Dot6 is a major regulator of cell size and a transcriptional activator of ribosome biogenesis in the opportunistic yeast Candida albicans
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ABSTRACT: In most species, size homeostasis appears to be exerted in late G1 phase as cells commit to division, called Start in yeast and the Restriction Point in metazoans. This size threshold couples cell growth to division and thereby establishes long-term size homeostasis. Our former investigations have shown that hundreds of genes markedly altered cell size under homeostatic growth conditions in the opportunistic yeast Candida albicans, but surprisingly only few of these overlapped with size control genes in the budding yeast Saccharomyces cerevisiae. Here, we investigated one of the divergent potent size regulators in C. albicans, the Myb-like HTH transcription factor Dot6. Our data demonstrated that Dot6 is a negative regulator of Start and also acts as a transcriptional activator of ribosome biogenesis (Ribi) genes. Genetic epistasis uncovered that Dot6 interacted with the master transcriptional regulator of the G1 machinery, SBF complex, but not with the Ribi and cell size regulators Sch9, Sfp1 and p38/Hog1. Dot6 was required for carbon-source modulation of cell size and it is regulated at the level of nuclear localization by TOR pathway. Our findings support a model where Dot6 acts as a hub that integrate directly growth cues via the TOR pathway to control the commitment to mitotic division at G1.
Project description:The basis for commitment to cell division in late G1 phase, called Start in yeast and the Restriction Point in metazoans, is a critical but still poorly understood aspect of eukaryotic cell proliferation. All eukaryotic cells must grow to a critical cell size before commitment to division occurs. This size threshold couples cell growth to division and thereby establishes long-term size homeostasis. Here, to address the problem of cell size control across different species, we performed the first quantitative survey of the size phenome in the pathogenic yeast Candida albicans by focusing on mutants of transcriptional regulators and kinases. We investigate one of the divergent size regulatory pathways in C. albicans, the p38/HOG MAPK module, to uncover a novel stress-independent function for the HOG module in size control. We show that Hog1 inhibits G1/S transcription to delay the G1/S transition and that Hog1 also controls ribosome biogenesis gene expression. These results suggest that Hog1 represents a critical and previously unsuspected nexus between the growth and division machineries. Our study thus delineates the overall genetic basis for size control in C. albicans and identifies, to our knowledge, the first specific conduit that couples cell growth to cell division.
Project description:This work reports the role of the transcription factor Ahr1 in mediating cell size control in the pathogenic yeast Candida albicans. To investigate the role of Ahr1 at Start, we performed a transcriptional profiling by comparing the transcriptome of G1 phase cells of both WT and ahr1 mutant strains.
Project description:Growth-dependent accumulation of the limiting SBF transcription factor, composed of Swi4 and Swi6, occurs in G1 phase in budding yeast and is limiting for commitment to division, termed Start. Here we measure size-dependence of Swi4 protein copy number under different genetic contexts using a quantitative scanning number and brightness technique. Mutation of SBF binding sites in the SWI4 promoter or disruption of SBF activation resulted in ~33-50% decrease in Swi4 accumulation rate and concordantly increased cell size at Start. Ectopic inducible expression of Swi4 in G1 phase cells increased production of Swi4 from the endogenous promoter, upregulated transcription of the G1/S regulon, and accelerated Start. Despite the potential for Swi4 positive feedback, G1 phase Swi4 accumulation was linear unless the Whi5 transcriptional repressor was inactivated. A threshold model in which Swi4 titrates SBF binding sites in G1/S promoters predicted the effects of nutrients, ploidy, and G1/S regulatory mutations on cell size. These results exemplify how transcription factor auto-production can refine a cell state transition.
