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N6-methyladenosine (m6A) RNA modification impacts mRNA fate primarily via reader proteins, which dictate processes in development, stress, and disease. Yet little is known about m6A function in Saccharomyces cerevisiae, which occurs solely during early meiosis. Here we perform a multifaceted analysis of the m6A reader protein Pho92/Mrb1. Cross-linking immunoprecipitation analysis reveals that Pho92 associates with the 3’end of meiotic mRNAs in both an m6A-dependent and independent manner. Within cells, Pho92 transitions from the nucleus to the cytoplasm, and associates with translating ribosomes. In the nucleus Pho92 associates with target loci through its interaction with transcriptional elongator Paf1C. Functionally, we show that Pho92 promotes and links protein synthesis to mRNA decay. As such, the Pho92-mediated m6A-mRNA decay is contingent on active translation and the CCR4-NOT complex. We propose that the m6A reader Pho92 is loaded co-transcriptionally to facilitate protein synthesis and subsequent decay of m6A modified transcripts, and thereby promotes meiosis.

The directionality of gene promoters—the ratio of protein-coding over divergent noncoding transcription—is highly variable. How promoter directionality is controlled remains poorly understood. Here, we show that the chromatin remodelling complex RSC and general regulatory factors (GRFs) dictate promoter directionality by attenuating divergent transcription relative to protein-coding transcription. At gene promoters that are highly directional, depletion of RSC leads to a relative increase in divergent noncoding transcription and thus to a decrease in promoter directionality. We find that RSC has a modest effect on nucleosome positioning upstream in promoters at the sites of divergent transcription. These promoters are also enriched for the binding of GRFs such as Reb1 and Abf1. Ectopic targeting of divergent transcription initiation sites with GRFs or the dCas9 DNA-binding protein suppresses divergent transcription. Our data suggest that RSC and GRFs play a pervasive role in limiting divergent transcription relative to coding direction transcription. We propose that any DNA-binding factor, when stably associated with cryptic transcription start sites, forms a barrier which represses divergent transcription, thereby promoting promoter directionality.

N6- methyladenosine (m6A) RNA modification impacts mRNA fate primarily via reader proteins, which dictate processes in development, stress, and disease. Yet little is known about m6A function in Saccharomyces cerevisiae, which occurs solely during early meiosis. Here, we perform a multifaceted analysis of the m6A reader protein Pho92/Mrb1. Cross-linking immunoprecipitation analysis reveals that Pho92 associates with the 3’end of meiotic mRNAs in both an m6A-dependent and independent manner. Within cells, Pho92 transitions from the nucleus to the cytoplasm, and associates with translating ribosomes. In the nucleus Pho92 associates with target loci through its interaction with transcriptional elongator Paf1C. Functionally, we show that Pho92 promotes and links protein synthesis to mRNA decay. As such, the Pho92-mediated m6A-mRNA decay is contingent on active translation and the CCR4-NOT complex. We propose that the m6A reader Pho92 is loaded co-transcriptionally to facilitate protein synthesis and subsequent decay of m6A modified transcripts, and thereby promotes meiosis.

Background The start and end sites of messenger RNAs (TSSs and TESs) are highly regulated, often in a cell-type-specific manner. Yet the contribution of transcript diversity in regulating gene expression remains largely elusive. We perform an integrative analysis of multiple highly synchronized cell-fate transitions and quantitative genomic techniques in Saccharomyces cerevisiae to identify regulatory functions associated with transcribing alternative isoforms. Results Cell-fate transitions feature widespread elevated expression of alternative TSS and, to a lesser degree, TES usage. These dynamically regulated alternative TSSs are located mostly upstream of canonical TSSs, but also within gene bodies possibly encoding for protein isoforms. Increased upstream alternative TSS usage is linked to various effects on canonical TSS levels, which range from co-activation to repression. We identified two key features linked to these outcomes: an interplay between alternative and canonical promoter strengths, and distance between alternative and canonical TSSs. These two regulatory properties give a plausible explanation of how locally transcribed alternative TSSs control gene transcription. Additionally, we find that specific chromatin modifiers Set2, Set3, and FACT play an important role in mediating gene repression via alternative TSSs, further supporting that the act of upstream transcription drives the local changes in gene transcription. Conclusions The integrative analysis of multiple cell-fate transitions suggests the presence of a regulatory control system of alternative TSSs that is important for dynamic tuning of gene expression. Our work provides a framework for understanding how TSS heterogeneity governs eukaryotic gene expression, particularly during cell-fate changes.

