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SUMMARYAlternative transcription start sites can affect transcript isoform diversity and translation levels. In a recently described form of gene regulation, coordinated transcriptional and translational interference results in transcript isoform-dependent changes in protein expression. Specifically, a long undecoded transcript isoform (LUTI) is transcribed from a gene-distal promoter, interfering with expression of the gene-proximal promoter. While transcriptional and chromatin features associated with LUTI expression have been described, the mechanism underlying LUTI-based transcriptional interference is not well understood. Using an unbiased genetic approach followed by integrated genomic analysis, we uncovered that the Swi/Snf chromatin remodeling complex is required for co-transcriptional nucleosome remodeling that leads to LUTI-based repression. We identified genes with tandem promoters that rely on Swi/Snf function for transcriptional interference during protein folding stress, including LUTI-regulated genes. To our knowledge, this study is the first to observe Swi/Snf’s direct involvement in gene repression via acistranscriptional interference mechanism.

The process of gametogenesis is orchestrated by a dynamic program of gene expression, where a vital subset constitutes the early meiotic genes (EMGs). In budding yeast, the transcription factor Ume6 represses EMG expression during mitosis. However, during mitosis to meiosis transition, EMGs are activated in response to the meiotic regulator Ime1 through its interaction with Ume6. While it is known that binding of Ime1 to Ume6 promotes EMG expression, the mechanism of EMG activation remains elusive. Two competing models have been proposed whereby Ime1 either forms a coactivator complex with Ume6 or promotes Ume6 degradation. Here, we resolve this controversy. First, we identify the set of genes that are directly regulated by Ume6, includingUME6itself. While Ume6 levels increase in response to Ime1, Ume6 degradation occurs much later in meiosis. Importantly, we found that depletion of Ume6 shortly before meiotic entry is detrimental to EMG activation and gamete formation, whereas tethering of Ume6 to a heterologous activation domain is sufficient to trigger EMG expression and produce viable gametes in the absence of Ime1. We conclude that Ime1 and Ume6 function as a coactivator complex. While Ume6 is indispensable for EMG expression, Ime1 primarily serves as a transactivator for Ume6.

SUMMARYThe mitosis to meiosis transition requires dynamic changes in gene expression, but whether and how the mitotic transcriptional machinery is regulated during this transition is unknown. In budding yeast, SBF and MBF transcription factors initiate the mitotic gene expression program. Here, we report two mechanisms that work together to restrict SBF activity during meiotic entry: repression of the SBF-specific Swi4 subunit through LUTI-based regulation and inhibition of SBF by Whi5, a homolog of the Rb tumor suppressor. We find that untimely SBF activation causes downregulation of early meiotic genes and delays meiotic entry. These defects are largely driven by the SBF-target G1 cyclins, which block the interaction between the central meiotic regulator Ime1 and its cofactor Ume6. Our study provides insight into the role ofSWI4LUTIin establishing the meiotic transcriptional program and demonstrates how the LUTI-based regulation is integrated into a larger regulatory network to ensure timely SBF activity.

This review describes the current models for how the subunit abundance of the Ndc80 complex, a key kinetochore component, is regulated in budding yeast and metazoan meiosis. The past decades of kinetochore research have established the Ndc80 complex to be a key microtubule interactor and a central hub for regulating chromosome segregation. Recent studies further demonstrate that Ndc80 is the limiting kinetochore subunit that dictates the timing of kinetochore activation in budding yeast meiosis. Here, we discuss the molecular circuits that regulate Ndc80 protein synthesis and degradation in budding yeast meiosis and compare the findings with those from metazoans. We envision the regulatory principles discovered in budding yeast to be conserved in metazoans, thereby providing guidance into future investigations on kinetochore regulation in human health and disease.