Project description:Commitment to cell division at the end of G1 phase, termed Start in the budding yeast Saccharomyces cerevisiae, is strongly influenced by nutrient availability. To identify newdominant activators of Start that might operate under different nutrient conditions, we screened a genome-wide ORF overexpression library for genes that bypass a Start arrest caused by absence of the G1 cyclin Cln3 and the transcriptional activator Bck2. We recovered a hypothetical gene YLR053c, renamed NRS1 for Nitrogen-responsive Start Regulator 1, which encodes a poorly characterized 108 amino acid microprotein. Endogenous NRS1 was nuclear-localized, restricted to poor nitrogen conditions, induced upon mTORC1 inhibition, and cell cycle-regulated with a peak at Start. NRS1 interacted genetically with SWI4 and SWI6, which encode the master G1/S transcription factor complex SBF. Correspondingly, NRS1 physically interacted with Swi4 and Swi6 and was localized to G1/S promoter DNA. NRS1 exhibited inherent transactivation activity and fusion of NRS1 to the SBF inhibitor Whi5 was sufficient to suppress other Start defects. NRS1 appears to be a recently evolved microprotein that rewires the G1/S transcriptional machinery under poor nutrient conditions.
Project description:Tissue development and homeostasis depend on the balance between growth and terminal differentiation, but the mechanisms coordinating these processes remain elusive. Accumulating evidence indicates that ribosome biogenesis (RiBi) and protein synthesis, two cellular processes sustaining growth, are tightly regulated and yet can be uncoupled during stem cell differentiation. Using the Drosophila adult female germline stem cell (GSC) and larval neuroblast (NB) systems, we show that Mei-P26 and Brat, two Drosophila TRIM-NHL paralogs, are responsible for uncoupling RiBi and protein synthesis during differentiation. In differentiating cells, Mei-P26 and Brat activate the Target of rapamycin (Tor) kinase to promote translation, while concomitantly repressing RiBi. Depletion of Mei-P26 or Brat results in defective terminal differentiation, which can be rescued by ectopic activation of Tor together with suppression of RiBi. Our results indicate that uncoupling RiBi and translation activities by TRIM-NHL activity creates the conditions required for terminal differentiation.
Project description:TORC1 is a structurally and functionally conserved multiprotein complex that regulates many aspects of eukaryote growth including the synthesis and assembly of ribosomes. The protein kinase activity of this complex is responsive to environmental cues and is potently inhibited by the natural product macrolide rapamycin. Insights into how TORC1 regulates growth have been provided with the recent identification of the rapamycin-sensitive phosphoproteome in yeast. Building on these data, we show here that Sch9, an AGC family kinase and direct substrate of TORC1, promotes ribosome biogenesis (ribi) and ribosomal protein (RP) gene expression via direct inhibitory phosphorylation of three transcription repressors, Stb3, Dot6 and Tod6. Dephosphorylation of these factors allows them to recruit the RPD3L histone deactelyase complex to ribi/RP gene promoters. Since rRNA and tRNA transcription are also under its control, Sch9 appears to be well positioned to coordinately regulate transcriptional aspects of ribosome biogenesis.
Project description:TORC1 is a structurally and functionally conserved multiprotein complex that regulates many aspects of eukaryote growth including the synthesis and assembly of ribosomes. The protein kinase activity of this complex is responsive to environmental cues and is potently inhibited by the natural product macrolide rapamycin. Insights into how TORC1 regulates growth have been provided with the recent identification of the rapamycin-sensitive phosphoproteome in yeast. Building on these data, we show here that Sch9, an AGC family kinase and direct substrate of TORC1, promotes ribosome biogenesis (ribi) and ribosomal protein (RP) gene expression via direct inhibitory phosphorylation of three transcription repressors, Stb3, Dot6 and Tod6. Dephosphorylation of these factors allows them to recruit the RPD3L histone deactelyase complex to ribi/RP gene promoters. Since rRNA and tRNA transcription are also under its control, Sch9 appears to be well positioned to coordinately regulate transcriptional aspects of ribosome biogenesis.