N6-methyladenosine (m6A) is the most prevalent modification of messenger RNA in mammals. To interrogate its functions and dynamics, there is a critical need to quantify m6A at three levels: site, gene and sample. Current approaches address these needs in a limited manner. Here we develop m6A-seq2, relying on multiplexed m6A-immunoprecipitation of barcoded and pooled samples. m6A-seq2 allows a big increase in throughput while reducing technical variability, requirements of input material and cost. m6A-seq2 is furthermore uniquely capable of providing sample-level relative quantitations of m6A, serving as an orthogonal alternative to mass spectrometry-based approaches. Finally, we develop a computational approach for gene-level quantitation of m6A. We demonstrate that using this metric, roughly 30% of the variability in RNA half life in mouse embryonic stem cells can be explained, establishing m6A as a main driver of RNA stability. m6A-seq2 thus provides an experimental and analytic framework for dissecting m6A-mediated regulation at three different levels.

Summary: LUTIs (Long Undecoded Transcript Isoforms) are 5′-extended and poorly translated mRNAs that can downregulate transcription from promoters more proximal to a gene’s coding sequence (CDS). In this protocol, polyA RNA is extracted from budding yeast cells undergoing highly synchronized meiosis. Using a combination of long-read direct RNA sequencing and transcript leader sequencing (TL-seq), meiosis-specific LUTIs are systematically identified. Following identification, TL-seq is used to quantify the abundance of both LUTI and the more canonical gene-proximal (PROX) transcripts.For complete details on the use and execution of this protocol, please refer to Tresenrider et al. (2021).

Intrinsic signals and cues from the external environment drive cell fate decisions. In budding yeast, the decision to enter meiosis is controlled by nutrient and mating-type signals that regulate expression of the master transcription factor for meiotic entry, IME1. How nutrient signals control IME1 expression remains poorly understood. Here we show that IME1 transcription is regulated by multiple sequence-specific transcription factors that mediate association of Tup1-Cyc8 co-repressor to its promoter. We find that at least eight transcription factors bind the IME1 promoter when nutrients are ample. Remarkably, association of these transcription factors is highly regulated by different nutrient cues. Mutant cells lacking three transcription factors (Sok2/Phd1/Yap6) displayed reduced Tup1-Cyc8 association, increased IME1 expression and earlier onset of meiosis. Our data demonstrate that the promoter of a master regulator is primed for rapid activation while repression by multiple transcription factors mediating Tup1-Cyc8 recruitment dictates the fate decision to enter meiosis.

Many long noncoding RNAs (lncRNAs) act in cis through transcription-coupled chromatin alterations that drive changes in local gene expression. How some cis-acting lncRNAs promote and others repress gene expression remains poorly understood. Here we report that in S. cerevisiae transcription levels of the lncRNA IRT2, located upstream in the promoter of the inducer of meiosis gene, regulate opposing chromatin and transcription states. Low IRT2 transcription displays enhancer RNA-like features. At these levels, IRT2 promotes histone exchange delivering acetylated histone H3 lysine 56 to chromatin thereby facilitating recruitment of a transcription factor and consequently activating transcription. Conversely, increasing IRT2 transcription enhances chromatin assembly and transcriptional repression. The opposing functions of IRT2 direct a regulatory circuit, which ensures that cells expressing opposite, but not one of either, mating-type loci enter meiosis. Our data demonstrate that the transcription levels of an lncRNA are key to controlling gene expression and cell fate outcomes.

SUMMARYLong Undecoded Transcript Isoforms (LUTIs) represent a class of non-canonical mRNAs that downregulate gene expression through the combined act of transcriptional and translational repression. While single gene studies revealed some important aspects of LUTI-based repression, how these features impact gene regulation at a global scale is unknown. By using transcript leader and direct RNA sequencing, here we identify 74 LUTI candidates that are expressed specifically during meiotic prophase. Translational repression of these candidates is ubiquitous and dependent on upstream open reading frames. However, LUTI-based transcriptional repression is highly variable. In only 50% of the cases, LUTI transcription causes downregulation of the protein-coding transcript isoform. Higher LUTI expression, enrichment of histone 3 lysine 36 trimethylation, and changes in nucleosome position are the strongest predictors of LUTI-based transcriptional repression. We conclude that LUTIs downregulate gene expression in a manner that integrates translational repression, chromatin state changes, and the magnitude of LUTI expression.