Gametogenesis is an evolutionarily conserved developmental program whereby a diploid progenitor cell undergoes meiosis and cellular remodeling to differentiate into haploid gametes, the precursors for sexual reproduction. Even in the simple eukaryotic organism Saccharomyces cerevisiae, the meiotic transcriptome is very rich and complex, thereby necessitating new tools for functional studies. Here, we report the construction of 5 stage-specific, inducible complementary DNA libraries from meiotic cells that represent over 84% of the genes found in the budding yeast genome. We employed computational strategies to detect endogenous meiotic transcript isoforms as well as library-specific gene truncations. Furthermore, we developed a robust screening pipeline to test the effect of each complementary DNA on competitive fitness. Our multiday proof-of-principle time course revealed 877 complementary DNAs that were detrimental for competitive fitness when overexpressed. The list included mitochondrial proteins that cause dose-dependent disruption of cellular respiration as well as library-specific gene truncations that expose a dominant negative effect on competitive growth. Together, these high-quality complementary DNA libraries provide an important tool for systematically identifying meiotic genes, transcript isoforms, and protein domains that are important for a specific biological function.

Nuclear pore complexes (NPCs) are large proteinaceous assemblies that mediate nuclear compartmentalization. NPCs undergo large-scale structural rearrangements during mitosis in metazoans and some fungi. However, our understanding of NPC remodeling beyond mitosis remains limited. Using time-lapse fluorescence microscopy, we discovered that NPCs undergo two mechanistically separable remodeling events during budding yeast meiosis in which parts or all of the nuclear basket transiently dissociate from the NPC core during meiosis I and II, respectively. Meiosis I detachment, observed for Nup60 and Nup2, is driven by Polo kinase-mediated phosphorylation of Nup60 at its interface with the Y-complex. Subsequent reattachment of Nup60-Nup2 to the NPC core is facilitated by a lipid-binding amphipathic helix in Nup60. Preventing Nup60-Nup2 reattachment causes misorganization of the entire nuclear basket in gametes. Strikingly, meiotic nuclear basket remodeling also occurs in the distantly related fission yeast, Schizosaccharomyces pombe. Our study reveals a conserved and developmentally programmed aspect of NPC plasticity, providing key mechanistic insights into the nuclear basket organization., Journal of Cell Biology, 222 (2), ISSN:0021-9525, ISSN:1540-8140

Neighboring sequences of a gene can influence its expression. In the phenomenon known as transcriptional interference, transcription at one region in the genome can repress transcription at a nearby region in cis. Transcriptional interference occurs at a number of eukaryotic loci, including the alcohol dehydrogenase (Adh) gene in Drosophila melanogaster. Adh is regulated by two promoters, which are distinct in their developmental timing of activation. It has been shown using transgene insertion that when the promoter distal from the Adh start codon is deleted, transcription from the proximal promoter becomes de-regulated. As a result, the Adh proximal promoter, which is normally active only during the early larval stages, becomes abnormally activated in adults. Whether this type of regulation occurs in the endogenous Adh context, however, remains unclear. Here, we employed the CRISPR/Cas9 system to edit the endogenous Adh locus and found that removal of the distal promoter also resulted in the untimely expression of the proximal promoter-driven mRNA isoform in adults, albeit at lower levels than previously reported. Importantly, transcription from the distal promoter was sufficient to repress proximal transcription in larvae, and the degree of this repression was dependent on the degree of distal promoter activity. Finally, upregulation of the distal Adh transcript led to the enrichment of histone 3 lysine 36 trimethylation over the Adh proximal promoter. We conclude that the endogenous Adh locus is developmentally regulated by transcriptional interference in a tunable manner.

Gametogenesis is a highly regulated and dynamic developmental program where a diploid progenitor cell differentiates into haploid gametes, the precursors for sexual reproduction. During meiosis, several pathways converge to initiate ploidy reduction and organelle remodelling to render gametes competent for zygote formation and subsequent organismal development. Additionally, meiosis inherently rejuvenates the newly formed gametes resulting in lifespan resetting. Here, we construct five stage-specific, inducible meiotic cDNA libraries that represent over 84% of the yeast genome. We employ computational strategies to detect stage-specific meiotic transcript isoforms in each library and develop a robust screening pipeline to test the effect of each cDNA on competitive fitness. Our multi-day proof-of-principle time course reveals gene isoforms that are important for competitive fitness as well as mitochondrial proteins that cause dose-dependent disruption of respiration. Together, these novel meiotic cDNA libraries provide an important resource for systematically studying meiotic genes and gene isoforms in future studies.HIGHLIGHTSConstruction of five stage-specific, inducible meiotic cDNA libraries in budding yeast that collectively represent 5563 genes, which is over 84% of the genomeAnalysis of the cDNA libraries reveal the presence of meiosis-specific transcript isoforms that are largely uncharacterizedDevelopment of a robust gain-of-function screening pipeline identifies previously characterized genes and novel gene isoforms important for competitive fitnessMulti-day proof-of-principle screen reveals mitochondrial proteins that cause dosage-specific respiration defects

