Family or career? I want both-the control of meiosis.

In this paper I describe my professional and personal journey in science. In the 20th century there were fewer women scientists than man scientists. My personal experience and opinion is that women avoided academic careers. How one can combine family and career is discussed. The interest in science and the interactions I had with prominent leading Yeast Scientists changed my point of view, I matured and developed an academic career. My research focused on how budding yeast cells chose to exit the cell cycle and enter meiosis. My journey started using classical Genetic techniques. The development of Genetic engineering techniques enabled us to verify models and elucidate how entry into meiosis is controlled.

Genetic Approaches to Study Meiosis and Meiosis-Specific Gene Expression in Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae has a long history as a model organism for studies of meiosis and the cell cycle. The popularity of this yeast as a model is in large part due to the variety of genetic and cytological approaches that can be effectively performed with the cells. Cultures of the cells can be induced to synchronously progress through meiosis and sporulation allowing large-scale gene expression and biochemical studies to be performed. Additionally, the spore tetrads resulting from meiosis make it possible to characterize the haploid products of meiosis allowing investigation of meiotic recombination and chromosome segregation. Here we describe genetic methods for analysis progression of S. cerevisiae through meiosis and sporulation with an emphasis on strategies for the genetic analysis of regulators of meiosis-specific genes.

High-Throughput Screening to Identify Regulators of Meiosis-Specific Gene Expression in Saccharomyces cerevisiae.

Meiosis and gamete formation are processes that are essential for sexual reproduction in all eukaryotic organisms. Multiple intracellular and extracellular signals feed into pathways that converge on transcription factors that induce the expression of meiosis-specific genes. Once triggered the meiosis-specific gene expression program proceeds in a cascade that drives progress through the events of meiosis and gamete formation. Meiosis-specific gene expression is tightly controlled by a balance of positive and negative regulatory factors that respond to a plethora of signaling pathways. The budding yeast Saccharomyces cerevisiae has proven to be an outstanding model for the dissection of gametogenesis owing to the sophisticated genetic manipulations that can be performed with the cells. It is possible to use a variety selection and screening methods to identify genes and their functions. High-throughput screening technology has been developed to allow an array of all viable yeast gene deletion mutants to be screened for phenotypes and for regulators of gene expression. This chapter describes a protocol that has been used to screen a library of homozygous diploid yeast deletion strains to identify regulators of the meiosis-specific IME1 gene.

A Computational Approach to Study Gene Expression Networks.

We describe a simple computational approach that can be used for the study and simulation of regulatory networks. The advantage of this approach is that it requires neither computational background nor exact quantitative data about the biological system under study. Moreover, it is suitable for examining alternative hypotheses about the structure of a biological network. We used a tool called BioNSi (Biological Network Simulator) that is based on a simple computational model, which can be easily integrated as part of the lab routine, in parallel to experimental work. One benefit of this approach is that it enables the identification of regulatory proteins, which are missing from the experimental work. We describe the general methodology for modeling a network's dynamics in the tool, and then give a point by point example for a specific known network, entry into meiosis in budding yeast.

The roles of the catalytic and noncatalytic activities of Rpd3L and Rpd3S in the regulation of gene transcription in yeast.

In budding yeasts, the histone deacetylase Rpd3 resides in two different complexes called Rpd3L (large) and Rpd3S (small) that exert opposing effects on the transcription of meiosis-specific genes. By introducing mutations that disrupt the integrity and function of either Rpd3L or Rpd3S, we show here that Rpd3 function is determined by its association with either of these complexes. Specifically, the catalytic activity of Rpd3S activates the transcription of the two major positive regulators of meiosis, IME1 and IME2, under all growth conditions and activates the transcription of NDT80 only during vegetative growth. In contrast, the effects of Rpd3L depends on nutrients; it represses or activates transcription in the presence or absence of a nitrogen source, respectively. Further, we show that transcriptional activation does not correlate with histone H4 deacetylation, suggesting an effect on a nonhistone protein. Comparison of rpd3-null and catalytic-site point mutants revealed an inhibitory activity that is independent of either the catalytic activity of Rpd3 or the integrity of Rpd3L and Rpd3S.

Multiple MAPK cascades regulate the transcription of IME1, the master transcriptional activator of meiosis in Saccharomyces cerevisiae.

