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1.  The transcriptome landscape of early maize meiosis 
BMC Plant Biology  2014;14:118.
A major step in the higher plant life cycle is the decision to leave the mitotic cell cycle and begin the progression through the meiotic cell cycle that leads to the formation of gametes. The molecular mechanisms that regulate this transition and early meiosis remain largely unknown. To gain insight into gene expression features during the initiation of meiotic recombination, we profiled early prophase I meiocytes from maize (Zea mays) using capillary collection to isolate meiocytes, followed by RNA-seq.
We detected ~2,000 genes as preferentially expressed during early meiotic prophase, most of them uncharacterized. Functional analysis uncovered the importance of several cellular processes in early meiosis. Processes significantly enriched in isolated meiocytes included proteolysis, protein targeting, chromatin modification and the regulation of redox homeostasis. The most significantly up-regulated processes in meiocytes were processes involved in carbohydrate metabolism. Consistent with this, many mitochondrial genes were up-regulated in meiocytes, including nuclear- and mitochondrial-encoded genes. The data were validated with real-time PCR and in situ hybridization and also used to generate a candidate maize homologue list of known meiotic genes from Arabidopsis.
Taken together, we present a high-resolution analysis of the transcriptome landscape in early meiosis of an important crop plant, providing support for choosing genes for detailed characterization of recombination initiation and regulation of early meiosis. Our data also reveal an important connection between meiotic processes and altered/increased energy production.
PMCID: PMC4032173  PMID: 24885405
Maize; Meiosis; Meiocytes; Mitochondria; RNA-seq; Transcriptome
2.  Transcriptomic landscape of prophase I sunflower male meiocytes 
Meiosis is a form of specialized cell division that generates gametes, allowing recombination of alleles and halving the chromosome number. Arabidopsis and maize are the plant models that have been most extensively studied to determine the genes involved in meiosis. Here we present an RNA-seq study in which gene expression in male meiocytes isolated during prophase I was compared to that in somatic tissues of the sunflower HA89 line. We sampled more than 490 million gene tags from these libraries, assembled them de novo into a sunflower transcriptome. We obtained expression data for 36,304 sunflower genes, of which 19,574 (54%) were differentially expressed (DE) between meiocytes and somatic tissue. We also determined the functional categories and metabolic pathways that are DE in these libraries. As expected, we found large differences between the meiotic and somatic transcriptomes, which is in accordance with previous studies in Arabidopsis and maize. Furthermore, most of the previously implicated meiotic genes were abundantly and DE in meiocytes and a large repertoire of transcription factors (TF) and genes related to silencing are expressed in the sunflower meiocytes. We detected TFs which appear to be exclusively expressed in meiocytes. Our results allow for a better understanding of the conservation and differences in the meiotic transcriptome of plants.
PMCID: PMC4059168  PMID: 24982667
meiosis; transcriptome; RNA-seq; meiocytes; sunflower; meiotic genes; HA89
3.  Characterization of Putative cis-Regulatory Elements in Genes Preferentially Expressed in Arabidopsis Male Meiocytes 
BioMed Research International  2014;2014:708364.
Meiosis is essential for plant reproduction because it is the process during which homologous chromosome pairing, synapsis, and meiotic recombination occur. The meiotic transcriptome is difficult to investigate because of the size of meiocytes and the confines of anther lobes. The recent development of isolation techniques has enabled the characterization of transcriptional profiles in male meiocytes of Arabidopsis. Gene expression in male meiocytes shows unique features. The direct interaction of transcription factors (TFs) with DNA regulatory sequences forms the basis for the specificity of transcriptional regulation. Here, we identified putative cis-regulatory elements (CREs) associated with male meiocyte-expressed genes using in silico tools. The upstream regions (1 kb) of the top 50 genes preferentially expressed in Arabidopsis meiocytes possessed conserved motifs. These motifs are putative binding sites of TFs, some of which share common functions, such as roles in cell division. In combination with cell-type-specific analysis, our findings could be a substantial aid for the identification and experimental verification of the protein-DNA interactions for the specific TFs that drive gene expression in meiocytes.
PMCID: PMC4163388  PMID: 25250331
4.  Characterization of a set of novel meiotically-active promoters in Arabidopsis 
BMC Plant Biology  2012;12:104.
Homologous recombination, together with selection, laid the foundation for traditional plant breeding. The recombination process that takes place during meiotic cell division is crucial for the creation of novel variations of highly desired traits by breeders. Gaining control over this process is important for molecular breeding to achieve more precise, large-scale and quicker plant improvement. As conventional ubiquitous promoters are neither tissue-specific nor efficient in driving gene expression in meiocytes, promoters with high meiotic activities are potential candidates for manipulating the recombination process. So far, only a few meiotically-active promoters have been reported. Recently developed techniques to profile the transcriptome landscape of isolated meiocytes provided the means to discover promoters from genes that are actively expressed in meiosis.
In a screen for meiotically-active promoters, we examined ten promoter sequences that are associated with novel meiotic candidate genes. Each promoter was tested by expressing a GFP reporter gene in Arabidopsis. Characterization of regulatory regions revealed that these meiotically-active promoters possessed conserved motifs and motif arrangement. Some of the promoters unite optimal properties which are invaluable for meiosis-directed studies such as delivering specific gene expression in early meiosis I and/or meiosis II. Furthermore, the examination of homologs of the corresponding genes within green plants points to a great potential of applying the information from Arabidopsis to other species, especially crop plants.
We identified ten novel meiotically-active promoters; which, along with their homologs, are prime candidates to specifically drive gene expression during meiosis in plants and can thus provide important tools for meiosis study and crop breeding.
PMCID: PMC3462685  PMID: 22776406
Meiosis; Homologous recombination; Promoter; GFP; cis-regulatory elements; Plant molecular breeding
5.  Identification of germ cell-specific genes in mammalian meiotic prophase 
BMC Bioinformatics  2013;14:72.
Mammalian germ cells undergo meiosis to produce sperm or eggs, haploid cells that are primed to meet and propagate life. Meiosis is initiated by retinoic acid and meiotic prophase is the first and most complex stage of meiosis when homologous chromosomes pair to exchange genetic information. Errors in meiosis can lead to infertility and birth defects. However, despite the importance of this process, germ cell-specific gene expression patterns during meiosis remain undefined due to difficulty in obtaining pure germ cell samples, especially in females, where prophase occurs in the embryonic ovary. Indeed, mixed signals from both germ cells and somatic cells complicate gonadal transcriptome studies.
We developed a machine-learning method for identifying germ cell-specific patterns of gene expression in microarray data from mammalian gonads, specifically during meiotic initiation and prophase. At 10% recall, the method detected spermatocyte genes and oocyte genes with 90% and 94% precision, respectively. Our method outperformed gonadal expression levels and gonadal expression correlations in predicting germ cell-specific expression. Top-predicted spermatocyte and oocyte genes were both preferentially localized to the X chromosome and significantly enriched for essential genes. Also identified were transcription factors and microRNAs that might regulate germ cell-specific expression. Finally, we experimentally validated Rps6ka3, a top-predicted X-linked spermatocyte gene. Protein localization studies in the mouse testis revealed germ cell-specific expression of RPS6KA3, mainly detected in the cytoplasm of spermatogonia and prophase spermatocytes.