Summary Long undecoded transcript isoforms (LUTIs) represent a class of non-canonical mRNAs that downregulate gene expression through the combined act of transcriptional and translational repression. While single gene studies revealed important aspects of LUTI-based repression, how these features affect gene regulation on a global scale is unknown. Using transcript leader and direct RNA sequencing, here, we identify 74 LUTI candidates that are specifically induced in meiotic prophase. Translational repression of these candidates appears to be ubiquitous and is dependent on upstream open reading frames. However, LUTI-based transcriptional repression is variable. In only 50% of the cases, LUTI transcription causes downregulation of the protein-coding transcript isoform. Higher LUTI expression, enrichment of histone 3 lysine 36 trimethylation, and changes in nucleosome position are the strongest predictors of LUTI-based transcriptional repression. We conclude that LUTIs downregulate gene expression in a manner that integrates translational repression, chromatin state changes, and the magnitude of LUTI expression., Graphical abstract, Highlights • Long-read RNA-seq reveals 5′-extended mRNAs as candidate LUTIs in yeast meiosis • Majority of the LUTIs are regulated by a cell-fate-instructive transcription factor • LUTI-based translational repression depends on uORFs • Chromatin changes and LUTI expression predict LUTI-based transcriptional repression, Tresenrider et al. conducted a genome-wide study to dissect an unconventional gene regulatory mechanism whereby transcription factors could function as repressors by inducing non-canonical mRNAs called LUTIs. Their findings reveal that LUTI-based gene repression depends on a combination of factors, including translational repression, chromatin state changes, and LUTI expression.

Yeast cells enter and undergo gametogenesis relatively asynchronously, making it technically challenging to perform stage-specific genomic and biochemical analyses. Cell-to-cell variation in the expression of the master regulator of entry into sporulation, IME1, has been implicated to be the underlying cause of asynchronous sporulation. Here, we find that timing of IME1 expression is of critical importance for inducing cells to undergo sporulation synchronously. When we force expression of IME1 from an inducible promoter in cells incubated in sporulation medium for 2 hr, the vast majority of cells exhibit synchrony during premeiotic DNA replication and meiotic divisions. Inducing IME1 expression too early or too late affects the synchrony of sporulation. Surprisingly, our approach for synchronous sporulation does not require growth in acetate-containing medium, but can be achieved in cells grown in rich medium until saturation. Our system requires solely IME1, because the expression of the N6-methyladenosine methyltransferase IME4, another key regulator of early sporulation, is controlled by IME1 itself. The approach described here can be combined easily with other stage-specific synchronization methods, and thereby applied to study specific stages of sporulation, or the complete sporulation program.

In eukaryotes, divergent transcription is a major source of noncoding RNAs. Recent studies have uncovered that in yeast, the transcription factor Rap1 restricts transcription in the divergent direction and thereby controls promoter directionality. Here, we summarize these findings, propose regulatory principles, and discuss the implications for eukaryotic gene regulation.

Summary Transcription through noncoding regions of the genome is pervasive. How these transcription events regulate gene expression remains poorly understood. Here, we report that, in S. cerevisiae, the levels of transcription through a noncoding region, IRT2, located upstream in the promoter of the inducer of meiosis, IME1, regulate opposing chromatin and transcription states. At low levels, the act of IRT2 transcription promotes histone exchange, delivering acetylated histone H3 lysine 56 to chromatin locally. The subsequent open chromatin state directs transcription factor recruitment and induces downstream transcription to repress the IME1 promoter and meiotic entry. Conversely, increasing transcription turns IRT2 into a repressor by promoting transcription-coupled chromatin assembly. The two opposing functions of IRT2 transcription shape a regulatory circuit, which ensures a robust cell-type-specific control of IME1 expression and yeast meiosis. Our data illustrate how intergenic transcription levels are key to controlling local chromatin state, gene expression, and cell fate outcomes., Graphical Abstract, Highlights • Transcription levels of the lncRNA IRT2 regulate opposing local transcription states • Low levels of IRT2 promote H3K56ac, chromatin disassembly, and TF recruitment • Increasing IRT2 levels promote chromatin assembly and transcriptional repression • IRT2 transcription shapes the regulatory circuit for cell-type-specific control of yeast meiosis, Moretto et al. demonstrate that transcription levels of a lncRNA named IRT2 can have opposing effects on local gene transcription, thereby controlling entry into yeast meiosis. Low levels of IRT2 promote H3K56ac, chromatin disassembly, transcription factor recruitment, and, thus, transcriptional activation. Increasing levels promote chromatin assembly and, consequently, transcriptional repression.

