Meiotic Cells Counteract Programmed Retrotransposon Activation via RNA-Binding Translational Repressor Assemblies

International audienceRetrotransposon proliferation poses a threat to germline integrity. While retrotransposons must be activated in developing germ cells in order to survive and propagate, how they are selectively activated in the context of meiosis is unclear. We demonstrate that the transcriptional activation of Ty3/Gypsy retrotransposons and host defense are controlled by master meiotic regulators. We show that budding yeast Ty3/Gypsy co-opts binding sites of the essential meiotic transcription factor Ndt80 upstream of the integration site, thereby tightly linking its transcriptional activation to meiotic progression. We also elucidate how yeast cells thwart Ty3/Gypsy proliferation by blocking translation of the retrotransposon mRNA using amyloid-like assemblies of the RNA-binding protein Rim4. In mammals, several inactive Ty3/Gypsy elements are undergoing domestication. We show that mammals utilize equivalent master meiotic regulators (Stra8, Mybl1, Dazl) to regulate Ty3/Gypsy-derived genes in developing gametes. Our findings inform how genes that are evolving from retrotransposons can build upon existing regulatory networks during domestication

A meiotic XPF-ERCC1-like complex recognizes joint molecule recombination intermediates to promote crossover formation

Meiotic crossover formation requires the stabilization of early recombination intermediates by a set of proteins and occurs within the environment of the chromosome axis, a structure important for the regulation of meiotic recombination events. The molecular mechanisms underlying and connecting crossover recombination and axis localization are elusive. Here, we identified the ZZS (Zip2–Zip4–Spo16) complex, required for crossover formation, which carries two distinct activities: one provided by Zip4, which acts as hub through physical interactions with components of the chromosome axis and the crossover machinery, and the other carried by Zip2 and Spo16, which preferentially bind branched DNA molecules in vitro. We found that Zip2 and Spo16 share structural similarities to the structure-specific XPF–ERCC1 nuclease, although it lacks endonuclease activity. The XPF domain of Zip2 is required for crossover formation, suggesting that, together with Spo16, it has a noncatalytic DNA recognition function. Our results suggest that the ZZS complex shepherds recombination intermediates toward crossovers as a dynamic structural module that connects recombination events to the chromosome axis. The identification of the ZZS complex improves our understanding of the various activities required for crossover implementation and is likely applicable to other organisms, including mammals

Concerted action of the MutL beta heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion

Gene conversions resulting from meiotic recombination are critical in shaping genome diversification and evolution. How the extent of gene conversions is regulated is unknown. Here we show that the budding yeast mismatch repair related MutL beta complex, Mlh1-Mlh2, specifically inter acts with the conserved meiotic Mer3 helicase, which recruits it to recombination hotspots, independently of mismatch recognition. This recruitment is essential to limit gene conversion tract lengths genome-wide, without affecting cross over formation. Contrary to expectations, Mer3 helicase activity, proposed to extend the displacement loop (D-loop) recombination intermediate, does not influence the length of gene conversion events, revealing non-catalytical roles of Mer3. In addition, both purified Mer3 and MutL beta preferentially recognize D-loops, providing a mechanism for limiting gene conversion in vivo. These findings show that MutL beta is an integral part of a new regulatory step of meiotic recombination, which has implications to prevent rapid allele fixation and hotspot erosion in populations

Data from: Mek1 down regulates Rad51 activity during yeast meiosis by phosphorylation of Hed1

During meiosis, programmed double strand breaks (DSBs) are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH) bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i) phosphorylation of Rad54 by Mek1 and (ii) binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1