Organelle tethers stabilize contact sites between organelles, yet cellular architecture is dynamically remodeled during cellular differentiation. Sawyer et al. demonstrate that a conserved CDK-like kinase, Ime2, promotes cellular remodeling through destruction of mitochondria–plasma membrane contact sites in yeast gametogenesis., Cellular differentiation involves remodeling cellular architecture to transform one cell type to another. By investigating mitochondrial dynamics during meiotic differentiation in budding yeast, we sought to understand how organelle morphogenesis is developmentally controlled in a system where regulators of differentiation and organelle architecture are known, but the interface between them remains unexplored. We analyzed the regulation of mitochondrial detachment from the cell cortex, a known meiotic alteration to mitochondrial morphology. We found that mitochondrial detachment is enabled by the programmed destruction of the mitochondria–endoplasmic reticulum–cortex anchor (MECA), an organelle tether that bridges mitochondria and the plasma membrane. MECA regulation is governed by a meiotic transcription factor, Ndt80, which promotes the activation of a conserved kinase, Ime2. We further present evidence for Ime2-dependent phosphorylation and degradation of MECA in a temporally controlled manner. Our study defines a key mechanism that coordinates mitochondrial morphogenesis with the landmark events of meiosis and demonstrates that cells can developmentally regulate tethering to induce organelle remodeling.

While retrotransposons must activate in developing gametes in order to survive and propagate, how a retrotransposon selectively activates in the context of meiosis is unclear. These mobile genetic elements can promote evolutionary innovations, but their unchecked proliferation is catastrophic for their host. Consequently, organisms have developed elegant mechanisms to counteract retrotransposon proliferation. In this manuscript, we demonstrate that both transcriptional activation of Ty3/Gypsy retrotransposons and host defense are controlled by master meiotic regulators. We show that Ty3 retrotransposons in budding yeast co-opt binding sites of the essential meiotic transcription factor Ndt80 upstream of the integration site, thereby tightly linking their transcriptional activation to meiotic progression. Our results raise the question of how yeast protects the genomes of its developing gametes from retrotransposon proliferation during meiosis. We elucidate how yeast cells thwart the Ty3/Gypsy retrotransposition life cycle by blocking translation of the retrotransposon mRNA using amyloid-like assemblies of the RNA-binding protein Rim4. In mammals, Gypsy elements have lost retrotransposition capacity. Building from our findings in yeast, we show that developing gametes in mammals also utilize essential meiotic transcription factors and assembly-forming RNA-binding proteins to regulate Gypsy-derived genes. Our findings inform how novel genes that are evolving from retrotransposons can build upon existing regulatory networks during domestication.

The kinetochore complex is a conserved machinery that connects chromosomes to spindle microtubules. During meiosis, the kinetochore is restructured to accommodate a specialized chromosome segregation pattern. In budding yeast, meiotic kinetochore remodeling is mediated by the temporal changes in the abundance of a single subunit called Ndc80. We have previously described the regulatory events that control the timely synthesis of Ndc80. Here, we report that Ndc80 turnover is also tightly regulated in meiosis: Ndc80 degradation is active in meiotic prophase, but not in metaphase I. Ndc80 degradation depends on the ubiquitin ligase APCAma1and is mediated by the proteasome. Importantly, Aurora B-dependent Ndc80 phosphorylation, a mark that has been previously implicated in correcting erroneous microtubule–kinetochore attachments, is essential for Ndc80 degradation in a microtubule-independent manner. The N-terminus of Ndc80, including a 27-residue sequence and Aurora B phosphorylation sites, is both necessary and sufficient for kinetochore protein degradation. Finally, defects in Ndc80 turnover predispose meiotic cells to chromosome mis-segregation. Our study elucidates the mechanism by which meiotic cells modulate their kinetochore composition through regulated Ndc80 degradation, and demonstrates that Aurora B-dependent regulation of kinetochores extends beyond altering microtubule attachments.

