Transcription Of Meiotic Genes Research Articles

R-Loop Formation in Meiosis: Roles in Meiotic Transcription-Associated DNA Damage.

Meiosis is specialized cell division during gametogenesis that produces genetically unique gametes via homologous recombination. Meiotic homologous recombination entails repairing programmed 200–300 DNA double-strand breaks generated during the early prophase. To avoid interference between meiotic gene transcription and homologous recombination, mammalian meiosis is thought to employ a strategy of exclusively transcribing meiotic or post-meiotic genes before their use. Recent studies have shown that R-loops, three-stranded DNA/RNA hybrid nucleotide structures formed during transcription, play a crucial role in transcription and genome integrity. Although our knowledge about the function of R-loops during meiosis is limited, recent findings in mouse models have suggested that they play crucial roles in meiosis. Given that defective formation of an R-loop can cause abnormal transcription and transcription-coupled DNA damage, the precise regulatory network of R-loops may be essential in vivo for the faithful progression of mammalian meiosis and gametogenesis.

Discovery of a chemical probe for PRDM9

PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36. Its normal expression is restricted to germ cells and attenuation of its activity results in altered meiotic gene transcription, impairment of double-stranded breaks and pairing between homologous chromosomes. There is growing evidence for a role of aberrant expression of PRDM9 in oncogenesis and genome instability. Here we report the discovery of MRK-740, a potent (IC50: 80 ± 16 nM), selective and cell-active PRDM9 inhibitor (Chemical Probe). MRK-740 binds in the substrate-binding pocket, with unusually extensive interactions with the cofactor S-adenosylmethionine (SAM), conferring SAM-dependent substrate-competitive inhibition. In cells, MRK-740 specifically and directly inhibits H3K4 methylation at endogenous PRDM9 target loci, whereas the closely related inactive control compound, MRK-740-NC, does not. The discovery of MRK-740 as a chemical probe for the PRDM subfamily of methyltransferases highlights the potential for exploiting SAM in targeting SAM-dependent methyltransferases.

Histone deacetylation promotes transcriptional silencing at facultative heterochromatin.

It is important to accurately regulate the expression of genes involved in development and environmental response. In the fission yeast Schizosaccharomyces pombe, meiotic genes are tightly repressed during vegetative growth. Despite being embedded in heterochromatin these genes are transcribed and believed to be repressed primarily at the level of RNA. However, the mechanism of facultative heterochromatin formation and the interplay with transcription regulation is not understood. We show genome-wide that HDAC-dependent histone deacetylation is a major determinant in transcriptional silencing of facultative heterochromatin domains. Indeed, mutation of class I/II HDACs leads to increased transcription of meiotic genes and accumulation of their mRNAs. Mechanistic dissection of the pho1 gene where, in response to phosphate, transient facultative heterochromatin is established by overlapping lncRNA transcription shows that the Clr3 HDAC contributes to silencing independently of SHREC, but in an lncRNA-dependent manner. We propose that HDACs promote facultative heterochromatin by establishing alternative transcriptional silencing.

Bdf1 Bromodomains Are Essential for Meiosis and the Expression of Meiotic-Specific Genes.

Bromodomain and Extra-terminal motif (BET) proteins play a central role in transcription regulation and chromatin signalling pathways. They are present in unicellular eukaryotes and in this study, the role of the BET protein Bdf1 has been explored in Saccharomyces cerevisiae. Mutation of Bdf1 bromodomains revealed defects on both the formation of spores and the meiotic progression, blocking cells at the exit from prophase, before the first meiotic division. This phenotype is associated with a massive deregulation of the transcription of meiotic genes and Bdf1 bromodomains are required for appropriate expression of the key meiotic transcription factor NDT80 and almost all the Ndt80-inducible genes, including APC complex components. Bdf1 notably accumulates on the promoter of Ndt80 and its recruitment is dependent on Bdf1 bromodomains. In addition, the ectopic expression of NDT80 during meiosis partially bypasses this dependency. Finally, purification of Bdf1 partners identified two independent complexes with Bdf2 or the SWR complex, neither of which was required to complete sporulation. Taken together, our results unveil a new role for Bdf1 –working independently from its predominant protein partners Bdf2 and the SWR1 complex–as a regulator of meiosis-specific genes.

Rpl22 is required for IME1 mRNA translation and meiotic induction in S. cerevisiae.