The choice between alternative developmental pathways is primarily controlled at the level of transcription. Induction of meiosis in budding yeasts in response to nutrient levels provides a system to investigate the molecular basis of cellular decision-making. In Saccharomyces cerevisiae, entry into meiosis depends on multiple signals converging upon IME1, the master transcriptional activator of meiosis. Here we studied the regulation of the cis-acting regulatory element Upstream Activation Signal (UAS)ru, which resides within the IME1 promoter. Guided by our previous data acquired using a powerful high-throughput screening system, here we provide evidence that UASru is regulated by multiple stimuli that trigger distinct signal transduction pathways as follows: (i) The glucose signal inhibited UASru activity through the cyclic AMP (cAMP/protein kinase A (PKA) pathway, targeting the transcription factors (TFs), Com2 and Sko1; (ii) high osmolarity activated UASru through the Hog1/mitogen-activated protein kinase (MAPK) pathway and its corresponding TF Sko1; (iii) elevated temperature increased the activity of UASru through the cell wall integrity pathway and the TFs Swi4/Mpk1 and Swi4/Mlp1; (iv) the nitrogen source repressed UASru activity through Sum1; and (v) the absence of a nitrogen source was detected and transmitted to UASru by the Kss1 and Fus3 MAPK pathways through their respective downstream TFs, Ste12/Tec1 and Ste12/Ste12 as well as by their regulators Dig1/2. These signaling events were specific to UASru; they did not affect the mating and filamentation response elements that are regulated by MAPK pathways. The complex regulation of UASru through all the known vegetative MAPK pathways is unique to S. cerevisiae and is specific for IME1, likely because it is the master regulator of gametogenesis.

The effective application of a discrete transition model to explore cell-cycle regulation in yeast.

BACKGROUND: Bench biologists often do not take part in the development of computational models for their systems, and therefore, they frequently employ them as "black-boxes". Our aim was to construct and test a model that does not depend on the availability of quantitative data, and can be directly used without a need for intensive computational background. RESULTS: We present a discrete transition model. We used cell-cycle in budding yeast as a paradigm for a complex network, demonstrating phenomena such as sequential protein expression and activity, and cell-cycle oscillation. The structure of the network was validated by its response to computational perturbations such as mutations, and its response to mating-pheromone or nitrogen depletion. The model has a strong predicative capability, demonstrating how the activity of a specific transcription factor, Hcm1, is regulated, and what determines commitment of cells to enter and complete the cell-cycle. CONCLUSION: The model presented herein is intuitive, yet is expressive enough to elucidate the intrinsic structure and qualitative behavior of large and complex regulatory networks. Moreover our model allowed us to examine multiple hypotheses in a simple and intuitive manner, giving rise to testable predictions. This methodology can be easily integrated as a useful approach for the study of networks, enriching experimental biology with computational insights.

Ectopic expression of human Cdk2 and its yeast homolog, Ime2, is deleterious to Saccharomyces cerevisiae.

Entry into and precise progression through the cell cycle depends on the sequential expression and activation of cyclin dependent kinases (CDK). In accord, CDK dysregulation is a hallmark of many cancers. The function of Cdk2 is still an enigma as in vitro studies revealed that it is required for S phase-entry, whereas in vivo studies showed that Cdk2 is not an essential gene. Moreover, unlike other Cdks, or its cyclin E regulator, Cdk2-overexpressing tumors were reported only in one type of tumor. In this report we used budding yeast as a tool to explore Cdk2 function. We showed that hCdk2 promoted S phase in cells carrying a temperature-sensitive mutation in yCDK1, albeit, only when expressed at low or moderate levels. Overexpression of hCdk2 resulted in a defect in the G1 to S transition and a reduction in viability. The same phenotypes were observed in cells overexpressing its yeast functional homolog, Ime2, which is a meiosis-specific CDK-like kinase. A genetic interaction with the DNA damage checkpoint was demonstrated by showing an increased toxicity of hCdk2 and Ime2 in RAD53-deleted cells, and delayed Rad53 activation in response to MMS treatment in cells overexpressing hCdk2 or Ime2.

Functional dissection of IME1 transcription using quantitative promoter-reporter screening.

Transcriptional regulation is a key mechanism that controls the fate and response of cells to diverse signals. Therefore, the identification of the DNA-binding proteins, which mediate these signals, is a crucial step in elucidating how cell fate is regulated. In this report, we applied both bioinformatics and functional genomic approaches to scrutinize the unusually large promoter of the IME1 gene in budding yeast. Using a recently described fluorescent protein-based reporter screen, reporter-synthetic genetic array (R-SGA), we assessed the effect of viable deletion mutants on transcription of various IME1 promoter-reporter genes. We discovered potential transcription factors, many of which have no perfect consensus site within the IME1 promoter. Moreover, most of the cis-regulatory sequences with perfect homology to known transcription factor (TF) consensus were found to be nonfunctional in the R-SGA analysis. In addition, our results suggest that lack of conservation may not discriminate against a TF regulatory role at a specific promoter. We demonstrate that Sum1 and Sok2, which regulate IME1, bind to nonperfect consensuses within nonconserved regions in the sensu stricto Saccharomyces strains. Our analysis supports the view that although comparative analysis can provide a useful guide, functional assays are required for accurate identification of TF-binding site interactions in complex promoters.