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.

Cellular differentiation depends on temporally controlled waves of gene activation and inactivation that ultimately transform one cell type into another. It is well established that transcription factor cascades coordinate the timely activation of gene expression clusters during development. In comparison, much less is understood about how gene repression events are coordinated with the transcription factor-driven waves of gene activation and how this repression is achieved at a mechanistic level. Using budding yeast as a model, we recently discovered a new gene regulatory event, whereby a central meiotic transcription factor induces the expression of an mRNA isoform to repress gene expression through an integrated transcriptional and translational mechanism. This new model could explain how gene activation and inactivation waves can be temporally coordinated. In this review, we discuss our findings and their potential implications.

Production of healthy gametes in meiosis relies on the quality control and proper distribution of both nuclear and cytoplasmic contents. Meiotic differentiation naturally eliminates age-induced cellular damage by an unknown mechanism. Using time-lapse fluorescence microscopy in budding yeast, we found that nuclear senescence factors – including protein aggregates, extrachromosomal ribosomal DNA circles, and abnormal nucleolar material – are sequestered away from chromosomes during meiosis II and subsequently eliminated. A similar sequestration and elimination process occurs for the core subunits of the nuclear pore complex in both young and aged cells. Nuclear envelope remodeling drives the formation of a membranous compartment containing the sequestered material. Importantly, de novo generation of plasma membrane is required for the sequestration event, preventing the inheritance of long-lived nucleoporins and senescence factors into the newly formed gametes. Our study uncovers a new mechanism of nuclear quality control and provides insight into its function in meiotic cellular rejuvenation., eLife digest The cells of living organisms accumulate damage as they age. Some of this age-associated damage is found around the organism’s DNA. However, when genetic material is passed on during sexual reproduction, newly born offspring avoid inheriting this age-induced damage. This ensures that the progeny are ‘re-set’ with a fresh lifespan that is independent from their parents’ age. A lot of what is known about aging has come from studying budding yeast. Yeast cells can undergo a process called meiosis and divide into four cells known as gametes, which are the equivalents of human sperm and egg. During meiosis, the structure that surrounds the cell’s genetic material – known as the nuclear membrane – remains intact, surrounding the DNA as it separates into four distinct parts. As the cell divides, age-associated factors that were originally present in the parent are not inherited by the gametes, but it remains unclear how this occurs. Now, King, Goodman et al. have investigated this process by attaching fluorescent labels to specific aging factors and tracking how they are distributed inside yeast cells undergoing meiosis. This revealed that age-associated factors were physically sequestered away from the inherited genetic material during meiosis. King, Goodman et al. found that as the nuclear membrane remodeled itself around the genetic material of the four gametes, the damage became confined to a fifth previously unknown membrane-bound compartment. Once outside of the gametes, the aging factors were then selectively destroyed by enzymes released from the parent cell. All cells age, and many of the mechanisms underlying these processes are similar across species and cell types. A better understanding of how cells age, and of the process by which gametes are able to sequester and eliminate age-induced damage, may help guide efforts to combat aging in other cells.

A hallmark of aging is the progressive accumulation of cellular damage. Age-induced damage arises due to a decrease in organelle function along with a decline in protein quality control. Although somatic tissues deteriorate with age, the germline must maintain cellular homeostasis in order to ensure the production of healthy progeny. While germline quality control has been primarily studied in multicellular organisms, recent evidence suggests the existence of gametogenesis-specific quality control mechanisms in unicellular eukaryotes, highlighting the evolutionary conservation of meiotic events beyond chromosome morphogenesis. Notably, budding yeast eliminates age-induced damage during meiotic differentiation, employing novel organelle and protein quality control mechanisms to produce young and healthy gametes. Similarly, organelle and protein quality control is present in metazoan gametogenesis, however, whether and how these mechanisms contribute to cellular rejuvenation requires further investigation. Here, we summarize recent findings that describe organelle and protein quality control in budding yeast gametogenesis, examine similar quality control mechanisms in metazoan development, and identify research directions that will improve our understanding of meiotic cellular rejuvenation.