We have demonstrated that, through the use of machine-learning methods, it is possible to detect germ cell-specific expression from gonadal microarray data. Results from this study improve our understanding of the transition from germ cells to meiocytes in the mammalian gonad. Further, this approach is applicable to other tissues for which isolating cell populations remains difficult.
PMCID: PMC3599307  PMID: 23445120
6.  Unravelling the proteomic profile of rice meiocytes during early meiosis 
Transfer of genetic traits from wild or related species into cultivated rice is nowadays an important aim in rice breeding. Breeders use genetic crosses to introduce desirable genes from exotic germplasms into cultivated rice varieties. However, in many hybrids there is only a low level of pairing (if existing) and recombination at early meiosis between cultivated rice and wild relative chromosomes. With the objective of getting deeper into the knowledge of the proteins involved in early meiosis, when chromosomes associate correctly in pairs and recombine, the proteome of isolated rice meiocytes has been characterized by nLC-MS/MS at every stage of early meiosis (prophase I). Up to 1316 different proteins have been identified in rice isolated meiocytes in early meiosis, being 422 exclusively identified in early prophase I (leptotene, zygotene, or pachytene). The classification of proteins in functional groups showed that 167 were related to chromatin structure and remodeling, nucleic acid binding, cell-cycle regulation, and cytoskeleton. Moreover, the putative roles of 16 proteins which have not been previously associated to meiosis or were not identified in rice before, are also discussed namely: seven proteins involved in chromosome structure and remodeling, five regulatory proteins [such as SKP1 (OSK), a putative CDK2 like effector], a protein with RNA recognition motifs, a neddylation-related protein, and two microtubule-related proteins. Revealing the proteins involved in early meiotic processes could provide a valuable tool kit to manipulate chromosome associations during meiosis in rice breeding programs. The data have been deposited to the ProteomeXchange with the PXD001058 identifier.
PMCID: PMC4109522  PMID: 25104955
prophase I; chromosome recognition; chromosome pairing; meiocytes proteome; SKP1 proteins; cytoskeleton; MS/MS
7.  A Novel Mouse Synaptonemal Complex Protein Is Essential for Loading of Central Element Proteins, Recombination, and Fertility 
PLoS Genetics  2011;7(5):e1002088.
The synaptonemal complex (SC) is a proteinaceous, meiosis-specific structure that is highly conserved in evolution. During meiosis, the SC mediates synapsis of homologous chromosomes. It is essential for proper recombination and segregation of homologous chromosomes, and therefore for genome haploidization. Mutations in human SC genes can cause infertility. In order to gain a better understanding of the process of SC assembly in a model system that would be relevant for humans, we are investigating meiosis in mice. Here, we report on a newly identified component of the murine SC, which we named SYCE3. SYCE3 is strongly conserved among mammals and localizes to the central element (CE) of the SC. By generating a Syce3 knockout mouse, we found that SYCE3 is required for fertility in both sexes. Loss of SYCE3 blocks synapsis initiation and results in meiotic arrest. In the absence of SYCE3, initiation of meiotic recombination appears to be normal, but its progression is severely impaired resulting in complete absence of MLH1 foci, which are presumed markers of crossovers in wild-type meiocytes. In the process of SC assembly, SYCE3 is required downstream of transverse filament protein SYCP1, but upstream of the other previously described CE–specific proteins. We conclude that SYCE3 enables chromosome loading of the other CE–specific proteins, which in turn would promote synapsis between homologous chromosomes.
Author Summary
Meiosis is a special type of cell division that takes place in the germ line of sexually reproducing diploid organisms. Major events during meiosis are the pairing, recombination, and segregation of homologous chromosomes. As a consequence, daughter cells are haploid and genetically diverse. Therefore, meiosis is of utmost importance for the life of sexually reproducing species as it maintains the species-specific chromosome number and generates genetic diversity within a species. Proper segregation of homologous chromosomes during meiosis requires homolog pairs to be physically linked. The synaptonemal complex (SC), a meiosis-specific structure conserved in evolution, is essential for this process. Defective assembly of the SC has deleterious effects on germ cells and can cause infertility in mice and humans. Here, we report on a newly identified protein component of the mammalian SC that we have named SYCE3. SYCE3 is strongly conserved among mammals. Using the mouse as a model system, we demonstrate that loss of SYCE3 leads to infertility in both sexes. Infertility is caused by disruption of meiosis due to the inability of Syce3−/− mice to assemble the central element of SCs. Our findings provide new insights into the complexity of SC assembly and its relevance to mammalian fertility.
PMCID: PMC3102746  PMID: 21637789
8.  Tomato Male sterile 1035 is essential for pollen development and meiosis in anthers 
Journal of Experimental Botany  2014;65(22):6693-6709.
This study demonstrated that tomato Male sterile 10 35 encodes a basic helix–loop–helix transcription factor involved in meiosis and tapetum development at the early stage of anther development.
Male fertility in flowering plants depends on proper cellular differentiation in anthers. Meiosis and tapetum development are particularly important processes in pollen production. In this study, we showed that the tomato male sterile (ms10 35) mutant of cultivated tomato (Solanum lycopersicum) exhibited dysfunctional meiosis and an abnormal tapetum during anther development, resulting in no pollen production. We demonstrated that Ms10 35 encodes a basic helix–loop–helix transcription factor that is specifically expressed in meiocyte and tapetal tissue from pre-meiotic to tetrad stages. Transgenic expression of the Ms10 35 gene from its native promoter complemented the male sterility of the ms10 35 mutant. In addition, RNA-sequencing-based transcriptome analysis revealed that Ms10 35 regulates 246 genes involved in anther development processes such as meiosis, tapetum development, cell-wall degradation, pollen wall formation, transport, and lipid metabolism. Our results indicate that Ms10 35 plays key roles in regulating both meiosis and programmed cell death of the tapetum during microsporogenesis.
PMCID: PMC4246194  PMID: 25262227
Anther; male sterility; meiosis; tapetum; tomato (Solanum lycopersicum).
9.  Identification of Arabidopsis Meiotic Cyclins Reveals Functional Diversification among Plant Cyclin Genes 
PLoS Genetics  2013;9(5):e1003508.
Meiosis is a modified cell division in which a single S-phase is followed by two rounds of chromosome segregation resulting in the production of haploid gametes. The meiotic mode of chromosome segregation requires extensive remodeling of the basic cell cycle machinery and employment of unique regulatory mechanisms. Cyclin-dependent kinases (CDKs) and cyclins represent an ancient molecular module that drives and regulates cell cycle progression. The cyclin gene family has undergone a massive expansion in angiosperm plants, but only a few cyclins were thoroughly characterized. In this study we performed a systematic immunolocalization screen to identify Arabidopsis thaliana A- and B-type cyclins expressed in meiosis. Many of these cyclins exhibit cell-type-specific expression in vegetative tissues and distinct subcellular localization. We found six A-type cyclins and a single B-type cyclin (CYCB3;1) to be expressed in male meiosis. Mutant analysis revealed that these cyclins contribute to distinct meiosis-related processes. While A2 cyclins are important for chromosome segregation, CYCB3;1 prevents ectopic cell wall formation. We further show that cyclin SDS does not contain a D-box and is constitutively expressed throughout meiosis. Analysis of plants carrying cyclin SDS with an introduced D-box motif determined that, in addition to its function in recombination, SDS acts together with CYCB3;1 in suppressing unscheduled cell wall synthesis. Our phenotypic and expression data provide extensive evidence that multiplication of cyclins is in plants accompanied by functional diversification.
