Transcription of a 5' extended mRNA isoform directs dynamic chromatin changes and interference of a downstream promoter.

Cell differentiation programs require dynamic regulation of gene expression. During meiotic prophase in Saccharomyces cerevisiae, expression of the kinetochore complex subunit Ndc80 is downregulated by a 5' extended long undecoded NDC80 transcript isoform. Here we demonstrate a transcriptional interference mechanism that is responsible for inhibiting expression of the coding NDC80 mRNA isoform. Transcription from a distal NDC80 promoter directs Set1-dependent histone H3K4 dimethylation and Set2-dependent H3K36 trimethylation to establish a repressive chromatin state in the downstream canonical NDC80 promoter. As a consequence, NDC80 expression is repressed during meiotic prophase. The transcriptional mechanism described here is rapidly reversible, adaptable to fine-tune gene expression, and relies on Set2 and the Set3 histone deacetylase complex. Thus, expression of a 5' extended mRNA isoform causes transcriptional interference at the downstream promoter. We demonstrate that this is an effective mechanism to promote dynamic changes in gene expression during cell differentiation

Kinetochore inactivation by expression of a repressive mRNA.

Differentiation programs such as meiosis depend on extensive gene regulation to mediate cellular morphogenesis. Meiosis requires transient removal of the outer kinetochore, the complex that connects microtubules to chromosomes. How the meiotic gene expression program temporally restricts kinetochore function is unknown. We discovered that in budding yeast, kinetochore inactivation occurs by reducing the abundance of a limiting subunit, Ndc80. Furthermore, we uncovered an integrated mechanism that acts at the transcriptional and translational level to repress NDC80 expression. Central to this mechanism is the developmentally controlled transcription of an alternate NDC80 mRNA isoform, which itself cannot produce protein due to regulatory upstream ORFs in its extended 5' leader. Instead, transcription of this isoform represses the canonical NDC80 mRNA expression in cis, thereby inhibiting Ndc80 protein synthesis. This model of gene regulation raises the intriguing notion that transcription of an mRNA, despite carrying a canonical coding sequence, can directly cause gene repression

Tight cooperation between Mot1p and NC2β in regulating genome-wide transcription, repression of transcription following heat shock induction and genetic interaction with SAGA

TATA-binding protein (TBP) is central to the regulation of eukaryotic transcription initiation. Recruitment of TBP to target genes can be positively regulated by one of two basal transcription factor complexes: SAGA or TFIID. Negative regulation of TBP promoter association can be performed by Mot1p or the NC2 complex. Recent evidence suggests that Mot1p, NC2 and TBP form a DNA-dependent protein complex. Here, we compare the functions of Mot1p and NC2βduring basal and activated transcription using the anchor-away technique for conditional nuclear depletion. Genome-wide expression analysis indicates that both proteins regulate a highly similar set of genes. Upregulated genes were enriched for SAGA occupancy, while downregulated genes preferred TFIID binding. Mot1p and NC2β depletion during heat shock resulted in failure to downregulate gene expression after initial activation, which was accompanied by increased TBP and RNA pol II promoter occupancies. Depletion of Mot1p or NC2β displayed preferential synthetic lethality with the TBP-interaction module of SAGA. Our results support the model that Mot1p and NC2β directly cooperate in vivo to regulate TBP function, and that they are involved in maintaining basal expression levels as well as in resetting gene expression after induction by stress

Nutrient Control of Yeast Gametogenesis Is Mediated by TORC1, PKA and Energy Availability