Neighboring sequences of a gene can influence its expression. In the phenomenon known as transcriptional interference, transcription at one region in the genome can repress transcription at a nearby region incis. Transcriptional interference occurs at a number of eukaryotic loci, including the alcohol dehydrogenase (Adh) gene inDrosophila melanogaster. Adhis regulated by two promoters, which are distinct in their developmental timing of activation. It has been shown using transgene insertion that when the promoter distal from theAdhstart codon is deleted, transcription from the proximal promoter becomes de-regulated. As a result, theAdhproximal promoter, which is normally active only during the early larval stages, becomes abnormally activated in adults. Whether this type of regulation occurs in the endogenousAdhcontext, however, remains unclear. Here, we employed the CRISPR/Cas9 system to edit the endogenousAdhlocus and found that removal of the distal promoter does also result in the untimely expression of the proximal promoter-driven mRNA isoform in adults, albeit at lower levels than previously reported. Importantly, we show that transcription from the distal promoter is sufficient to repress proximal transcription in larvae and that the degree of this repression depends on the degree of distal promoter activity. Finally, repression of the endogenousAdhproximal promoter is associated with the enrichment of histone 3 lysine 36 trimethylation (H3K36me3), a chromatin mark necessary for transcription-coupled gene repression in yeast. We conclude that the endogenousAdhlocus is developmentally regulated by transcriptional interference in a tunable manner.

Cellular differentiation involves remodeling cellular architecture to transform one cell type to another. By investigating mitochondrial dynamics during meiotic differentiation in budding yeast, we sought to understand how organelle morphogenesis is developmentally controlled in a system where regulators of differentiation as well as organelle architecture are known, but the interface between them remains unexplored. We found that mitochondria abruptly detach from the cell cortex shortly before segregating into gametes. Mitochondrial detachment is enabled by the programmed destruction of themitochondria-endoplasmic reticulum-cortexanchor (MECA), an organelle tether that forms contact sites between mitochondria and the plasma membrane. MECA regulation is governed by a meiotic transcription factor, Ndt80, which promotes the activation of a conserved kinase, Ime2. We found that MECA undergoes Ime2-dependent phosphorylation. Furthermore, Ime2 promotes MECA degradation in a temporally controlled manner. Our study defines a key mechanism that coordinates mitochondrial morphogenesis with the landmark events of meiosis and demonstrates that cells can developmentally regulate tethering to induce organelle remodeling.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene NDC80 during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in other yeast strains and growth conditions.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene NDC80 during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in other yeast strains and growth conditions.

We recently described an unconventional mode of gene regulation in budding yeast by which transcriptional and translational interference were used in an integrated manner to down-regulate protein expression. Developmentally timed transcriptional interference inhibited production of a well translated mRNA isoform and resulted in the production of an mRNA isoform containing inhibitory upstream open reading frames (uORFs) that blocked translation of the ORF. Transcriptional interference and uORF-based translational repression are established mechanisms outside of yeast, but whether this type of integrated regulation was conserved was unknown. Here we find that, indeed, a similar type of regulation occurs at the locus for the human oncogene MDM2. We observe evidence of transcriptional interference between the two MDM2 promoters, which produce a poorly translated distal promoter-derived uORF-containing mRNA isoform and a well-translated proximal promoter-derived transcript. Down-regulation of distal promoter activity markedly up-regulates proximal promoter-driven expression and results in local reduction of histone H3K36 trimethylation. Moreover, we observe that this transcript toggling between the two MDM2 isoforms naturally occurs during human embryonic stem cell differentiation programs.

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.