Author Summary
The alteration of haploid and diploid cell generations during the sexual life cycle requires meiosis, a specialized cell division that enables the formation of haploid gametes from diploid cells. Meiosis occurs only once during the life cycle, and the transition from the mitotic to meiotic mode of chromosome partitioning requires extensive remodeling of the cell cycle machinery. The cell cycle progression is driven by cyclin-dependent kinases and associated cyclins that regulate CDK activity and confer substrate specificity. Cyclin gene families have undergone a massive expansion in plants, which has raised the question of whether some of these cyclins evolved specific meiotic functions. We systematically analyzed two cyclin gene families in Arabidopsis to identify plant cyclins that are meiotically expressed. We found in total eight cyclins to be expressed in male meiotic cells, and functional characterization revealed their involvement in diverse meiotic processes. Interestingly, none of the cyclins appear to be essential for meiotic progression, indicating that plant meiosis is governed by unorthodox cell cycle regulators.
PMCID: PMC3649987  PMID: 23671425
10.  A Novel RNA-Recognition-Motif Protein Is Required for Premeiotic G1/S-Phase Transition in Rice (Oryza sativa L.) 
PLoS Genetics  2011;7(1):e1001265.
The molecular mechanism for meiotic entry remains largely elusive in flowering plants. Only Arabidopsis SWI1/DYAD and maize AM1, both of which are the coiled-coil protein, are known to be required for the initiation of plant meiosis. The mechanism underlying the synchrony of male meiosis, characteristic to flowering plants, has also been unclear in the plant kingdom. In other eukaryotes, RNA-recognition-motif (RRM) proteins are known to play essential roles in germ-cell development and meiosis progression. Rice MEL2 protein discovered in this study shows partial similarity with human proline-rich RRM protein, deleted in Azoospermia-Associated Protein1 (DAZAP1), though MEL2 also possesses ankyrin repeats and a RING finger motif. Expression analyses of several cell-cycle markers revealed that, in mel2 mutant anthers, most germ cells failed to enter premeiotic S-phase and meiosis, and a part escaped from the defect and underwent meiosis with a significant delay or continued mitotic cycles. Immunofluorescent detection revealed that T7 peptide-tagged MEL2 localized at cytoplasmic perinuclear region of germ cells during premeiotic interphase in transgenic rice plants. This study is the first report of the plant RRM protein, which is required for regulating the premeiotic G1/S-phase transition of male and female germ cells and also establishing synchrony of male meiosis. This study will contribute to elucidation of similarities and diversities in reproduction system between plants and other species.
Author Summary
Meiosis is a pivotal event to produce haploid spores and gametes in all sexually reproducing species and is a fundamentally different type of cell cycle from mitosis. Thus, the molecular mechanisms to switch the cell cycle from mitosis to meiosis have been studied by many researchers. In yeast and metazoans, RNA-binding proteins are known to play important roles in the post-transcriptional regulation of genes implicated in the meiotic entry and meiosis. In contrast, in the plant kingdom, the mechanisms to control the meiotic entry have largely remained elusive. In this study, we discover a novel RNA-recognition-motif (RRM) protein in rice (Oryza sativa L.), designated MEL2, and demonstrate that MEL2 is required for the faithful transition of germ cells from mitosis to meiotic cell cycle. Rice MEL2 shows partial similarity with human DAZAP1, which is an RRM protein and relates to Azoospermia syndrome in human, while there are critical structural differences between germline-specific RRM proteins of mammals and plants. Our findings will lead the molecular-biological studies of plant meiotic entry to the next steps and will enable a comparison of the systems of meiotic entry between animals and plants.
PMCID: PMC3017114  PMID: 21253568
11.  Sequencing-based large-scale genomics approaches with small numbers of isolated maize meiocytes 
High-throughput sequencing has become the large-scale approach of choice to study global gene expression and the distribution of specific chromatin marks and features. However, the limited availability of large amounts of purified cells made it very challenging to apply sequencing-based techniques in plant meiosis research in the past. In this paper, we describe a method to isolate meiocytes from maize anthers and detailed protocols to successfully perform RNA-seq, smRNA-seq, H3K4me3-ChIP-seq, and DNA bisulfite conversion sequencing with 5000–30,000 isolated maize male meiotic cells. These methods can be adjusted for other flowering plant species as well.
PMCID: PMC3933774  PMID: 24611068
meiocytes; meiosis; Illumina sequencing; RNA-seq; ChIP-seq; DNA methylation; small RNA; maize
12.  AtMND1 is required for homologous pairing during meiosis in Arabidopsis 
Pairing of homologous chromosomes at meiosis is an important requirement for recombination and balanced chromosome segregation among the products of meiotic division. Recombination is initiated by double strand breaks (DSBs) made by Spo11 followed by interaction of DSB sites with a homologous chromosome. This interaction requires the strand exchange proteins Rad51 and Dmc1 that bind to single stranded regions created by resection of ends at the site of DSBs and promote interactions with uncut DNA on the homologous partner. Recombination is also considered to be dependent on factors that stabilize interactions between homologous chromosomes. In budding yeast Hop2 and Mnd1 act as a complex to promote homologous pairing and recombination in conjunction with Rad51 and Dmc1.
We have analyzed the function of the Arabidopsis orthologue of the budding yeast MND1 gene (AtMND1). Loss of AtMND1 did not affect normal vegetative development but caused fragmentation and missegregation of chromosomes in male and female meiosis, formation of inviable gametes, and sterility. Analysis of the Atmnd1 Atspo11-1 double mutant indicated that chromosome fragmentation in Atmnd1 was suppressed by loss of Atspo11-1. Fluorescence in situ hybridization (FISH) analysis showed that homologous pairing failed to occur and homologues remained apart throughout meiosis. AtMND1 showed strong expression in meiocytes as revealed by RNA in situs.
We conclude that AtMND1 is required for homologous pairing and is likely to play a role in the repair of DNA double strand breaks during meiosis in Arabidopsis, thus showing conservation of function with that of MND1 during meiosis in yeast.
PMCID: PMC1557525  PMID: 16872528
13.  The Arabidopsis thaliana PARTING DANCERS Gene Encoding a Novel Protein Is Required for Normal Meiotic Homologous RecombinationD⃞ 
Molecular Biology of the Cell  2006;17(3):1331-1343.