An amendment to this paper has been published and can be accessed via the original article.

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.

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.

Cell size varies greatly between cell types, yet within a specific cell type and growth condition, cell size is narrowly distributed. Why maintenance of a cell-type specific cell size is important remains poorly understood. Here we show that growing budding yeast and primary mammalian cells beyond a certain size impairs gene induction, cell-cycle progression, and cell signaling. These defects are due to the inability of large cells to scale nucleic acid and protein biosynthesis in accordance with cell volume increase, which effectively leads to cytoplasm dilution. We further show that loss of scaling beyond a certain critical size is due to DNA becoming limiting. Based on the observation that senescent cells are large and exhibit many of the phenotypes of large cells, we propose that the range of DNA:cytoplasm ratio that supports optimal cell function is limited and that ratios outside these bounds contribute to aging.

Cell fate decisions are controlled by multiple cell-intrinsic and -extrinsic factors. In budding yeast, the decision to enter gametogenesis or sporulation is dictated by nutrient availability and mating type. Recently, we showed that in diploid cells harbouring opposite mating types (MATa and MATα), the protein kinase A (PKA) and target of rapamycin complex I (TORC1) signalling pathways integrate at the promoter of the master regulatory transcription factor IME1 to control sporulation via nutrient availability (Weidberg, et al. 2016). In cells with a single mating type (MATa or MATα), however, IME1 is repressed by transcription through the IME1 promoter of a long non-coding RNA called IRT1, which prevents this cell type from undergoing sporulation. Here, we investigated the role of nutrient signalling in mating-type control of IME1. We find that expression of IRT1, like IME1 itself, depends on nutrient availability and the activities of PKA and TORC1. IRT1 transcription is repressed when nutrients are ample and TORC1 and PKA are active. In contrast, inhibition of PKA and TORC1 is sufficient to recruit Rme1 to the IRT1 promoter and induce IRT1-mediated repression of IME1. Finally, we provide evidence that IRT1 and IME1 are co-repressed by the Tup1-Cyc8 complex when nutrients are ample. Thus, in cells with a single mating-type nutrient availability regulates mating-type repression of IME1 and sporulation. Our results indicate that there is a hierarchy between nutrient and mating-type signals in controlling the decision to enter sporulation.

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 Research, European 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 Institute

Gametogenesis is a fundamental aspect of sexual reproduction in eukaryotes. In the unicellular fungiSaccharomyces cerevisiae(budding yeast) andSchizosaccharomyces pombe(fission yeast), where this developmental programme has been extensively studied, entry into gametogenesis requires the convergence of multiple signals on the promoter of a master regulator. Starvation signals and cellular mating-type information promote the transcription of cell fate inducers, which in turn initiate a transcriptional cascade that propels a unique type of cell division, meiosis, and gamete morphogenesis. Here, we will provide an overview of how entry into gametogenesis is initiated in budding and fission yeast and discuss potential conserved features in the germ cell development of higher eukaryotes.

Summary Many active eukaryotic gene promoters exhibit divergent noncoding transcription, but the mechanisms restricting expression of these transcripts are not well understood. Here, we demonstrate how a sequence-specific transcription factor represses divergent noncoding transcription at highly expressed genes in yeast. We find that depletion of the transcription factor Rap1 induces noncoding transcription in a large fraction of Rap1-regulated gene promoters. Specifically, Rap1 prevents transcription initiation at cryptic promoters near its binding sites, which is uncoupled from transcription regulation in the protein-coding direction. We further provide evidence that Rap1 acts independently of previously described chromatin-based mechanisms to repress cryptic or divergent transcription. Finally, we show that divergent transcription in the absence of Rap1 is elicited by the RSC chromatin remodeler. We propose that a sequence-specific transcription factor limits access of basal transcription machinery to regulatory elements and adjacent sequences that act as divergent cryptic promoters, thereby providing directionality toward productive transcription., Graphical Abstract, Highlights • Expression of divergent noncoding RNAs is repressed by Rap1 • Rap1 prevents initiation of divergent noncoding transcription near its binding sites • Rap1 provides directionality toward productive transcription, Wu et al. characterize how divergent noncoding RNAs are repressed at highly expressed gene promoters in yeast. The authors identified that the sequence-specific transcription factor Rap1 restricts transcription initiation in the divergent direction locally near its binding site, thereby controlling promoter directionality.