BackgroundThe transition from mitotic cell division to meiotic development in S. cerevisiae requires induction of a transient transcription program that is initiated by Ime1-dependent destruction of the repressor Ume6. Although IME1 mRNA is observed in vegetative cultures, Ime1 protein is not suggesting the presence of a regulatory system restricting translation to meiotic cells.ResultsThis study demonstrates that IME1 mRNA translation requires Rpl22A and Rpl22B, eukaryotic-specific ribosomal protein paralogs of the 60S large subunit. In the absence of Rpl22 function, IME1 mRNA synthesis is normal in cultures induced to enter meiosis. However, Ime1 protein production is reduced and the Ume6 repressor is not destroyed in rpl22 mutant cells preventing early meiotic gene induction resulting in a pre-meiosis I arrest. This role for Rpl22 is not a general consequence of mutating non-essential large ribosomal proteins as strains lacking Rpl29 or Rpl39 execute meiosis with nearly wild-type efficiencies. Several results indicate that Rpl22 functions by enhancing IME1 mRNA translation. First, the Ime1 protein synthesized in rpl22 mutant cells demonstrates the same turnover rate as in wild-type cultures. In addition, IME1 transcript is found in polysome fractions isolated from rpl22 mutant cells indicating that mRNA nuclear export and ribosome association occurs. Finally, deleting the unusually long 5′UTR restores Ime1 levels and early meiotic gene transcription in rpl22 mutants suggesting that Rpl22 enhances translation through this element. Polysome profiles revealed that under conditions of high translational output, Rpl22 maintains high free 60S subunit levels thus preventing halfmer formation, a translation species indicative of mRNAs bound by an unpaired 40S subunit. In addition to meiosis, Rpl22 is also required for invasive and pseudohyphal growth.ConclusionsThese findings indicate that Rpl22A and Rpl22B are required to selectively translate IME1 mRNA that is required for meiotic induction and subsequent gametogenesis. In addition, our results imply a more general role for Rpl22 in cell fate switches responding to environmental nitrogen signals.

Fhl1 gene of the fission yeast regulates transcription of meiotic genes and nitrogen starvation response, downstream of the TORC1 pathway.

Environmental changes, such as nutrient limitation or starvation induce different signal transducing pathways, which require coordinated cooperation of several genes. Our previous data revealed that the fhl1 fork-head type transcription factor of the fission yeast could be involved in sporulation, which was typically induced under poor conditions. Since the exact role of Fhl1 in this process was not known, we wanted to identify its downstream targets and to investigate its possible cooperation with another known regulator of sporulation. Gene expression and Northern blot analysis of the fhl1∆ mutant strain revealed the target genes involved in mating and sporulation. Our results also showed that Fhl1 could regulate nutrient sensing, the transporter and permease genes. Since the majority of these genes belonged to the nitrogen starvation response, the possible cooperation of fhl1 and tor2 was also investigated. Comparison of their microarray data and the expression of fhl1 + from a strong promoter in the tor2-ts mutant cells suggested that one part of the target genes are commonly regulated by Fhl1 and Tor2. Since the expression of fhl1 + from a strong promoter could rescue rapamycin and temperature sensitivity and suppressed the hyper-sporulation defect of the tor2-ts mutant cells, we believe that Fhl1 acts in TOR signaling, downstream of Tor2. Thus, this work shed light on certain novel details of the regulation of the sexual processes and a new member of the TOR pathway, but further experiments are needed to confirm the involvement of Fhl1 in nutrient sensing.

The Chromatin Protein DUET/MMD1 Controls Expression of the Meiotic Gene TDM1 during Male Meiosis in Arabidopsis.

Meiosis produces haploid cells essential for sexual reproduction. In yeast, entry into meiosis activates transcription factors which trigger a transcriptional cascade that results in sequential co-expression of early, middle and late meiotic genes. However, these factors are not conserved, and the factors and regulatory mechanisms that ensure proper meiotic gene expression in multicellular eukaryotes are poorly understood. Here, we report that DUET/MMD1, a PHD finger protein essential for Arabidopsis male meiosis, functions as a transcriptional regulator in plant meiosis. We find that DUET-PHD binds H3K4me2 in vitro, and show that this interaction is critical for function during meiosis. We also show that DUET is required for proper microtubule organization during meiosis II, independently of its function in meiosis I. Remarkably, DUET protein shows stage-specific expression, confined to diplotene. We identify two genes TDM1 and JAS with critical functions in cell cycle transitions and spindle organization in male meiosis, as DUET targets, with TDM1 being a direct target. Thus, DUET is required to regulate microtubule organization and cell cycle transitions during male meiosis, and functions as a direct transcription activator of the meiotic gene TDM1. Expression profiling showed reduced expression of a subset comprising about 12% of a known set of meiosis preferred genes in the duet mutant. Our results reveal the action of DUET as a transcriptional regulator during male meiosis in plants, and suggest that transcription of meiotic genes is under stagewise control in plants as in yeast.