Recent studies of meiotic recombination in the budding yeast and the model plant Arabidopsis thaliana indicate that meiotic crossovers (COs) occur through two genetic pathways: the interference-sensitive pathway and the interference-insensitive pathway. However, few genes have been identified in either pathway. Here, we describe the identification of the PARTING DANCERS (PTD) gene, as a gene with an elevated expression level in meiocytes. Analysis of two independently generated transferred DNA insertional lines in PTD showed that the mutants had reduced fertility. Further cytological analysis of male meiosis in the ptd mutants revealed defects in meiosis, including reduced formation of chiasmata, the cytological appearance of COs. The residual chiasmata in the mutants were distributed randomly, indicating that the ptd mutants are defective for CO formation in the interference-sensitive pathway. In addition, transmission electron microscopic analysis of the mutants detected no obvious abnormality of synaptonemal complexes and apparently normal late recombination nodules at the pachytene stage, suggesting that the mutant's defects in bivalent formation were postsynaptic. Comparison to other genes with limited sequence similarity raises the possibility that PTD may present a previously unknown function conserved in divergent eukaryotic organisms.
PMCID: PMC1382321  PMID: 16394097
14.  Mouse Y-Linked Zfy1 and Zfy2 Are Expressed during the Male-Specific Interphase between Meiosis I and Meiosis II and Promote the 2nd Meiotic Division 
PLoS Genetics  2014;10(6):e1004444.
Mouse Zfy1 and Zfy2 encode zinc finger transcription factors that map to the short arm of the Y chromosome (Yp). They have previously been shown to promote meiotic quality control during pachytene (Zfy1 and Zfy2) and at the first meiotic metaphase (Zfy2). However, from these previous studies additional roles for genes encoded on Yp during meiotic progression were inferred. In order to identify these genes and investigate their function in later stages of meiosis, we created three models with diminishing Yp and Zfy gene complements (but lacking the Y-long-arm). Since the Y-long-arm mediates pairing and exchange with the X via their pseudoautosomal regions (PARs) we added a minute PAR-bearing X chromosome derivative to enable formation of a sex bivalent, thus avoiding Zfy2-mediated meiotic metaphase I (MI) checkpoint responses to the unpaired (univalent) X chromosome. Using these models we obtained definitive evidence that genetic information on Yp promotes meiosis II, and by transgene addition identified Zfy1 and Zfy2 as the genes responsible. Zfy2 was substantially more effective and proved to have a much more potent transactivation domain than Zfy1. We previously established that only Zfy2 is required for the robust apoptotic elimination of MI spermatocytes in response to a univalent X; the finding that both genes potentiate meiosis II led us to ask whether there was de novo Zfy1 and Zfy2 transcription in the interphase between meiosis I and meiosis II, and this proved to be the case. X-encoded Zfx was also expressed at this stage and Zfx over-expression also potentiated meiosis II. An interphase between the meiotic divisions is male-specific and we previously hypothesised that this allows meiosis II critical X and Y gene reactivation following sex chromosome silencing in meiotic prophase. The interphase transcription and meiosis II function of Zfx, Zfy1 and Zfy2 validate this hypothesis.
Author Summary
The mouse Y chromosome genes Zfy1 and Zfy2 were first identified in the late 1980s during the search for the gene on the Y that triggers male development; they encode proteins that regulate the expression of other genes to which they bind via a ‘zinc finger’ domain. We have now discovered that these genes play important roles during spermatogenesis. Zfy2 proved to be essential for the efficient operation of a ‘checkpoint’ during the first meiotic division that identifies and kills cells that would otherwise produce sperm with an unbalanced chromosome set. Female meiosis, which does not have an equivalent checkpoint, generates a significant proportion of eggs with an unbalanced chromosome set. In the present study we show that Zfy2 also has a major role in ensuring that the second meiotic division occurs, with Zfy1 and a related gene, Zfx, on the X chromosome providing some support. In order to fulfil this function all three genes are expressed in the ‘interphase’ stage between the two divisions. In female meiosis there is no interphase stage between the two meiotic divisions but in this case essential functions during the divisions are supported by stored RNAs, so an interphase is not needed.
PMCID: PMC4072562  PMID: 24967676
15.  Programmed fluctuations in sense/antisense transcript ratios drive sexual differentiation in S. pombe 
Strand-specific RNA sequencing of S. pombe reveals a highly structured programme of ncRNA expression at over 600 loci. Functional investigations show that this extensive ncRNA landscape controls the complex programme of sexual differentiation in S. pombe.
The model eukaryote S. pombe features substantial numbers of ncRNAs many of which are antisense regulatory transcripts (ARTs), ncRNAs expressed on the opposing strand to coding sequences.Individual ARTs are generated during the mitotic cycle, or at discrete stages of sexual differentiation to downregulate the levels of proteins that drive and coordinate sexual differentiation.Antisense transcription occurring from events such as bidirectional transcription is not simply artefactual ‘chatter', it performs a critical role in regulating gene expression.
Regulation of the RNA profile is a principal control driving sexual differentiation in the fission yeast Schizosaccharomyces pombe. Before transcription, RNAi-mediated formation of heterochromatin is used to suppress expression, while post-transcription, regulation is achieved via the active stabilisation or destruction of transcripts, and through at least two distinct types of splicing control (Mata et al, 2002; Shimoseki and Shimoda, 2001; Averbeck et al, 2005; Mata and Bähler, 2006; Xue-Franzen et al, 2006; Moldon et al, 2008; Djupedal et al, 2009; Amorim et al, 2010; Grewal, 2010; Cremona et al, 2011).
Around 94% of the S. pombe genome is transcribed (Wilhelm et al, 2008). While many of these transcripts encode proteins (Wood et al, 2002; Bitton et al, 2011), the majority have no known function. We used a strand-specific protocol to sequence total RNA extracts taken from vegetatively growing cells, and at different points during a time course of sexual differentiation. The resulting data redefined existing gene coordinates and identified additional transcribed loci. The frequency of reads at each of these was used to monitor transcript abundance.
Transcript levels at 6599 loci changed in at least one sample (G-statistic; False Discovery Rate <5%). 4231 (72.3%), of which 4011 map to protein-coding genes, while 809 loci were antisense to a known gene. Comparisons between haploid and diploid strains identified changes in transcript levels at over 1000 loci.
At 354 loci, greater antisense abundance was observed relative to sense, in at least one sample (putative antisense regulatory transcripts—ARTs). Since antisense mechanisms are known to modulate sense transcript expression through a variety of inhibitory mechanisms (Faghihi and Wahlestedt, 2009), we postulated that the waves of antisense expression activated at different stages during meiosis might be regulating protein expression.
To ask whether transcription factors that drive sense-transcript levels influenced ART production, we performed RNA-seq of a pat1.114 diploid meiosis in the absence of the transcription factors Atf21 and Atf31 (responsible for late meiotic transcription; Mata et al, 2002). Transcript levels at 185 ncRNA loci showed significant changes in the knockout backgrounds. Although meiotic progression is largely unaffected by removal of Atf21 and Atf31, viability of the resulting spores was significantly diminished, indicating that Atf21- and Atf31-mediated events are critical to efficient sexual differentiation.