Transcription regulation in eukaryotes involves rapid recruitment and proper assembly of transcription factors at gene promoters. To determine the dynamics of the transcription machinery on DNA, we used a differential chromatin immunoprecipitation procedure coupled to whole-genome microarray detection in Saccharomyces cerevisiae. We find that TATA-binding protein (TBP) turnover is low at RNA polymerase I (Pol I) promoters. Whereas RNA polymerase III (Pol III) promoters represent an intermediate case, TBP turnover is high at RNA polymerase II (Pol II) promoters. Within these promoters, the highest turnover correlates with binding of the Spt-Ada-Gcn5 acetyltransferase complex (SAGA) coactivator, Mot1p dependence and presence of a canonical TATA box. In contrast, slow turnover Pol II promoters depend on TFIID and on the gene-specific factor, Rap1p. Together this shows that TBP turnover is regulated by protein factors rather than DNA sequence and argues that TBP turnover is an important determinant in regulating gene expression.

Promoter recognition by TATA-binding protein (TBP) is an essential step in the initiation of RNA polymerase II (pol II) mediated transcription. Genetic and biochemical studies in yeast have shown that Mot1p and NC2 play important roles in inhibiting TBP activity. To understand how TBP activity is regulated in a genome-wide manner, we profiled the binding of TBP, NC2, Mot1p, TFIID, SAGA, and pol II across the yeast genome using chromatin immunoprecipitation (ChIP)–chip for cells in exponential growth and during reprogramming of transcription. We find that TBP, NC2, and Mot1p colocalize at transcriptionally active pol II core promoters. Relative binding of NC2α and Mot1p is higher at TATA promoters, whereas NC2β has a preference for TATA-less promoters. In line with the ChIP–chip data, we isolated a stable TBP–NC2–Mot1p–DNA complex from chromatin extracts. ATP hydrolysis releases NC2 and DNA from the Mot1p–TBP complex. In vivo experiments indicate that promoter dissociation of TBP and NC2 is highly dynamic, which is dependent on Mot1p function. Based on these results, we propose that NC2 and Mot1p cooperate to dynamically restrict TBP activity on transcribed promoters.

We previously showed that the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex is recruited to the activated HXT2 and HXT4 genes and plays a role in the association of TBP-associated factors. Using the HXT2 and HXT4 genes, we now present evidence for a functional link between Snf1p-dependent activation, recruitment of the SAGA complex, histone H3 removal, and H3 acetylation. Recruitment of the SAGA complex is dependent on the release of Ssn6p-Tup1p repression by Snf1p. In addition, we found that the Gcn5p subunit of the SAGA complex preferentially acetylates histone H3K18 on the HXT2 and HXT4 promoters and that Gcn5p activity is required for removal of histone H3 from the HXT4 promoter TATA region. In contrast, histone H3 removal from the HXT2 promoter does not require Gcn5p. In conclusion, although similar protein complexes are involved, induction of HXT2 and HXT4 displays important mechanistic differences.

Met-overaccumulating mutants provide a powerful genetic tool for examining both the regulation of the Met biosynthetic pathway and in vivo developmental responses of gene expression to altered Met levels. We have previously reported the identification of two Arabidopsis thaliana Met over-accumulation (mto) mutants, mto1-1 and mto2-1, that carry mutations in the genes encoding cystathionine gamma-synthase (CGS) and threonine synthase (TS), respectively. A third mutant, mto3-1, has recently been reported to carry a mutation in the gene encoding S-adenosylmethionine synthetase 3 (SAMS3). Here, we report the isolation of a new ethionine-resistant A. thaliana mutant that over-accumulates soluble Met approximately 20-fold in young rosettes. The causal mutation was determined to be a single, recessive mutation that was mapped to chromosome 3. Sequence analysis identified a single nucleotide change in the gene encoding SAMS3 that was distinct from the mto3-1 mutation and altered the amino acid sequence of the enzyme active site. This mutation was therefore referred to as mto3-2. Although Met over-accumulation in the mto3-2 mutant was similar to that in the mto2-1 mutant, CGS mRNA levels did not respond to the mto3-2 mutation and were similar to that in equivalent wild-type plants.