Cell fate choices are tightly controlled by the interplay between intrinsic and extrinsic signals, and gene regulatory networks. In Saccharomyces cerevisiae, the decision to enter into gametogenesis or sporulation is dictated by mating type and nutrient availability. These signals regulate the expression of the master regulator of gametogenesis, IME1. Here we describe how nutrients control IME1 expression. We find that protein kinase A (PKA) and target of rapamycin complex I (TORC1) signalling mediate nutrient regulation of IME1 expression. Inhibiting both pathways is sufficient to induce IME1 expression and complete sporulation in nutrient-rich conditions. Our ability to induce sporulation under nutrient rich conditions allowed us to show that respiration and fermentation are interchangeable energy sources for IME1 transcription. Furthermore, we find that TORC1 can both promote and inhibit gametogenesis. Down-regulation of TORC1 is required to activate IME1. However, complete inactivation of TORC1 inhibits IME1 induction, indicating that an intermediate level of TORC1 signalling is required for entry into sporulation. Finally, we show that the transcriptional repressor Tup1 binds and represses the IME1 promoter when nutrients are ample, but is released from the IME1 promoter when both PKA and TORC1 are inhibited. Collectively our data demonstrate that nutrient control of entry into sporulation is mediated by a combination of energy availability, TORC1 and PKA activities that converge on the IME1 promoter.Jane Coffin Childs Memorial Fund for Medical ResearchEuropean Molecular Biology Organization (EMBO Long-term Fellowship)Weizmann Institute of Science (Israel National Postdoctoral Program for Advancing Women in Science)National Institutes of Health (U.S.) (NIH grant GM62207)Francis Crick Institut

Single cell quantification of <i>IME1</i>.

<p>(A) Representative images used for the analyses of <i>IME1</i> and <i>ACT1</i> transcript levels in single cells. Cells harbouring <i>tpk1-as</i> (FW1762) were grown in YPD overnight and were either shifted to SPO, or diluted into fresh YPD plus rapamycin, 1NM-PP1 or 1NM-PP1/rapamycin. Cells were fixed, hybridized with probes directed against <i>IME1</i> (AF594) and <i>ACT1</i> (Cy5), and were imaged (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006075#sec018" target="_blank">Materials and Methods</a> for details). <i>ACT1</i> was used as an internal control and only <i>ACT1</i> positive cells were selected for the analysis. (B) Mean of <i>IME1</i> and <i>ACT1</i> transcripts’ number among single cells as described in A. Cells harbouring <i>tpk1-as</i> (FW1762) were grown in YPD overnight, diluted into fresh YPD plus 1NM-PP1 or 1NM-PP1/ rapamycin and samples were taken at the indicated time points. Cells were imaged and quantified (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006075#sec018" target="_blank">Materials and Methods</a> for details). At least, 60 cells (n = 60) were quantified per time point. The standard error of the mean of at least two biological experiments is shown. (C) Distribution of <i>IME1</i> and <i>ACT1</i> transcripts among single cells grown in YPD as described in A and B. Cells were binned in five classes of transcript levels (0 to 5; 6 to 10; 11 to 15; 16 to 20 and 21 or more transcripts per cell) and the percentages of each class contributing to the total population are indicated. At least, 60 cells (n = 60) were quantified per time point. The standard error of the mean of at least two biological experiments is shown. (D-G) Similar to C except that cells were grown in YPD overnight and transferred to YPD plus rapamycin (D), YPD plus 1NM-PP1 (E), YPD plus 1NMPP and rapamycin (F), or SPO (G). Samples were taken at the indicated time points. Cells were binned in five classes of transcript levels (0 to 5; 6 to 10; 11 to 15; 16 to 20 and 21 or more transcripts per cell) and the percentages of each class contributing to the total population are indicated.</p

Scheme of how PKA and TORC1 signalling controls <i>IME1</i> and entry into sporulation.

<p>Scheme of how PKA and TORC1 signalling controls <i>IME1</i> and entry into sporulation.</p

Sch9 controls <i>IME1</i> and entry into sporulation.