A switch from a gradient to a threshold mode in the regulation of a transcriptional cascade promotes robust execution of meiosis in budding yeast.

Tight regulation of developmental pathways is of critical importance to all organisms, and is achieved by a transcriptional cascade ensuring the coordinated expression of sets of genes. We aimed to explore whether a strong signal is required to enter and complete a developmental pathway, by using meiosis in budding yeast as a model. We demonstrate that meiosis in budding yeast is insensitive to drastic changes in the levels of its consecutive positive regulators (Ime1, Ime2, and Ndt80). Entry into DNA replication is not correlated with the time of transcription of the early genes that regulate this event. Entry into nuclear division is directly regulated by the time of transcription of the middle genes, as premature transcription of their activator NDT80, leads to a premature entry into the first meiotic division, and loss of coordination between DNA replication and nuclear division. We demonstrate that Cdk1/Cln3 functions as a negative regulator of Ime2, and that ectopic expression of Cln3 delays entry into nuclear division as well as NDT80 transcription. Because Ime2 functions as a positive regulator for premeiotic DNA replication and NDT80 transcription, as well as a negative regulator of Cdk/Cln, we suggest that a double negative feedback loop between Ime2 and Cdk1/Cln3 promotes a bistable switch from the cell cycle to meiosis. Moreover, our results suggest a regulatory mode switch that ensures robust meiosis as the transcription of the early meiosis-specific genes responds in a graded mode to Ime1 levels, whereas that of the middle and late genes as well as initiation of DNA replication, are regulated in a threshold mode.

Human Cdk2 is a functional homolog of budding yeast Ime2, the meiosis-specific Cdk-like kinase.

Progression through the cell cycle depends on sequential activation of Cyclin-Dependent Kinase(s). In this report we use budding-yeast meiosis as a tool to elucidate the specific functions of mammalian Cdks. Yeast meiosis is regulated by both Cdc28 (yCdk1) and Ime2 (a meiosis-specific Cdk-like kinase). We show that human Cdk2 is a functional homolog for most of Ime2 functions. It promotes efficient and timely entry into premeiotic DNA replication and the first nuclear division, as well as the regulated transcription of IME1 and the early meiosis-specific genes. We show that this effect is specific, and that neither mice Cdk1, nor mice Cdk4 can suppress ime2. We show that Cdk1 is a functional homolog of Cdc28 that also suppresses one of its meiotic functions, namely inhibiting the transcription of IME1. Cdk2, on the other hand, show dominant negative effects on entry into the cell cycle, most probably by inhibiting the function of Cdc28. Finally, we show that in the meiotic pathway Cdk4 functions as a transcriptional activator.

Faithful modeling of transient expression and its application to elucidating negative feedback regulation.

Modeling and analysis of genetic regulatory networks is essential both for better understanding their dynamic behavior and for elucidating and refining open issues. We hereby present a discrete computational model that effectively describes the transient and sequential expression of a network of genes in a representative developmental pathway. Our model system is a transcriptional cascade that includes positive and negative feedback loops directing the initiation and progression through meiosis in budding yeast. The computational model allows qualitative analysis of the transcription of early meiosis-specific genes, specifically, Ime2 and their master activator, Ime1. The simulations demonstrate a robust transcriptional behavior with respect to the initial levels of Ime1 and Ime2. The computational results were verified experimentally by deleting various genes and by changing initial conditions. The model has a strong predictive aspect, and it provides insights into how to distinguish among and reason about alternative hypotheses concerning the mode by which negative regulation through Ime1 and Ime2 is accomplished. Some predictions were validated experimentally, for instance, showing that the decline in the transcription of IME1 depends on Rpd3, which is recruited by Ime1 to its promoter. Finally, this general model promotes the analysis of systems that are devoid of consistent quantitative data, as is often the case, and it can be easily adapted to other developmental pathways.

The Saccharomyces cerevisiae GSK-3 beta homologs.

