter the quiescent state. We conclude that posttranscriptional regulation of mRNAs plays a crucial role in the transition in and out of quiescence.

The Forkhead transcription factor Hcm1 regulates chromosome segregation genes and fills the S-phase gap in the transcriptional circuitry of the cell cycle.

Transcription patterns shift dramatically as cells transit from one phase of the cell cycle to another. To better define this transcriptional circuitry, we collected new microarray data across the cell cycle of budding yeast. The combined analysis of these data with three other cell cycle data sets identifies hundreds of new highly periodic transcripts and provides a weighted average peak time for each transcript. Using these data and phylogenetic comparisons of promoter sequences, we have identified a late S-phase-specific promoter element. This element is the binding site for the forkhead protein Hcm1, which is required for its cell cycle-specific activity. Among the cell cycle-regulated genes that contain conserved Hcm1-binding sites, there is a significant enrichment of genes involved in chromosome segregation, spindle dynamics, and budding. This may explain why Hcm1 mutants show 10-fold elevated rates of chromosome loss and require the spindle checkpoint for viability. Hcm1 also induces the M-phase-specific transcription factors FKH1, FKH2, and NDD1, and two cell cycle-specific transcriptional repressors, WHI5 and YHP1. As such, Hcm1 fills a significant gap in our understanding of the transcriptional circuitry that underlies the cell cycle.

Genetic interactions between mediator and the late G1-specific transcription factor Swi6 in Saccharomyces cerevisiae.

Swi6 associates with Swi4 to activate HO and many other late G(1)-specific transcripts in budding yeast. Genetic screens for suppressors of SWI6 mutants have been carried out. A total of 112 of these mutants have been identified and most fall into seven complementation groups. Six of these genes have been cloned and identified and they all encode subunits of the mediator complex. These mutants restore transcription to the HO-lacZ reporter in the absence of Swi6 and have variable effects on other Swi6 target genes. Deletions of other nonessential mediator components have been tested directly for suppression of, or genetic interaction with, swi6. Mutations in half of the known subunits of mediator show suppression and/or growth defects in combination with swi6. These phenotypes are highly variable and do not correlate with a specific module of the mediator. Mutations in tail module components sin4 and pgd1 showed both growth defects and suppression when combined with swi6, but a third tail component, gal11, showed neither. A truncated form of the essential Srb7 mediator subunit also suppresses swi6 mutations and shows a defect in recruitment of the tail module components Sin4, Pgd1, and Gal11 to the mediator complex.

Conserved homeodomain proteins interact with MADS box protein Mcm1 to restrict ECB-dependent transcription to the M/G1 phase of the cell cycle.

Two homeodomain proteins, Yox1 and Yhp1, act as repressors at early cell cycle boxes (ECBs) to restrict their activity to the M/G1 phase of the cell cycle in budding yeast. These proteins bind to Mcm1 and to a typical homeodomain binding site. The expression of Yox1 is periodic and directly correlated with its binding to, and repression of, ECB activity. The absence of Yox1 and Yhp1 or the constitutive expression of Yox1 leads to the loss of cell-cycle regulation of ECB activity. Therefore, the cell-cycle-regulated expression of these repressors defines the interval of ECB-dependent transcription. Twenty-eight genes, including MCM2-7, CDC6, SWI4, CLN3, and a number of genes required during late M phase have been identified that are coordinately regulated by this pathway.

Characterization of the ECB binding complex responsible for the M/G(1)-specific transcription of CLN3 and SWI4.

The transcription factor Mcm1 is regulated by adjacent binding of a variety of different factors regulating the expression of cell-type-specific, cell cycle-specific, and metabolic genes. In this work, we investigate a new class of Mcm1-regulated promoters that are cell cycle regulated and peak in late M-early G(1) phase of the cell cycle via a promoter element referred to as an early cell cycle box (ECB). Gel filtration experiments indicate that the ECB-specific DNA binding complex is over 200 kDa in size and includes Mcm1 and at least one additional protein. Using DNase I footprinting in vitro, we have observed protection of the ECB elements from the CLN3, SWI4, CDC6, and CDC47 promoters, which includes protection of the 16-bp palindrome to which Mcm1 dimers are known to bind as well as protection of extended flanking sequences. These flanking sequences influence the stability and the variety of complexes that form on the ECB elements, and base substitutions in the protected flank affect transcriptional activity of the element. Chromatin immunoprecipitations show that Mcm1 binds in vivo to ECB elements throughout the cell cycle and that binding is sensitive to carbon source changes.