If changes to relative antisense/sense transcript levels during a particular phase of sexual differentiation were to regulate protein expression, then the continued presence of the antisense at points in the differentiation programme where it would normally be absent should abolish protein function during this phase. We tested this hypothesis at four loci representing the three means of antisense production: convergent gene expression, improper termination and nascent transcription from an independent locus. Induction of the natural antisense transcripts that opposed spo4+, spo6+ and dis1+ (Figures 3 and 7) in trans from a heterologous locus phenocopied a loss of function of the target protein. ART overexpression decreased Dis1 protein levels. Antisense transcription opposing spk1+ originated from improper termination of the sense ups1+ transcript on the opposite strand (Figure 3B, left locus). Expression of either the natural full-length ups1+ transcript or a truncated version, restricted to the portion of ups1+ overlapping spk1+ (Figure 3, orange transcripts) in trans from a heterologous locus phenocopied the spk1.Δ differentiation deficiency. Convergent transcription from a neighbouring gene on the opposing strand is, therefore, an effective mechanism to generate RNAi-mediated (below) silencing in fission yeast. Further analysis of the data revealed, for many loci, substantial changes in UTR length over the course of meiosis, suggesting that UTR dynamics may have an active role in regulating gene expression by controlling the transcriptional overlap between convergent adjacent gene pairs.
The RNAi machinery (Grewal, 2010) was required for antisense suppression at each of the dis1, spk1, spo4 and spo6 loci, as antisense to each locus had no impact in ago1.Δ, dcr1.Δ and rdp1.Δ backgrounds. We conclude that RNAi control has a key role in maintaining the fidelity of sexual differentiation in fission yeast. The histone H3 methyl transferase Clr4 was required for antisense control from a heterologous locus.
Thus, a significant portion of the impact of ncRNA upon sexual differentiation arises from antisense gene silencing. Importantly, in contrast to the extensively characterised ability of the RNAi machinery to operate in cis at a target locus in S. pombe (Grewal, 2010), each case of gene silencing generated here could be achieved in trans by expression of the antisense transcript from a single heterologous locus elsewhere in the genome.
Integration of an antibiotic marker gene immediately downstream of the dis1+ locus instigated antisense control in an orientation-dependent manner. PCR-based gene tagging approaches are widely used to fuse the coding sequences of epitope or protein tags to a gene of interest. Not only do these tagging approaches disrupt normal 3′UTR controls, but the insertion of a heterologous marker gene immediately downstream of an ORF can clearly have a significant impact upon transcriptional control of the resulting fusion protein. Thus, PCR tagging approaches can no longer be viewed as benign manipulations of a locus that only result in the production of a tagged protein product.
Repression of Dis1 function by gene deletion or antisense control revealed a key role this conserved microtubule regulator in driving the horsetail nuclear migrations that promote recombination during meiotic prophase.
Non-coding transcripts have often been viewed as simple ‘chatter', maintained solely because evolutionary pressures have not been strong enough to force their elimination from the system. Our data show that phenomena such as improper termination and bidirectional transcription are not simply interesting artifacts arising from the complexities of transcription or genome history, but have a critical role in regulating gene expression in the current genome. Given the widespread use of RNAi, it is reasonable to anticipate that future analyses will establish ARTs to have equal importance in other organisms, including vertebrates.
These data highlight the need to modify our concept of a gene from that of a spatially distinct locus. This view is becoming increasingly untenable. Not only are the 5′ and 3′ ends of many genes indistinct, but that this lack of a hard and fast boundary is actively used by cells to control the transcription of adjacent and overlapping loci, and thus to regulate critical events in the life of a cell.
Strand-specific RNA sequencing of S. pombe revealed a highly structured programme of ncRNA expression at over 600 loci. Waves of antisense transcription accompanied sexual differentiation. A substantial proportion of ncRNA arose from mechanisms previously considered to be largely artefactual, including improper 3′ termination and bidirectional transcription. Constitutive induction of the entire spk1+, spo4+, dis1+ and spo6+ antisense transcripts from an integrated, ectopic, locus disrupted their respective meiotic functions. This ability of antisense transcripts to disrupt gene function when expressed in trans suggests that cis production at native loci during sexual differentiation may also control gene function. Consistently, insertion of a marker gene adjacent to the dis1+ antisense start site mimicked ectopic antisense expression in reducing the levels of this microtubule regulator and abolishing the microtubule-dependent ‘horsetail' stage of meiosis. Antisense production had no impact at any of these loci when the RNA interference (RNAi) machinery was removed. Thus, far from being simply ‘genome chatter', this extensive ncRNA landscape constitutes a fundamental component in the controls that drive the complex programme of sexual differentiation in S. pombe.
PMCID: PMC3738847  PMID: 22186733
antisense; meiosis; ncRNA; S. pombe; siRNA
16.  A novel method to follow meiotic progression in Arabidopsis using confocal microscopy and 5-ethynyl-2′-deoxyuridine labeling 
Plant Methods  2014;10(1):33.
Meiosis progression in the more recent past has been investigated using 5-bromo-2′-deoxyuridine (BrdU) uptake by S-phase meiocytes undergoing DNA replication. BrdU uptake is detected by reaction with BrdU antibody followed by epifluorescent microscopy examination of chromosome spreads and/or squashes. We here report using confocal microscopic examination of intact meiocytes in conjunction with the new thymidine analog 5-ethynyl-2′-deoxyuridine (EdU). The simplicity of the EdU detection coupled with confocal examination of anthers provides a more exact temporal description of meiotic prophase I progression in Arabidopsis and opens up the possibility of examining the coordination of microsporocyte development with the other tissues of the anther.
Using our time course protocol, we have determined the duration of wild type Arabidopsis leptotene to be 5 h, zygotene -6 h, pachytene -10 h and a diplotene duration of approximately 1 h. We estimate G2 duration to be approximately 7 h based on the timing of the initial appearance of EdU signal in early leptotene meiocytes. In addition we have found that DNA replication in meiocytes is not done synchronously with the associated tapetal layer of cells. The EdU labeling suggests that S-phase replication of meiocyte DNA precedes the duplication of tapetal cell DNA.
The increased number of meiotic staging criteria that can be assessed in our confocal analysis, as compared to chromosome spreading or squashing, makes the identification of even the early and late portions of the prophase I substages attainable. This enhanced staging coupled with the ability to easily generate large data sets at hourly time points makes it possible to more exactly determine substage duration and to detect modest temporal abnormalities involving meiocyte entrance into and/or exit from leptotene, zygotene and pachytene. Confocal analysis also makes it possible to study the relationships between different cell types within the flower bud as meiosis proceeds.
Electronic supplementary material
The online version of this article (doi:10.1186/1746-4811-10-33) contains supplementary material, which is available to authorized users.
PMCID: PMC4203904  PMID: 25337148
Arabidopsis; Meiosis; S-phase; Prophase I; Confocal microscopy; EdU labeling; Meiocyte filament; Tapetal cells; Time course; Multi-criteria meiotic staging; DNA replication
17.  Analysis of anther transcriptomes to identify genes contributing to meiosis and male gametophyte development in rice 
BMC Plant Biology  2011;11:78.