<p>(A) <i>IME1</i> promoter activity was measured in strains harbouring <i>tpk1-as</i> and <i>pIME1-LacZ</i> (control, FW1976) and <i>sch9</i>Δ (FW2498). Cells were grown in YPD overnight shifted to SPO and samples were taken at the indicated time points. β-galactosidase activity was measured using a quantitative liquid ortho-Nitrophenyl-β-galactoside (ONPG) assay (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006075#sec018" target="_blank">Materials and Methods</a> for details). The promoter activities are displayed in Miller Units, and the standard error of the mean of at least two biological experiments is shown. (B) Similar to A except that cells were diluted into YPD with 1NM-PP1 or 1NM-PP1 plus rapamycin. (C) <i>IME1</i> transcript distribution among single cells in control (FW1762), <i>tco89</i>Δ (FW1966) and <i>sch9</i>Δ (FW2437) strains (all in <i>tpk1-as</i> background). After 8 hours treatment with 1NM-PP1, cells were fixed, hybridized with probes directed against <i>IME1</i> (AF594) and <i>ACT1</i> (Cy5). Finally, cells were imaged and transcript levels were quantified (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006075#sec018" target="_blank">Materials and Methods</a> for details). <i>ACT1</i> was used as an internal control and only cells positive for <i>ACT1</i> were selected for the analysis. At least 120 cells (n = 120) were quantified. The error bars represent the standard error of the mean of two biological experiments. (D) Same treatments and strains as A and B, but here the percentage of cells that underwent meiotic divisions (MI+MII) was determined after 48 hours of treatment.</p

Tup1 binds, represses, and mediates nutrient control of the <i>IME1</i> promoter.

<p>(A) Data taken from <i>Rizzo et al</i>. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006075#pgen.1006075.ref039" target="_blank">39</a>] showing the nucleosome distribution at the <i>IME1</i> locus in control (closed circles) and <i>tup1</i>Δ mutant (open squares) cells. The x-axis shows the coordinates of the <i>IME1</i> locus at chromosome X in kilobases (kb), and y-axis shows the nucleosome occupancy score as described in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006075#pgen.1006075.ref039" target="_blank">39</a>]. The position of each dot or point on the graph represents the coordinate of the nucleosome dyad center at the <i>IME1</i> locus. Regions lacking dots are depleted for nucleosomes. (B) Binding of Tup1 to the <i>IME1</i> promoter measured by chromatin immunoprecipitation. Diploid cells harbouring <i>tpk1-as</i> (control, FW1762) and <i>tpk1-as</i> plus Tup1 tagged at the C-terminus with 3xV5 (FW3078) were grown in rich medium (YPD) to mid-log and cross-linked with formaldehyde. Tup1 was immunoprecipitated from chromatin extracts. The recovered DNA was quantified by real-time PCR with 9 different primer sets across the <i>IME1</i> promoter and gene. Signals were normalized to the silent mating type locus (<i>HMR</i>), which does not bind Tup1. The error bars represent the standard error of the mean of two biological experiments. (C) Tup1 binding to the <i>IME1</i> promoter was measured by chromatin immunoprecipitation in control (FW3078) and <i>tco89</i>Δ (FW3096) cells. Cells were grown in YPD and shifted to YPD and were either untreated or treated with rapamycin, 1NM-PP1 or both compounds. Tup1 tagged with 3xV5 epitope was immunoprecipitated from chromatin extracts. The recovered DNA was quantified by real-time PCR with primer set five corresponding to middle of the <i>IME1</i> promoter. Signals were normalized to the silent mating type locus (<i>HMR</i>), which does not bind Tup1. The error bars represent the standard error of the mean of two biological experiments. (D) <i>IME1</i> promoter activity upon depletion of Tup1. Cells harbouring <i>IME1</i> promoter fused to LacZ (<i>pIME1-LacZ</i>) and expressing either Tup1 fused to the auxin induced degron (<i>TUP1-AID</i>) (FW3188) or <i>TUP1-AID</i> together with <i>pTEF1-osTIR1</i> (FW3184) were grown in YPD overnight. Cells were diluted to fresh YPD, either untreated or treated with indole-3-acetic acid (<i>IAA</i>) (500 μM), and samples were taken at the indicated time points. β-galactosidase activity was measured using a quantitative liquid ortho-Nitrophenyl-β-galactoside (ONPG) assay (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006075#sec018" target="_blank">Materials and Methods</a> for details). The promoter activities are displayed in Miller Units, and the standard error of the mean of at least two biological experiments is shown. (E) Comparison of <i>IME1</i> promoter activity during different treatments and growth conditions. Diploid cells harbouring <i>tpk1-as</i> and <i>pIME1-LacZ</i> (FW1976) were grown overnight in YPD, and diluted to YPD with 1NM-PP1 and rapamycin or cells were washes with water before transferred to sporulation medium. Diploid cells harbouring <i>TUP1-AID</i> and <i>pTEF1-osTIR1</i> (FW3188) were grown and treated as described D. Samples were taken at the indicated time points, and β-galactosidase activity was measured as described in D.</p