The ultimate success of cell division relies on the accurate partitioning of the genetic material. Errors in this process occur in nearly all tumors and are the leading cause of miscarriages and congenital birth defects in humans. Two cell divisions, mitosis and meiosis, use common as well as unique mechanisms to ensure faithful chromosome segregation. In mitosis, alternating rounds of DNA replication and chromosome segregation preserves the chromosome complement of the progenitor cell. In contrast, during meiosis two consecutive rounds of nuclear division, meiosis I and meiosis II, follow a single round of DNA replication to reduce the chromosome complement by half. Meiosis likely evolved through changes to the mitotic cell division program. This review will focus on the recent findings describing the modifications that transform mitosis into meiosis.

The purpose of this study is to reveal and to test the relationshipsbetween dependent variable critical thinking ability and threeindependent variables such as gender, academic achievement and fieldachievement of the students of the Faculty of Education, theDepartment of Fine Arts Education (FE-DFAE). This study is adescriptive research and based on the relational measurement model.The data of personal information of the students has been collected viaquestionnaires and to be able to measure critical thinking abilities ofthe students of the FE-DFAE the California Critical ThinkingDispositions Inventory (CCTDI) has been applied. While the targetpopulation of this study covers whole FE-DFAE students from academicyear of 2011-2012 -spring semester, the size of the sampling group islimited to 204 students. The result of this research shows that there isno significant relationship between critical thinking abilities of thestudents of the FE-DFAE and their genders. Beside that it has beendiscovered that the tendencies of truth seeking and open minded levelof female students of music department are higher than male studentsof the same department. It has been also discovered that there is nosignificant relationship between critical thinking abilities and academic,field achievements among the student of the music department. On thecontrary, among the fine arts students the significant and positiverelationship has been found between critical thinking abilities andacademic success. Also a positive and lower level significance has beenfound between critical thinking and their field studies of fine artsstudents. As a result, among the fine arts students, there is significantand positive relationship between their critical thinking abilities andtheir academic successes, which are at higher level than thoserelationships among the music department students. Unfortunately, itcan be concluded that the student of the Adnan Menderes Universityand especially the students who are teacher candidates have lack ofcritical thinking abilities. One of the primary purposes of universityeducation is to educate the students to make them gained inquisitiveand analytically thinking abilities. Therefore, it is crucial that theteachers of tomorrow have to furnished with all those abilities to be ableto teach and pass the critical thinking abilities to their students. Bu çalışmada, Eğitim Fakültesi Güzel Sanatlar Eğitimi Bölümü öğrencilerinin eleştirel düşünme eğilimleri ile cinsiyetleri, alan başarıları ve akademik başarıları arasındaki ilişkinin ortaya konması amaçlanmıştır. Çalışma, ilişkisel tarama modeline dayalı, betimsel bir araştırmadır. Çalışmada veri toplama aracı olarak kişisel bilgi formu ve Güzel Sanatlar Eğitimi Bölümü öğrencilerinin eleştirel düşünme eğilimlerini ortaya koymak amacıyla ‘California Eleştirel Düşünme Eğilimleri Ölçeği’ (CCTDI) kullanılmıştır. Çalışmanın evrenini, Eğitim Fakültesi Güzel Sanatlar Eğitimi Bölümlerinde öğrenim görmekte olan öğrenciler oluştururken örneklemini 2011-2012 bahar yarıyılında Adnan Menderes Üniversitesi Eğitim Fakültesi Müzik Eğitimi Anabilim Dalı ve Resim-iş Anabilim Dalında öğrenim görmekte olan 204 öğrenci oluşturmaktadır. Araştırmanın sonucunda, Eğitim Fakültesi Güzel Sanatlar Eğitimi Bölümü öğrencilerinin eleştirel düşünme eğilimlerinin, alanlarına ve cinsiyetlerine göre anlamlı farklılık göstermediği; ancak müzik bölümünde okuyan kız öğrencilerin açık fikirlilik ve doğruyu arama düzeylerinin erkeklere oranla önemli derecede yüksek olduğu, müzik öğrencilerinin eleştirel düşünme eğilimleri ile akademik başarıları ve alan başarıları arasında önemli ilişki olmadığı belirlenmiştir. Resim öğrencilerinin eleştirel düşünme eğilimleri ile akademik başarıları arasında pozitif yönde yüksek anlamlı ilişki, alan başarıları arasında pozitif yönde düşük anlamlı ilişki olduğu saptanırken müzik öğrencilerinin ise eleştirel düşünme eğilimleri ile akademik başarıları ve alan başarıları arasında anlamlı bir ilişki olmadığı belirlenmiştir. Üniversite öğrencilerinin özellikle de öğretmen adaylarının eleştirel düşünme eğilimine sahip olmamalarının dikkat çekici olduğu düşünülmektedir. Sonuç olarak üniversitelerin en başta gelen ortak amaçlarından biri de analitik düşünme becerisine sahip olan, sorgulayan, merak eden bireyler yetiştirmektir. Araştırmanın sonuçları incelendiğinde öğretmen adaylarının eleştirel düşünme beceri ve eğilimine sahip olmasının meslek hayatlarında bu becerileri ve eğilimleri kendi öğrencilerine de kazandırmaları bakımından hayati bir önemesahip olduğu düşünülmektedir.