In flowering plants, the anther is the site of male gametophyte development. Two major events in the development of the male germline are meiosis and the asymmetric division in the male gametophyte that gives rise to the vegetative and generative cells, and the following mitotic division in the generative cell that produces two sperm cells. Anther transcriptomes have been analyzed in many plant species at progressive stages of development by using microarray and sequence-by synthesis-technologies to identify genes that regulate anther development. Here we report a comprehensive analysis of rice anther transcriptomes at four distinct stages, focusing on identifying regulatory components that contribute to male meiosis and germline development. Further, these transcriptomes have been compared with the transcriptomes of 10 stages of rice vegetative and seed development to identify genes that express specifically during anther development.
Transcriptome profiling of four stages of anther development in rice including pre-meiotic (PMA), meiotic (MA), anthers at single-celled (SCP) and tri-nucleate pollen (TPA) revealed about 22,000 genes expressing in at least one of the anther developmental stages, with the highest number in MA (18,090) and the lowest (15,465) in TPA. Comparison of these transcriptome profiles to an in-house generated microarray-based transcriptomics database comprising of 10 stages/tissues of vegetative as well as reproductive development in rice resulted in the identification of 1,000 genes specifically expressed in anther stages. From this sub-set, 453 genes were specific to TPA, while 78 and 184 genes were expressed specifically in MA and SCP, respectively. The expression pattern of selected genes has been validated using real time PCR and in situ hybridizations. Gene ontology and pathway analysis of stage-specific genes revealed that those encoding transcription factors and components of protein folding, sorting and degradation pathway genes dominated in MA, whereas in TPA, those coding for cell structure and signal transduction components were in abundance. Interestingly, about 50% of the genes with anther-specific expression have not been annotated so far.
Not only have we provided the transcriptome constituents of four landmark stages of anther development in rice but we have also identified genes that express exclusively in these stages. It is likely that many of these candidates may therefore contribute to specific aspects of anther and/or male gametophyte development in rice. In addition, the gene sets that have been produced will assist the plant reproductive community in building a deeper understanding of underlying regulatory networks and in selecting gene candidates for functional validation.
PMCID: PMC3112077  PMID: 21554676
18.  Analysis of Meiosis in SUN1 Deficient Mice Reveals a Distinct Role of SUN2 in Mammalian Meiotic LINC Complex Formation and Function 
PLoS Genetics  2014;10(2):e1004099.
LINC complexes are evolutionarily conserved nuclear envelope bridges, composed of SUN (Sad-1/UNC-84) and KASH (Klarsicht/ANC-1/Syne/homology) domain proteins. They are crucial for nuclear positioning and nuclear shape determination, and also mediate nuclear envelope (NE) attachment of meiotic telomeres, essential for driving homolog synapsis and recombination. In mice, SUN1 and SUN2 are the only SUN domain proteins expressed during meiosis, sharing their localization with meiosis-specific KASH5. Recent studies have shown that loss of SUN1 severely interferes with meiotic processes. Absence of SUN1 provokes defective telomere attachment and causes infertility. Here, we report that meiotic telomere attachment is not entirely lost in mice deficient for SUN1, but numerous telomeres are still attached to the NE through SUN2/KASH5-LINC complexes. In Sun1−/− meiocytes attached telomeres retained the capacity to form bouquet-like clusters. Furthermore, we could detect significant numbers of late meiotic recombination events in Sun1−/− mice. Together, this indicates that even in the absence of SUN1 telomere attachment and their movement within the nuclear envelope per se can be functional.
Author Summary
Correct genome haploidization during meiosis requires tightly regulated chromosome movements that follow a highly conserved choreography during prophase I. Errors in these movements cause subsequent meiotic defects, which typically lead to infertility. At the beginning of meiotic prophase, chromosome ends are tethered to the nuclear envelope (NE). This attachment of telomeres appears to be mediated by well-conserved membrane spanning protein complexes within the NE (LINC complexes). In mouse meiosis, the two main LINC components SUN1 and SUN2 were independently described to localize at the sites of telomere attachment. While SUN1 has been demonstrated to be critical for meiotic telomere attachment, the precise role of SUN2 in this context, however, has been discussed controversially in the field. Our current study was targeted to determine the factual capacity of SUN2 in telomere attachment and chromosome movements in SUN1 deficient mice. Remarkably, although telomere attachment is impaired in the absence of SUN1, we could find a yet undescribed SUN1-independent telomere attachment, which presumably is mediated by SUN2 and KASH5. This SUN2 mediated telomere attachment is stable throughout prophase I and functional in moving telomeres within the NE. Thus, our results clearly indicate that SUN1 and SUN2, at least partially, fulfill redundant meiotic functions.
PMCID: PMC3937131  PMID: 24586178
19.  The Cell Cycle Timing of Centromeric Chromatin Assembly in Drosophila Meiosis Is Distinct from Mitosis Yet Requires CAL1 and CENP-C 
PLoS Biology  2012;10(12):e1001460.
The centromeric histone CENP-A is incorporated at different cell cycle phases during somatic mitosis, meiosis I and meiosis II in Drosophila melanogaster.
CENP-A (CID in flies) is the histone H3 variant essential for centromere specification, kinetochore formation, and chromosome segregation during cell division. Recent studies have elucidated major cell cycle mechanisms and factors critical for CENP-A incorporation in mitosis, predominantly in cultured cells. However, we do not understand the roles, regulation, and cell cycle timing of CENP-A assembly in somatic tissues in multicellular organisms and in meiosis, the specialized cell division cycle that gives rise to haploid gametes. Here we investigate the timing and requirements for CID assembly in mitotic tissues and male and female meiosis in Drosophila melanogaster, using fixed and live imaging combined with genetic approaches. We find that CID assembly initiates at late telophase and continues during G1 phase in somatic tissues in the organism, later than the metaphase assembly observed in cultured cells. Furthermore, CID assembly occurs at two distinct cell cycle phases during male meiosis: prophase of meiosis I and after exit from meiosis II, in spermatids. CID assembly in prophase I is also conserved in female meiosis. Interestingly, we observe a novel decrease in CID levels after the end of meiosis I and before meiosis II, which correlates temporally with changes in kinetochore organization and orientation. We also demonstrate that CID is retained on mature sperm despite the gross chromatin remodeling that occurs during protamine exchange. Finally, we show that the centromere proteins CAL1 and CENP-C are both required for CID assembly in meiosis and normal progression through spermatogenesis. We conclude that the cell cycle timing of CID assembly in meiosis is different from mitosis and that the efficient propagation of CID through meiotic divisions and on sperm is likely to be important for centromere specification in the developing zygote.
Author Summary
Centromeres are regions of eukaryotic chromosomes that recruit the kinetochores and are essential for faithful segregation of DNA during all cell divisions. The centromere-specific histone H3 variant CENP-A accumulates at the centromere, defining this region, and is maintained throughout cellular generations by epigenetic mechanisms in most eukaryotes. Previous studies have discovered many factors regulating both the maintenance and assembly of CENP-A at centromeres during mitosis in cultured cells, but the mode of regulation of CENP-A assembly during meiosis and mitosis in animal tissues is unknown. In this study, we use Drosophila melanogaster as an organismal model to investigate the timing and requirements for assembly of CID, the fly CENP-A homolog. We find that that CID is loaded at centromeres during telophase/G1 phase in brain stem and nonstem cells. In male meiosis, CID is loaded in two phases, during the first stages of meiosis I and after the second meiotic division. Meiosis I loading time is also conserved in females. We also report an unprecedented drop in CID levels after meiosis I and before meiosis II, which correlates with the timing of kinetochore reorientation. Additionally, we find that two essential centromere proteins (CAL1 and CENP-C) are necessary for CID assembly and chromosome segregation during meiosis. Our data demonstrate novel differential timing for CENP-A assembly during mitosis and meiosis in the whole organism.