Gcn5p-dependent acetylation induces degradation of the meiotic transcriptional repressor Ume6p

Ume6p represses early meiotic gene transcription in Saccharomyces cerevisiae by recruiting the Rpd3p histone deacetylase and chromatin-remodeling proteins. Ume6p repression is relieved in a two-step destruction process mediated by the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. The first step induces partial Ume6p degradation when vegetative cells shift from glucose- to acetate-based medium. Complete proteolysis happens only upon meiotic entry. Here we demonstrate that the first step in Ume6p destruction is controlled by its acetylation and deacetylation by the Gcn5p acetyltransferase and Rpd3p, respectively. Ume6p acetylation occurs in medium lacking dextrose and results in a partial destruction of the repressor. Preventing acetylation delays Ume6p meiotic destruction and retards both the transient transcription program and execution of the meiotic nuclear divisions. Conversely, mimicking acetylation induces partial destruction of Ume6p in dextrose medium and accelerates meiotic degradation by the APC/C. These studies reveal a new mechanism by which acetyltransferase activity induces gene expression through targeted destruction of a transcriptional repressor. These findings also demonstrate an important role for nonhistone acetylation in the transition between mitotic and meiotic cell division.

Repression of meiotic genes by antisense transcription and by Fkh2 transcription factor in Schizosaccharomyces pombe.

In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the “unspliced” signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression.

Meiosis-Specific Destruction of the Ume6p Repressor by the Cdc20-Directed APC/C

Meiotic development in yeast requires the coordinated induction of transient waves of gene transcription. The present study investigates the regulation of Ume6p, a mitotic repressor of the "early" class of meiosis-specific genes. Western blot analysis revealed that Ume6p is destroyed early in meiosis by Cdc20p, an activator of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. This control appears direct as Cdc20p and Ume6p associate in vivo and APC/C(Cdc20) ubiquitylates Ume6p in vitro. Inactivating Cdc20p, or stabilizing Ume6p through mutation, prevented meiotic gene transcription and meiotic progression. During mitotic cell division, Ume6p is protected from destruction by protein kinase A phosphorylation of Cdc20p. Complete elimination of Ume6p in meiotic cells requires association with the meiotic inducer Ime1p. These results indicate that Ume6p degradation is required for normal meiotic gene induction and meiotic progression. These findings demonstrate a direct connection between the transcription machinery and ubiquitin-mediated proteolysis that is developmentally regulated.

Arg-Pro-X-Ser/Thr Is a Consensus Phosphoacceptor Sequence for the Meiosis-Specific Ime2 Protein Kinase in Saccharomyces cerevisiae

Ime2 is a meiosis-specific protein kinase in Saccharomyces cerevisiae that is functionally related to cyclin-dependent kinase. Although Ime2 regulates multiple steps in meiosis, only a few of its substrates have been identified. Here we show that Ime2 phosphorylates Sum1, a repressor of meiotic gene transcription, on Thr-306. Ime2 protein kinase assays with Sum1 mutants and synthetic peptides define a consensus Arg-Pro-X-Ser/Thr motif that is required for efficient phosphorylation by Ime2. The carboxyl residue adjacent to the phosphoacceptor (+1 position) also influences the efficiency of Ime2 phosphorylation with alanine being a preferred residue. This information has predictive value in identifying new potential Ime2 targets as shown by the ability of Ime2 to phosphorylate Sgs1 and Gip1 in vitro and could be important in differentiating mitotic and meiotic regulatory pathways.

A histone H3 methyltransferase controls epigenetic events required for meiotic prophase

Epigenetic modifications of histones regulate gene expression and chromatin structure. Here we show that Meisetz (meiosis-induced factor containing a PR/SET domain and zinc-finger motif) is a histone methyltransferase that is important for the progression of early meiotic prophase. Meisetz transcripts are detected only in germ cells entering meiotic prophase in female fetal gonads and in postnatal testis. Notably, Meisetz has catalytic activity for trimethylation, but not mono- or dimethylation, of lysine 4 of histone H3, and a transactivation activity that depends on its methylation activity. Mice in which the Meisetz gene is disrupted show sterility in both sexes due to severe impairment of the double-stranded break repair pathway, deficient pairing of homologous chromosomes and impaired sex body formation. In Meisetz-deficient testis, trimethylation of lysine 4 of histone H3 is attenuated and meiotic gene transcription is altered. These findings indicate that meiosis-specific epigenetic events in mammals are crucial for proper meiotic progression.