Production of haploid gametes from diploid progenitor cells is mediated by a specialized cell division, meiosis, where two divisions, meiosis I and II, follow a single S phase. Errors in progression from meiosis I to meiosis II lead to aneuploid and polyploid gametes, but the regulatory mechanisms controlling this transition are poorly understood. Here, we demonstrate that the conserved kinase Ime2 regulates the timing and order of the meiotic divisions by controlling translation. Ime2 coordinates translational activation of a cluster of genes at the meiosis I–meiosis II transition, including the critical determinant of the meiotic chromosome segregation pattern CLB3. We further show that Ime2 mediates translational control through the meiosis-specific RNA-binding protein Rim4. Rim4 inhibits translation of CLB3 during meiosis I by interacting with the 5′ untranslated region (UTR) of CLB3. At the onset of meiosis II, Ime2 kinase activity rises and triggers a decrease in Rim4 protein levels, thereby alleviating translational repression. Our results elucidate a novel developmentally regulated translational control pathway that establishes the meiotic chromosome segregation pattern., American Cancer Society (Post-doctoral Fellowship), Virginia and D.K. Ludwig Fund for Cancer Research (Post-doctoral Fellowship), National Institutes of Health (U.S.) (Grant GM62207)

International audience; The cell-fate decision leading to gametogenesis is essential for sexual reproduction. In S. cerevisiae, only diploid MATa/α but not haploid MATa or MATα cells undergo gametogenesis, known as sporulation. We find that transcription of two long noncoding RNAs (lncRNAs) mediates mating-type control of sporulation. In MATa or MATα haploids, expression of IME1, the central inducer of gametogenesis, is inhibited in cis by transcription of the lncRNA IRT1, located in the IME1 promoter. IRT1 transcription recruits the Set2 histone methyltransferase and the Set3 histone deacetylase complex to establish repressive chromatin at the IME1 promoter. Inhibiting expression of IRT1 and an antisense transcript that antagonizes the expression of the meiotic regulator IME4 allows cells expressing the haploid mating type to sporulate with kinetics that are indistinguishable from that of MATa/α diploids. Conversely, expression of the two lncRNAs abolishes sporulation in MATa/α diploids. Thus, transcription of two lncRNAs governs mating-type control of gametogenesis in yeast.

Chromatin immunoprecipitation combined with DNA microarrays (ChIP-chip) is a powerful technique to detect in vivo protein–DNA interactions. Due to low yields, ChIP assays of transcription factors generally require amplification of immunoprecipitated genomic DNA. Here, we present an adapted linear amplification method that involves two rounds of T7 RNA polymerase amplification (double-T7). Using this we could successfully amplify as little as 0.4 ng of ChIP DNA to sufficient amounts for microarray analysis. In addition, we compared the double-T7 method to the ligation-mediated polymerase chain reaction (LM-PCR) method in a ChIP-chip of the yeast transcription factor Gsm1p. The double-T7 protocol showed lower noise levels and stronger binding signals compared to LM-PCR. Both LM-PCR and double-T7 identified strongly bound genomic regions, but the double-T7 method increased sensitivity and specificity to allow detection of weaker binding sites.

Affinity tagging has been used in many global studies towards protein function. We describe a highly efficient system for in vivo biotinylation of transcription factors in the yeast Saccharomyces cerevisiae, which is based on the bacterial BirA biotin ligase. The strength of the biotin–streptavidin interaction was exploited to improve detection of in vivo protein–DNA complexes in chromatin immunoprecipitation (ChIP) experiments. In a test system using the biotin-tagged LexA DNA-binding protein, we found that stringent washing conditions resulted in a strong improvement of the signal-to-noise ratios. Yeast strains with chromosomally integrated versions of tagged transcription factor genes were generated using N- or C-terminal biotin-tagging cassettes. ChIP experiments with biotinylated Rbp3p, a RNA polymerase II subunit, showed that Rbp3p-binding could even be detected at weakly expressed genes. Other methods failed to detect RNA polymerase II binding at such genes. Our results show that biotinylation of yeast transcription factors improves the detection of in vivo protein–DNA complexes.