Chromosome segregation and the repair of DNA double-strand breaks (DSBs) require cohesin, the protein complex that mediates sister chromatid cohesion. Cohesion requires both a chromatin binding step and a subsequent tethering step called cohesion generation. Here we provide insight into how cohesion generation is restricted to S phase but can be activated in G2/M by a DSB in budding yeast. We show that Wpl1p inhibits cohesion in G2/M. A DSB counteracts Wpl1p and stimulates cohesion generation by first inducing the phosphorylation of the Mcd1p subunit of cohesin. This phosphorylation activates Eco1p-dependent acetylation of Mcd1p, which in turn antagonizes Wpl1p. Previous studies show that Eco1p antagonizes Wpl1p in S phase by acetylating the Smc3p subunit of cohesin. We show that Mcd1p and Smc3p acetylation antagonize Wpl1p only in their proper context. Thus, Eco1p antagonizes Wpl1p in distinct ways to modulate cohesion generation during the cell cycle and after DNA damage.

Chromosome segregation, transcriptional regulation, and repair of DNA double-strand breaks require the cohesin protein complex. Cohesin holds the replicated chromosomes (sister chromatids) together to mediate sister chromatid cohesion. The mechanism of how cohesion is established is unknown. We found that in budding yeast, the head domain of the Smc3p subunit of cohesin is acetylated by the Eco1p acetyltransferase at two evolutionarily conserved residues, promoting the chromatin-bound cohesin to tether sister chromatids. Smc3p acetylation is induced in S phase after the chromatin loading of cohesin and is suppressed in G 1 and G 2 /M. Smc3 head acetylation and its cell cycle regulation provide important insights into the biology and mechanism of cohesion establishment.

Cohesin, the protein complex that mediates sister chromatid cohesion, is required for faithful chromosome segregation and efficient repair of double-strand breaks (DSBs). Cohesion generation is normally restricted to S phase. However, in G2/M, a DSB activates cohesion generation near the DSB and genome-wide. Here, using budding yeast, we show that DSB-induced cohesion occurs when cohesin contains the kleisin subunit, Mcd1 (Scc1), but not when Mcd1 is replaced by its meiotic isoform, Rec8. We exploit this divergence to demonstrate that serine 83 of Mcd1 and the Chk1 kinase are critical determinants for DSB-induced cohesion. We propose that a DSB in G2/M activates Mec1 (ATR), which in turn stimulates Chk1-dependent phosphorylation of Mcd1 at serine 83. Serine 83 phosphorylation promotes chromatin-bound cohesin to become cohesive.

Faithful chromosome segregation and repair of DNA double-strand breaks (DSBs) require cohesin, the protein complex that mediates sister-chromatid cohesion. Cohesion between sister chromatids is thought to be generated only during ongoing DNA replication by an obligate coupling between cohesion establishment factors such as Eco1 (Ctf7) and the replisome. Using budding yeast, we challenge this model by showing that cohesion is generated by an Eco1-dependent but replication-independent mechanism in response to DSBs in G 2 /M. Furthermore, our studies reveal that Eco1 has two functions: a cohesive activity and a conserved acetyltransferase activity, which triggers the generation of cohesion in response to the DSB and the DNA damage checkpoint. Finally, the DSB-induced cohesion is not limited to broken chromosomes but occurs also on unbroken chromosomes, suggesting that the DNA damage checkpoint through Eco1 provides genome-wide protection of chromosome integrity.