Ume1p Represses Meiotic Gene Transcription in Saccharomyces cerevisiae through Interaction with the Histone Deacetylase Rpd3p

Ume1p is a member of a conserved protein family including RbAp48 that associates with histone deacetylases. Consistent with this finding, Ume1p is required for the full repression of a subset of meiotic genes during vegetative growth in budding yeast. In addition to mitotic cell division, this report describes a new role for Ume1p in meiotic gene repression in precommitment sporulating cultures returning to vegetative growth. However, Ume1p is not required to re-establish repression as part of the meiotic transient transcription program. Mutational analysis revealed that two conserved domains (NEE box and a WD repeat motif) are required for Ume1p-dependent repression. Co-immunoprecipitation studies revealed that both the NEE box and the WD repeat motif are essential for normal Rpd3p binding. Finally, Ume1p-Rpd3p association is dependent on the global co-repressor Sin3p. Moreover, this activity was localized to one of the four paired amphipathic-helix domains of Sin3p shown previously to be required for transcriptional repression. These findings support a model that Ume1p binding to Rpd3p is required for its repression activity. In addition, these results suggest that Rpd3-Ume1p-Sin3p comprises an interdependent complex required for mediating transcriptional repression.

Identification of the Sin3-binding site in Ume6 defines a two-step process for conversion of Ume6 from a transcriptional repressor to an activator in yeast.

The DNA-binding protein Ume6 is required for both repression and activation of meiosis-specific genes, through interaction with the Sin3 corepressor and Rpd3 histone deacetylase and the meiotic activator Ime1. Here we show that fusion of a heterologous activation domain to Ume6 is unable to convert it into a constitutive activator of early meiotic gene transcription, indicating that an additional function is needed to overcome repression at these promoters. Mutations in UME6 allowing the fusion to activate lie in a predicted amphipathic alpha helix and specifically disrupt interaction with Sin3 but not with Teal, an activator of Ty transcription also found to interact with Ume6 in a two-hybrid screen. The mutations cause a loss of repression by Ume6 and precisely identify the Ume6 Sin3-binding domain, which we show interacts with the paired amphipathic helix 2 region of Sin3. Analysis of these mutants indicates that conversion of Ume6 to an activator involves two genetically distinct steps that act to relieve Sin3-mediated repression and provide an activation domain to Ume6. The mutants further demonstrate that premature expression and lack of subsequent rerepression of Ume6-Sin3-regulated genes are not deleterious to meiotic progression and suggest that the essential role of Sin3 in meiosis is independent of Ume6. The model for Ume6 function arising from these studies indicates that Ume6 is similar in many respects to metazoan regulators that utilize Sin3, such as the Myc-Mad-Max system and nuclear hormone receptors, and provides new insights into the control of transcriptional repression and activation by the Ume6-URS1 regulatory complex in yeast.

Induction of meiosis in Saccharomyces cerevisiae depends on conversion of the transcriptional represssor Ume6 to a positive regulator by its regulated association with the transcriptional activator Ime1.

The transcription of meiosis-specific genes, as well as the initiation of meiosis, in the budding yeast Saccharomyces cerevisiae depends on IME1. IME1 encodes a transcriptional activator which lacks known DNA binding motifs. In this study we have determined the mode by which Ime1 specifically activates the transcription of meiotic genes. We demonstrate that Ime1 is recruited to the promoters of meiotic genes by interacting with a DNA-binding protein, Ume6. This association between Ime1 and Ume6 depends on both starvation and the activity of a protein kinase, encoded by RIM11 In the absence of Ime1, Ume6 represses the transcription of meiotic genes. However, in the presence of Ime1, or when Ume6 is fused in frame to the Gal4 activation domain, Ume6 is converted from a repressor to an activator, resulting in the transcription of meiosis-specific genes and the formation of asci.

Identification of negative regulatory genes that govern the expression of early meiotic genes in yeast.

Mutations in Saccharomyces cerevisiae have been identified that derepress early meiotic genes functioning in separable pathways required for normal meiotic development. The phenotypes of these ume (unscheduled meiotic gene expression) mutations suggest that their wild-type alleles encode negative regulators acting downstream of both the cell-type and nutritional controls of meiosis. These newly defined loci do not affect either general transcription or transcription of meiotic genes expressed later in meiosis and spore formation.