Yeast cells carry four homologs of GSK-3beta, RIM11, MCK1, MRK1 and YGK3. The significant homologs are RIM11 and MCK1 that presumably arose from a recent genome duplication followed by a rapid divergence. Accordingly, these homologs phosphorylate specific substrates. Rim11 is essential for entry into meiosis, whereas Mck1 is essential for growth at elevated and low temperatures. Both kinases transmit nutrient signals, but Mck1 transmits additional signals including stress signals such as, temperature, osmotic shock and Ca(2+). Consequently, Mck1 plays a role in multiple functions, including cell wall integrity, meiosis and centromere function. The other two homologs, MRK1 and YGK3 that belong to the RIM11 and MCK1 phylogenetic trees, respectively, show no distinct phenotype. These paralogs posses redundant roles, though less important, with Rim11 and Mck1 functions. This review summarizes the cellular roles of these kinases, their mode of regulation, and the signals that they transmit.

Aym1, a mouse meiotic gene identified by virtue of its ability to activate early meiotic genes in the yeast Saccharomyces cerevisiae.

Our understanding of the molecular mechanisms that operate during differentiation of mitotically dividing spermatogonia cells into spermatocytes lags way behind what is known about other differentiating systems. Given the evolutionary conservation of the meiotic process, we screened for mouse proteins that could specifically activate early meiotic promoters in Saccharomyces cerevisiae yeast cells, when fused to the Gal4 activation domain (Gal4AD). Our screen yielded the Aym1 gene that encodes a short peptide of 45 amino acids. We show that a Gal4AD-AYM1 fusion protein activates expression of reporter genes through the promoters of the early meiosis-specific genes IME2 and HOP1, and that this activation is dependent on the DNA-binding protein Ume6. Aym1 is transcribed predominantly in mouse primary spermatocytes and in gonads of female embryos undergoing the corresponding meiotic divisions. Aym1 immunolocalized to nuclei of primary spermatocytes and oocytes and to specific type A spermatogonia cells, suggesting it might play a role in the processes leading to meiotic competence. The potential functional relationship between AYM1 and yeast proteins that regulate expression of early meiotic genes is discussed.

The in vivo activity of Ime1, the key transcriptional activator of meiosis-specific genes in Saccharomyces cerevisiae, is inhibited by the cyclic AMP/protein kinase A signal pathway through the glycogen synthase kinase 3-beta homolog Rim11.

Phosphorylation is the main mode by which signals are transmitted to key regulators of developmental pathways. The glycogen synthase kinase 3 family plays pivotal roles in the development and well-being of all eukaryotic organisms. Similarly, the budding yeast homolog Rim11 is essential for the exit of diploid cells from the cell cycle and for entry into the meiotic developmental pathway. In this report we show that in vivo, in cells grown in a medium promoting vegetative growth with acetate as the sole carbon source (SA medium), Rim11 phosphorylates Ime1, the master transcriptional activator required for entry into the meiotic cycle and for the transcription of early meiosis-specific genes. We demonstrate that in the presence of glucose, the kinase activity of Rim11 is inhibited. This inhibition could be due to phosphorylation on Ser-5, Ser-8, and/or Ser-12 because in the rim11S5AS8AS12A mutant, Ime1 is incorrectly phosphorylated in the presence of glucose and cells undergo sporulation. We further show that this nutrient signal is transmitted to Rim11 and consequently to Ime1 by the cyclic AMP/protein kinase A signal transduction pathway. Ime1 is phosphorylated in SA medium on at least two residues, Tyr-359 and Ser-302 and/or Ser-306. Ser-302 and Ser-306 are part of a consensus site for the mammalian homolog of Rim11, glycogen synthase kinase 3-beta. Phosphorylation on Tyr-359 but not Ser-302 or Ser-306 is essential for the transcription of early meiosis-specific genes and sporulation. We show that Tyr-359 is phosphorylated by Rim11.

Glucose and nitrogen regulate the switch from histone deacetylation to acetylation for expression of early meiosis-specific genes in budding yeast.

In eukaryotes, the switch between alternative developmental pathways is mainly attributed to a switch in transcriptional programs. A major mode in this switch is the transition between histone deacetylation and acetylation. In budding yeast, early meiosis-specific genes (EMGs) are repressed in the mitotic cell cycle by active deacetylation of their histones. Transcriptional activation of these genes in response to the meiotic signals (i.e., glucose and nitrogen depletion) requires histone acetylation. Here we follow how this regulated switch is accomplished, demonstrating the existence of two parallel mechanisms. (i) We demonstrate that depletion of glucose and nitrogen leads to a transient replacement of the histone deacetylase (HDAC) complex on the promoters of EMG by the transcriptional activator Ime1. The occupancy by either component occurs independently of the presence or absence of the other. Removal of the HDAC complex depends on the protein kinase Rim15, whose activity in the presence of nutrients is inhibited by protein kinase A phosphorylation. (ii) In the absence of glucose, HDAC loses its ability to repress transcription, even if this repression complex is directly bound to a promoter. We show that this relief of repression depends on Ime1, as well as on the kinase activity of Rim11, a glycogen synthase kinase 3beta homolog that phosphorylates Ime1. We further show that the glucose signal is transmitted through Rim11. In cells expressing the constitutive active rim11-3SA allele, HDAC repression in glucose medium is impaired.