Sister chromatid cohesion is established during S phase and maintained until anaphase. The cohesin complex (Mcd1p/Scc1p, Smc1p, Smc3p Irr1p/Scc3p in budding yeast) serves a structural role as it is required at all times when cohesion exists. Pds5p colocalizes temporally and spatially with cohesin on chromosomes but is thought to serve as a regulator of cohesion maintenance during mitosis. In contrast, Ctf7p/Eco1p is required during S phase for establishment but is not required during mitosis. Here we provide genetic and biochemical evidence that the pathways of cohesion establishment and maintenance are intimately linked. Our results show that mutants in ctf7 and pds5 are synthetically lethal. Moreover, over-expression of either CTF7 or PDS5 exhibits reciprocal suppression of the other mutant's temperature sensitivity. The suppression by CTF7 is specific for pds5 mutants as CTF7 over-expression increases the temperature sensitivity of an mcd1 mutant but has no effect on smc1 or smc3 mutants. Three additional findings provide new insights into the process of cohesion establishment. First, over-expression of ctf7 alleles deficient in acetylase activity exhibit significantly reduced suppression of the pds5 mutant but exacerbated toxicity to the mcd1 mutant. Second, using chromosome spreads and chromatin immuno-precipitation, we find either cohesin complex or Pds5p chromosomal localization is altered in ctf7 mutants. Finally, biochemical analysis reveals that Ctf7p and Pds5p coimmunoprecipitate, which physically links these regulators of cohesion establishment and maintenance. We propose a model whereby Ctf7p and Pds5p cooperate to facilitate efficient establishment by mediating changes in cohesin complex on chromosomes after its deposition.

The postreplicative repair of double-strand breaks (DSBs) is thought to require sister chromatid cohesion, provided by the cohesin complex along the chromosome arms. A further specialized role for cohesin in DSB repair is suggested by its de novo recruitment to regions of DNA damage in mammals. Here, we show in budding yeast that a single DSB induces the formation of a approximately 100 kb cohesin domain around the lesion. Our analyses suggest that the primary DNA damage checkpoint kinases Mec1p and Tel1p phosphorylate histone H2AX to generate a large domain, which is permissive for cohesin binding. Cohesin binding to the phospho-H2AX domain is enabled by Mre11p, a component of a critical repair complex, and Scc2p, a component of the cohesin loading machinery that is necessary for sister chromatid cohesion. We also provide evidence that the DSB-induced cohesin domain functions in postreplicative repair.

During meiosis, a single round of DNA replication is followed by two consecutive rounds of nuclear divisions called meiosis I and meiosis II. In meiosis I, homologous chromosomes segregate, while sister chromatids remain together. Determining how this unusual chromosome segregation behavior is established is central to understanding germ cell development. Here we show that preventing microtubule–kinetochore interactions during premeiotic S phase and prophase I is essential for establishing the meiosis I chromosome segregation pattern. Premature interactions of kinetochores with microtubules transform meiosis I into a mitosis-like division by disrupting two key meiosis I events: coorientation of sister kinetochores and protection of centromeric cohesin removal from chromosomes. Furthermore we find that restricting outer kinetochore assembly contributes to preventing premature engagement of microtubules with kinetochores. We propose that inhibition of microtubule–kinetochore interactions during premeiotic S phase and prophase I is central to establishing the unique meiosis I chromosome segregation pattern., Howard Hughes Medical Institute, National Institutes of Health (U.S.) (grant GM62207), Jane Coffin Childs Memorial Fund for Medical Research, American Cancer Society

Gametogenesis is a process whereby a germ cell differentiates into haploid gametes. We found that, in budding yeast, replicatively aged cells remove age-induced cellular damage during gametogenesis. Importantly, gametes of aged cells have the same replicative potential as those derived from young cells, indicating that life span resets during gametogenesis. Here, we explore the potential mechanisms responsible for gametogenesis-induced rejuvenation and discuss putative analogous mechanisms in higher eukaryotes., National Institutes of Health (U.S.) (grant GM62207)