PMCID: PMC3531500  PMID: 23300382
20.  Onset of meiosis in the chicken embryo; evidence of a role for retinoic acid 
Meiosis in higher vertebrates shows a dramatic sexual dimorphism: germ cells enter meiosis and arrest at prophase I during embryogenesis in females, whereas in males they enter mitotic arrest during embryogenesis and enter meiosis only after birth. Here we report the molecular analysis of meiosis onset in the chicken model and provide evidence for conserved regulation by retinoic acid.
Meiosis in the chicken embryo is initiated late in embryogenesis (day 15.5), relative to gonadal sex differentiation (from day 6). Meiotic germ cells are first detectable only in female gonads from day 15.5, correlating with the expression of the meiosis marker, SCP3. Gonads isolated from day 10.5 female embryos and grown in serum-free medium could still initiate meiosis at day 16.5, suggesting that this process is controlled by an endogenous clock in the germ cells themselves, and/or that germ cells are already committed to meiosis at the time of explantation. Early commitment is supported by the analysis of chicken STRA8, a pre-meiotic marker shown to be essential for meiosis in mouse. Chicken STRA8 is expressed female-specifically from embryonic day 12.5, preceding morphological evidence of meiosis at day 15.5. Previous studies have shown that, in the mouse embryo, female-specific induction of STRA8 and meiosis are triggered by retinoic acid. A comprehensive analysis of genes regulating retinoic acid metabolism in chicken embryos reveals dynamic expression in the gonads. In particular, the retinoic acid-synthesising enzyme, RALDH2, is expressed in the left ovarian cortex at the time of STRA8 up-regulation, prior to meiosis.
This study presents the first molecular analysis of meiosis onset in an avian embryo. Although aspects of avian meiosis differ from that of mammals, a role for retinoic acid may be conserved.
PMCID: PMC2564928  PMID: 18799012
21.  Genome-Wide Crossover Distribution in Arabidopsis thaliana Meiosis Reveals Sex-Specific Patterns along Chromosomes 
PLoS Genetics  2011;7(11):e1002354.
In most species, crossovers (COs) are essential for the accurate segregation of homologous chromosomes at the first meiotic division. Their number and location are tightly regulated. Here, we report a detailed, genome-wide characterization of the rate and localization of COs in Arabidopsis thaliana, in male and female meiosis. We observed dramatic differences between male and female meiosis which included: (i) genetic map length; 575 cM versus 332 cM respectively; (ii) CO distribution patterns: male CO rates were very high at both ends of each chromosome, whereas female CO rates were very low; (iii) correlations between CO rates and various chromosome features: female CO rates correlated strongly and negatively with GC content and gene density but positively with transposable elements (TEs) density, whereas male CO rates correlated positively with the CpG ratio. However, except for CpG, the correlations could be explained by the unequal repartition of these sequences along the Arabidopsis chromosome. For both male and female meiosis, the number of COs per chromosome correlates with chromosome size expressed either in base pairs or as synaptonemal complex length. Finally, we show that interference modulates the CO distribution both in male and female meiosis.
Author Summary
Reciprocal exchanges of genetic material (crossovers) between homologous chromosomes ensure their proper segregation to generate gametes. Their number and location along chromosomes are tightly regulated. We localized precisely the position of 13,535 crossovers in more than 3,000 plants of Arabidopsis thaliana. While A. thaliana is a hermaphrodite plant with male and female meiosis occurring in the same flower and thus with the same genome, we observed dramatic differences in the distribution and the rate of crossovers along chromosomes in male and female meiosis. On average, chromosomes recombine 1.7 times more in male than in female meiosis. Moreover, male CO rates are very high at both ends of each chromosome, whereas female CO rates are very low. Finally, for the first time in a eukaryote, we show that the correlations between CO rates and various chromosome features differ in male and female meiosis. Female CO rates correlated strongly and negatively with GC content and gene density but positively with transposable elements density, whereas male CO rates correlated positively with the CpG ratio. However, most of the correlations could be explained by the structure of the Arabidopsis genome.
PMCID: PMC3207851  PMID: 22072983
22.  Mouse Pachytene Checkpoint 2 (Trip13) Is Required for Completing Meiotic Recombination but Not Synapsis 
PLoS Genetics  2007;3(8):e130.
In mammalian meiosis, homologous chromosome synapsis is coupled with recombination. As in most eukaryotes, mammalian meiocytes have checkpoints that monitor the fidelity of these processes. We report that the mouse ortholog (Trip13) of pachytene checkpoint 2 (PCH2), an essential component of the synapsis checkpoint in Saccharomyces cerevisiae and Caenorhabditis elegans, is required for completion of meiosis in both sexes. TRIP13-deficient mice exhibit spermatocyte death in pachynema and loss of oocytes around birth. The chromosomes of mutant spermatocytes synapse fully, yet retain several markers of recombination intermediates, including RAD51, BLM, and RPA. These chromosomes also exhibited the chiasmata markers MLH1 and MLH3, and okadaic acid treatment of mutant spermatocytes caused progression to metaphase I with bivalent chromosomes. Double mutant analysis demonstrated that the recombination and synapsis genes Spo11, Mei1, Rec8, and Dmc1 are all epistatic to Trip13, suggesting that TRIP13 does not have meiotic checkpoint function in mice. Our data indicate that TRIP13 is required after strand invasion for completing a subset of recombination events, but possibly not those destined to be crossovers. To our knowledge, this is the first model to separate recombination defects from asynapsis in mammalian meiosis, and provides the first evidence that unrepaired DNA damage alone can trigger the pachytene checkpoint response in mice.
Author Summary
It is critical that the chromosomes carried by sperm and eggs contain faithful representations of the genome of the individual that produced them. During the process of meiosis, the maternal and paternal copies of each chromosome “synapse” with each other (become tightly associated), exchange genetic material via the process of recombination, then separate into daughter cells in the first of two meiotic cell divisions. The intricate chromosome behavior is subject to errors, so most organisms have evolved meiotic “checkpoints” that monitor fidelity of chromosome synapsis and repair of DNA damage. These checkpoints cause defective cells to self destruct rather than generate defective sperm or eggs. We studied the effects of deleting mouse Trip13, a gene that in distant organisms plays a key role in meiotic checkpoint control. These experiments revealed that instead of having a checkpoint role, Trip13 is required for one of the two major classes of recombination in meiosis that is required for repairing broken DNA molecules. The chromosomes still synapsed normally, but animals were sterile due to massive death of oocytes and spermatocytes. These results indicate that, in addition to a checkpoint that responds to failed synapsis, one exists to specifically detect unrepaired DNA damage that is due to failed recombination.