The role and regulation of the preRC component Cdc6 in the initiation of premeiotic DNA replication.

In all eukaryotes, the initiation of DNA replication is regulated by the ordered assembly of DNA/protein complexes on origins of DNA replication. In this report, we examine the role of Cdc6, a component of the prereplication complex, in the initiation of premeiotic DNA replication in budding yeast. We show that in the meiotic cycle, Cdc6 is required for DNA synthesis and sporulation. Moreover, similarly to the regulation in the mitotic cell cycle, Cdc6 is specifically degraded upon entry into the meiotic S phase. By contrast, chromatin-immunoprecipitation analysis reveals that the origin-bound Cdc6 is stable throughout the meiotic cycle. Preliminary evidence suggests that this protection reflects a change in chromatin structure that occurs in meiosis. Using the cdc28-degron allele, we show that depletion of Cdc28 leads to stabilization of Cdc6 in the mitotic cycle, but not in the meiotic cycle. We show physical association between Cdc6 and the meiosis-specific hCDK2 homolog Ime2. These results suggest that under meiotic conditions, Ime2, rather than Cdc28, regulates the stability of Cdc6. Chromatin-immunoprecipitation analysis reveals that similarly to the mitotic cell cycle, Mcm2 binds origins in G1 and meiotic S phases, and at the end of the second meiotic division, it is gradually removed from chromatin.

Transcriptional regulation of meiosis in budding yeast.

Initiation of meiosis in Saccharomyces cerevisiae is regulated by mating type and nutritional conditions that restrict meiosis to diploid cells grown under starvation conditions. Specifically, meiosis occurs in MATa/MATalpha cells shifted to nitrogen depletion media in the absence of glucose and the presence of a nonfermentable carbon source. These conditions lead to the expression and activation of Ime 1, the master regulator of meiosis. IME1 encodes a transcriptional activator recruited to promoters of early meiosis-specific genes by association with the DNA-binding protein, Ume6. Under vegetative growth conditions these genes are silent due to recruitment of the Sin3/Rpd3 histone deacetylase and Isw2 chromatin remodeling complexes by Ume6. Transcription of these meiotic genes occurs following histone acetylation by Gcn5. Expression of the early genes promote entry into the meiotic cycle, as they include genes required for premeiotic DNA synthesis, synapsis of homologous chromosomes, and meiotic recombination. Two of the early meiosis specific genes, a transcriptional activator, Ndt80, and a CDK2 homologue, Ime2, are required for the transcription of middle meiosis-specific genes that are involved with nuclear division and spore formation. Spore maturation depends on late genes whose expression is indirectly dependent on Ime1, Ime2, and Ndt80. Finally, phosphorylation of Imel by Ime2 leads to its degradation, and consequently to shutting down of the meiotic transcriptional cascade. This review is focusing on the regulation of gene expression governing initiation and progression through meiosis.

Ime2, a meiosis-specific kinase in yeast, is required for destabilization of its transcriptional activator, Ime1.

In the budding yeast Saccharomyces cerevisiae, entry into meiosis and its successful completion depend on two positive regulators, Ime1 and Ime2. Ime1 is a transcriptional activator that is required for transcription of IME2, a serine/threonine protein kinase. We show that in vivo Ime2 associates with Ime1, that in vitro Ime2 phosphorylates Ime1, and that in living cells the stability of Ime1 depends on Ime2. Diploid cells with IME2 deleted show an increase in the level of Ime1, whereas haploid cells overexpressing IME2 show a decrease in the stability of Ime1. Furthermore, the level of Ime1 depends on the kinase activity of Ime2. Using a mutation in one of the ATPase subunits of the proteasome, RPT2, we demonstrate that Ime1, amino acids 270 to 360, is degraded by the 26S proteasome. We also show that Ime2 itself is an extremely unstable protein whose expression in vegetative cultures is toxic. We propose that a negative-feedback loop ensures that the activity of Ime1 will be restricted to a narrow window.