TORC1 activity is required for sporulation.

<p>(A) Cells (FW1762) were treated with different concentrations of rapamycin, and doubling times (left panel) as well as the fraction of cells that underwent meiosis (right panel) were quantified. Left panel, cells were grown in YPD, shifted to YPD plus 0, 5, 20, or 1000 ng/ml rapamycin and doubling times were measured during exponential growth. Right panel, cells were diluted into YPD plus PKA inhibitors and treated with different concentrations of rapamycin as indicated. DAPI masses were counted after 48 hours of treatment. (B) Control (FW1762) and <i>KOG-AID</i>/<i>pTEF1-osTIR1</i> (FW1904) cells harbouring <i>tpk1-as</i> were grown in YPD overnight, diluted into fresh YPD and treated with 1NM-PP1, rapamycin or IAA. The nuclei number in cells was counted after 48 hours of treatment by DAPI staining, and percentage of cells that underwent meiosis (MI+MII) was quantified. (C) Quantification of <i>IME1</i> mRNA levels in control (FW1762) and <i>KOG1-AID</i>/<i>pTEF1-osTIR1</i> (FW1904) cells harbouring <i>tpk1-as</i> and treated with 1NM-PP1. <i>KOG1-AID</i>/<i>pTEF1-osTIR1</i> cells were also treated with IAA. Samples were taken at the indicated time points. Total RNA was isolated, reverse transcribed, and <i>IME1</i> mRNA levels were measured by quantitative PCR. Signals were normalized to <i>ACT1</i> levels. The standard error of the mean of at least two biological experiments is shown. (D) Percentage of cells that underwent meiotic divisions (MI+MII) was determined in gene deletion strains, all harbouring <i>tpk1-as</i> and <i>pIME1-LacZ</i> (FW1976, control). The following gene deletion mutants were used for the analyses: control (FW1976), <i>tco89</i>Δ (FW2154), <i>gtr1</i>Δ (FW2164) or <i>tor1</i>Δ (FW2162). Samples were grown in YPD medium, fixed, and DAPI masses were counted at 48 hours after treatment with 1NM-PP1 or with 1NM-PP1 and rapamycin. (E) <i>IME1</i> promoter activity was measured in strains described in D. Cells were grown in YPD overnight, diluted into YPD plus 1NMPP1 and/or rapamycin, and samples were taken after 0, 4, 8, and 12 hours. β-galactosidase activity was measured using a quantitative liquid ortho-Nitrophenyl-β-galactoside (ONPG) assay (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006075#sec018" target="_blank">Materials and Methods</a> for details). The promoter activities are displayed in Miller Units, and the standard error of the mean of at least two biological experiments is shown. (F) <i>IME1</i> promoter activity was measured as described in E for control (FW1976) and <i>tco89</i>Δ (FW2154) strains. Cells were grown in YPD overnight, diluted into YPD plus 1NMPP1 and/or rapamycin, and samples were taken after 0, 2, 4, 6, 8, 10, 12 and 24 hours. (G) Kinetics of meiotic division (MI+MII) of strains and treatments described in F. Samples were taken at the indicated time points, fixed, and DAPI masses were counted.</p