PMCID: PMC1941754  PMID: 17696610
23.  A Conserved E2F6-Binding Element in Murine Meiosis-Specific Gene Promoters1 
Biology of Reproduction  2008;79(5):921-930.
During gametogenesis, germ cells must undergo meiosis in order to become viable haploid gametes. Successful completion of this process is dependent upon the expression of genes whose protein products function specifically in meiosis. Failure to express these genes in meiotic cells often results in infertility, whereas aberrant expression in somatic cells may lead to mitotic catastrophe. The mechanisms responsible for regulating the timely expression of meiosis-specific genes have not been fully elucidated. Here we demonstrate that E2F6, a member of the E2F family of transcription factors, is essential for the repression of the newly identified meiosis-specific gene, Slc25a31 (also known as Ant4, Aac4), in somatic cells. This discovery, along with previous studies, prompted us to investigate the role of E2F6 in the regulation of meiosis-specific genes in general. Interestingly, the core E2F6-binding element (TCCCGC) was highly conserved in the proximal promoter regions of 19 out of 24 (79.2%) meiosis-specific genes. This was significantly higher than the frequency found in the promoters of all mouse genes (15.4%). In the absence of E2F6, only a portion of these meiosis-specific genes was derepressed in somatic cells. However, endogenous E2F6 bound to the promoters of these meiosis-specific genes regardless of whether they required E2F6 for their repression in somatic cells. Further, E2F6 overexpression was capable of reducing their transcription. These findings indicate that E2F6 possesses a broad ability to bind to and regulate the meiosis-specific gene population..
Meiosis-specific genes often harbor an E2F6-binding element in their proximal promoter regions upon which E2F6 binds and reduces expression, indicating that E2F6 may broadly serve as a repressor of meiosis-specific genes in somatic cells.
PMCID: PMC2715002  PMID: 18667754
E2F6; gene regulation; meiosis; repression; Slc25a31
24.  corona Is Required for Higher-Order Assembly of Transverse Filaments into Full-Length Synaptonemal Complex in Drosophila Oocytes 
PLoS Genetics  2008;4(9):e1000194.
The synaptonemal complex (SC) is an intricate structure that forms between homologous chromosomes early during the meiotic prophase, where it mediates homolog pairing interactions and promotes the formation of genetic exchanges. In Drosophila melanogaster, C(3)G protein forms the transverse filaments (TFs) of the SC. The N termini of C(3)G homodimers localize to the Central Element (CE) of the SC, while the C-termini of C(3)G connect the TFs to the chromosomes via associations with the axial elements/lateral elements (AEs/LEs) of the SC. Here, we show that the Drosophila protein Corona (CONA) co-localizes with C(3)G in a mutually dependent fashion and is required for the polymerization of C(3)G into mature thread-like structures, in the context both of paired homologous chromosomes and of C(3)G polycomplexes that lack AEs/LEs. Although AEs assemble in cona oocytes, they exhibit defects that are characteristic of c(3)G mutant oocytes, including failure of AE alignment and synapsis. These results demonstrate that CONA, which does not contain a coiled coil domain, is required for the stable ‘zippering’ of TFs to form the central region of the Drosophila SC. We speculate that CONA's role in SC formation may be similar to that of the mammalian CE proteins SYCE2 and TEX12. However, the observation that AE alignment and pairing occurs in Tex12 and Syce2 mutant meiocytes but not in cona oocytes suggests that the SC plays a more critical role in the stable association of homologs in Drosophila than it does in mammalian cells.
Author Summary
Meiosis is a specialized type of cell division that is needed to produce sperm and egg cells, which carry only half the number of chromosomes of other cells in the body. Meiosis is required for reproduction, but abnormalities in chromosome number caused by errors in the process of meiosis are responsible for many birth defects and mental retardation syndromes in humans. The fruit fly, Drosophila melanogaster, is an excellent organism in which to study meiosis because of the powerful genetic and microscopic techniques that can be implemented with it. Early in meiosis, homologous chromosomes are joined together by an elaborate protein structure called the synaptonemal complex (SC) that plays a critical role in both holding homologous chromosomes together and in facilitating a process known as meiotic recombination. In this study, we have found a protein called Corona that is required for the formation of the SC. Our data show that Corona is required for the proper localization of the SC protein C(3)G. In the absence of Corona, C(3)G fails to polymerize and form the central region of the SC. Increasing our understanding of SC assembly and function will lead to a better understanding of the mechanism for proper chromosome segregation during meiosis.
PMCID: PMC2529403  PMID: 18802461
25.  Estrogenic Exposure Alters the Spermatogonial Stem Cells in the Developing Testis, Permanently Reducing Crossover Levels in the Adult 
PLoS Genetics  2015;11(1):e1004949.
Bisphenol A (BPA) and other endocrine disrupting chemicals have been reported to induce negative effects on a wide range of physiological processes, including reproduction. In the female, BPA exposure increases meiotic errors, resulting in the production of chromosomally abnormal eggs. Although numerous studies have reported that estrogenic exposures negatively impact spermatogenesis, a direct link between exposures and meiotic errors in males has not been evaluated. To test the effect of estrogenic chemicals on meiotic chromosome dynamics, we exposed male mice to either BPA or to the strong synthetic estrogen, ethinyl estradiol during neonatal development when the first cells initiate meiosis. Although chromosome pairing and synapsis were unperturbed, exposed outbred CD-1 and inbred C3H/HeJ males had significantly reduced levels of crossovers, or meiotic recombination (as defined by the number of MLH1 foci in pachytene cells) by comparison with placebo. Unexpectedly, the effect was not limited to cells exposed at the time of meiotic entry but was evident in all subsequent waves of meiosis. To determine if the meiotic effects induced by estrogen result from changes to the soma or germline of the testis, we transplanted spermatogonial stem cells from exposed males into the testes of unexposed males. Reduced recombination was evident in meiocytes derived from colonies of transplanted cells. Taken together, our results suggest that brief exogenous estrogenic exposure causes subtle changes to the stem cell pool that result in permanent alterations in spermatogenesis (i.e., reduced recombination in descendent meiocytes) in the adult male.
Author Summary
During the past several decades, the incidence of human male reproductive abnormalities such as hypospadias, undescended testicles, testicular cancer, and low sperm counts has increased. Environmental factors—and in particular, exposure to environmental estrogens—have been implicated as contributing factors and, indeed, developmental exposure to a range of estrogenic chemicals induces similar defects in male rodents. Given the wide variety of ‘weak’ estrogenic chemicals found in everyday products, understanding how estrogenic exposures affect sperm production has direct human relevance. Here we show that brief exposure of newborn male mice to exogenous estrogen affects the developing spermatogonial stem cells of the testis and this, in turn, permanently alters spermatogenesis in the adult. Specifically, estrogens adversely affect meiotic recombination, a process that is essential for the production of haploid gametes. Subtle changes in the levels of recombination increase the incidence of meiotic errors, resulting in the elimination of cells before they become sperm. Thus, in addition to their other potential effects on the developing brain and reproductive tract, our results suggest that estrogenic exposures can act to reduce sperm production by affecting the spermatogonial stem cell pool of the developing testis.
PMCID: PMC4304829  PMID: 25615633

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