Aborting meiosis allows recombination in sterile diploid yeast hybrids

International audienceHybrids between diverged lineages contain novel genetic combinations but an impaired meiosis often makes them evolutionary dead ends. Here, we explore to what extent an aborted meiosis followed by a return-to-growth (RTG) promotes recombination across a panel of 20 Saccharomyces cerevisiae and S. paradoxus diploid hybrids with different genomic structures and levels of sterility. Genome analyses of 275 clones reveal that RTG promotes recombination and generates extensive regions of loss-of-heterozygosity in sterile hybrids with either a defective meiosis or a heavily rearranged karyotype, whereas RTG recombination is reduced by high sequence divergence between parental subgenomes. The RTG recombination preferentially arises in regions with low local heterozygosity and near meiotic recombination hotspots. The loss-of-heterozygosity has a profound impact on sexual and asexual fitness, and enables genetic mapping of phenotypic differences in sterile lineages where linkage analysis would fail. We propose that RTG gives sterile yeast hybrids access to a natural route for genome recombination and adaptation

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Hed1 T40 is a direct target of Mek1 phosphorylation.

<p>(A) Alignment of Hed1 phosphorylated amino acids (indicated in red) detected by MS analysis of phosphopeptides isolated after induction of <i>NDT80</i> in ySZ07. Numbers indicate amino acid positions. The line indicates the Mek1 consensus, RXXT. (B) Relationship of phosphosites to functional domains of Hed1 determined by [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006226#pgen.1006226.ref048" target="_blank">48</a>]. The pink and orange boxes indicate domains required for ssDNA binding and Hed1 interaction, respectively. (C) Specificity of the Hed1 α-pT40 antibody for Hed1 phosphorylated on threonine 40. The <i>dmc1Δ hed1Δ</i> (NH942), <i>dmc1Δ</i> (NH942::pNH302²), <i>dmc1Δ hed1-T40A</i> (NH942::pNH302-T40A²) and <i>mek1Δ</i> (NH729) diploids were incubated in Spo medium for 6 hr at 30°C and probed with α-Hed1 antibodies to detect total Hed1 protein or α-pT40 antibodies to detect Hed1 phosphorylated on T40. Arp7 was used as loading control [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006226#pgen.1006226.ref049" target="_blank">49</a>]. (D) Kinase reactions containing GST-Mek1-as, furfuryl (Fu)-ATPγS and recombinant GST-Hed1 or GST-Hed1-3A (both purified from <i>E</i>. <i>coli</i>) were fractionated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Phosphorylation was detected using the semi-synthetic epitope system [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006226#pgen.1006226.ref050" target="_blank">50</a>]. In this assay, GST-Mek1-as specifically transfers thiophosphates onto its substrates using Fu-ATPγS. Reaction with <i>p</i>-nitrobenzylmesylate (PNBM) converts the thiophosphates into epitopes that are recognized by a thiophosphate ester monoclonal antibody. The vertical white line indicates the juxtaposition of non-adjacent lanes from the same gel. The horizontal white lines indicate the same samples fractionated on different gels and probed with the indicated antibodies. GST-Mek1-as and GST-Hed1 were detected using α-GST and α-Hed1 antibodies, respectively. (E) <i>In vitro</i> phosphorylation of Hed1 T40 by GST-Mek1-as. Kinase reactions were performed as in Panel D except that Fu-ATP was used and Hed1 phospho-T40 was detected using the α-pT40 antibodies.</p

Physical analysis of recombination at the <i>HIS4-LEU2</i> hotspot.

<p>(A) DNA was isolated at the indicated timepoints, digested with XhoI and probed as described in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006226#pgen.1006226.ref059" target="_blank">59</a>] to detect DSBs and COs. For the WT, a darker exposure is shown on the left to show the DSBs. “EC” indicates bands resulting from ectopic recombination [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006226#pgen.1006226.ref042" target="_blank">42</a>]. (B) DNA from the same time course shown in A was cut with XhoI and NgoMIV to detect CO and NCO recombinants at the <i>HIS4-LEU2</i> hotspot. (C) Quantification of DSBs from two independent timecourses, one of which is shown in A. Error bars indicate the range. There was no 6 hr timepoint in the second timecourse. (D) Quantification of CO1 +CO2 as in Panel C. (E) Quantification of ECs as in Panel C. (F) Quantification of CO2 from two independent timecourses, one of which is shown in B. (G) Quantification of NCO1 as in Panel F.</p