Project description:This model is decribed in the article:
Dilution and titration of cell-cycle regulators may control cell size in budding yeast
Frank S. Heldt, Reece Lunstone, John J. Tyson, Bela Novak
PLoS Comput Biol, October 2018, 14(10), e1006548, doi: 10.1371/journal.pcbi.1006548
Abstract:
The size of a cell sets the scale for all biochemical processes within it, thereby affecting cellular fitness and survival. Hence, cell size needs to be kept within certain limits and relatively constant over multiple generations. However, how cells measure their size and use this information to regulate growth and division remains controversial. Here, we present two mechanistic mathematical models of the budding yeast (S. cerevisiae) cell cycle to investigate competing hypotheses on size control: inhibitor dilution and titration of nuclear sites. Our results suggest that an inhibitor-dilution mechanism, in which cell growth dilutes the transcriptional inhibitor Whi5 against the constant activator Cln3, can facilitate size homeostasis. This is achieved by utilising a positive feedback loop to establish a fixed size threshold for the START transition, which efficiently couples cell growth to cell cycle progression. Yet, we show that inhibitor dilution cannot reproduce the size of mutants that alter the cell’s overall ploidy and WHI5 gene copy number. By contrast, size control through titration of Cln3 against a constant number of genomic binding sites for the transcription factor SBF recapitulates both size homeostasis and the size of these mutant strains. Moreover, this model produces an imperfect ‘sizer’ behaviour in G1 and a ‘timer’ in S/G2/M, which combine to yield an ‘adder’ over the whole cell cycle; an observation recently made in experiments. Hence, our model connects these phenomenological data with the molecular details of the cell cycle, providing a systems-level perspective of budding yeast size control.
Project description:This model is decribed in the article:
Dilution and titration of cell-cycle regulators may control cell size in budding yeast
Frank S. Heldt, Reece Lunstone, John J. Tyson, Bela Novak
PLoS Comput Biol, October 2018, 14(10), e1006548, doi: 10.1371/journal.pcbi.1006548
Abstract:
The size of a cell sets the scale for all biochemical processes within it, thereby affecting cellular fitness and survival. Hence, cell size needs to be kept within certain limits and relatively constant over multiple generations. However, how cells measure their size and use this information to regulate growth and division remains controversial. Here, we present two mechanistic mathematical models of the budding yeast (S. cerevisiae) cell cycle to investigate competing hypotheses on size control: inhibitor dilution and titration of nuclear sites. Our results suggest that an inhibitor-dilution mechanism, in which cell growth dilutes the transcriptional inhibitor Whi5 against the constant activator Cln3, can facilitate size homeostasis. This is achieved by utilising a positive feedback loop to establish a fixed size threshold for the START transition, which efficiently couples cell growth to cell cycle progression. Yet, we show that inhibitor dilution cannot reproduce the size of mutants that alter the cell’s overall ploidy and WHI5 gene copy number. By contrast, size control through titration of Cln3 against a constant number of genomic binding sites for the transcription factor SBF recapitulates both size homeostasis and the size of these mutant strains. Moreover, this model produces an imperfect ‘sizer’ behaviour in G1 and a ‘timer’ in S/G2/M, which combine to yield an ‘adder’ over the whole cell cycle; an observation recently made in experiments. Hence, our model connects these phenomenological data with the molecular details of the cell cycle, providing a systems-level perspective of budding yeast size control.
Project description:In the unicellular eukaryote Saccharomyces cerevisiae, Cln3–cyclin-dependent kinase activity enables Start, the irreversible commitment to the cell division cycle. However, the concentration of Cln3 has been paradoxically considered to remain constant during G1, due to the presumed scaling of its production rate with cell size dynamics. Measuring metabolic and biosynthetic activity during cell cycle progression in single cells, we found that cells exhibit pulses in their protein production rate. Rather than scaling with cell size dynamics, these pulses follow the intrinsic metabolic dynamics, peaking around Start. Using a viral- based bicistronic construct and targeted proteomics to measure Cln3 at the single-cell and population levels, we show that the differential scaling between protein production and cell size leads to a temporal increase in Cln3 concentration, and passage through Start. This differential scaling causes Start in both daughter and mother cells across growth conditions. Thus, uncou- pling between two fundamental physiological parameters drives cell cycle commitment.