Identification and Functional Characterization of Genes Involved in the Control of Meiotic Crossover Frequency.

Les crossing-overs (CO) sont issus d’échange réciproque de matériel génétique entre les chromosomes homologues. Les COs produisent de la diversité génétique et sont essentiels chez la plupart des eucaryotes, pour la distribution équilibrée des chromosomes lors de la méiose. Malgré leur importance, et un large excès de précurseurs moléculaires, le nombre de CO est très limité dans la grande majorité des espèces (Typiquement 1 à 4 par paire de chromosomes). Cela suggère que les COs sont étroitement régulés, mais les mécanismes sous-jacents sont mal connus. Pour identifier les gènes qui limitent la formation des CO, l’équipe a mené un crible génétique chez Arabidopsis thaliana. Ces travaux ont mené à l’identification de plusieurs facteurs anti-CO, définissant trois voies : (i) L’hélicase FANCM et ses co-facteurs ; (ii) L’AAA-ATPase FIDGETIN-LIKE-1 (FIGL1) ; (iii) Le complexe RECQ4-Topoisomerase 3α-RMI1.Le premier objectif de ma thèse est d’explorer les relations entre ces trois voies en s’attachant aux questions suivantes ; (1) Jusqu’où peut-on augmenter la recombinaison en combinant les mutations dans FANCM, FIGL1 et RECQ4 ? Nous avons montré que la plus forte augmentation de recombinaison était obtenue dans recq4 figl1, atteignant 7,5 fois la fréquence du sauvage en moyenne sur le génome. (2) Quel est la distribution de ces extra-COs ? L’augmentation de recombinaison n’est pas homogène le long du génome : Les fréquences de CO augmente fortement des centromères vers les télomères, avec les plus hautes fréquences observées dans les régions distales. (3) La modification des fréquences de recombinaison est-elle identique lors des méioses mâles et femelle ? Chez le sauvage, la fréquence de recombinaison est plus élevée lors de la méiose mâle que femelle. Au contraire, la recombinaison femelle devient plus élevée que la recombinaison mâle chez les mutants recq4 et recq4 figl1. Ceci suggère que des contraintes qui s’appliquent sur la formation des CO lors de la méiose femelle sont relâchées chez ces mutants. En poursuivant le crible génétique, un nouveau mutant hyper-recombinant a été identifié. Le second objectif de ma thèse fut d’identifier et de caractériser fonctionnellement le gène correspondant. Une cartographie génétique et des études d’interactions protéine-protéine, ont mené à l’identification d’un facteur qui interagit directement avec FIGL1 et semble former un complexe conservé depuis les plantes jusqu’au mammifères. Nous avons baptisé cette protéine FLIP (Fidgetin-like-1 interacting protein). Les fréquences de recombinaisons sont augmentées dans flip-1, confirmant que FLIP1 limite la formation des COs. Des études d’épistasie ont montré que FLIP et FIGL1 agissent dans la même voie. De plus les protéines FIGL1/FLIP d’Arabidopsis ou humaine, interagissent avec RAD51 et DMC1, les deux protéines qui catalyse une étape clef de la recombinaison, l’invasion d’un ADN homologue. Finalement, dans flip comme dans figl1, la dynamique de DMC1 est modifiée. Nous proposons donc un modèle dans lequel le complexe FLIP-FIGL1 régule négativement l’activité de RAD51/DMC1 pour limiter la formation des COs. L’étude du complexe conservé FLIP-FIGL1 a mis en évidence un nouveau mode de régulation de la recombinaison, qui agit vraisemblablement à l’étape clé de l’échange de brin homologue. De plus, l’augmentation des CO sans précédent obtenues chez recq4 figl1 peut être d’un grand intérêt pour l’amélioration des plantes en permettant de diversité de nouvelles combinaisons alléliques.Meiotic crossovers (CO) are formed by reciprocal exchange of genetic material between the homologous chromosomes. CO generate genetic diversity and are essential for the proper segregation of chromosomes during meiosis in most eukaryotes. Despite their significance and a large excess of CO precursors, CO number is very low in vast majority of species (typically one to three per chromosome pair). This indicates that COs are tightly regulated but the underlying mechanisms of this limit remain elusive. In order to identify genes that limit COs, a genetic screen was performed in Arabidopsis thaliana. This led to the identification and characterization of several anti-CO factors belonging to three different pathways: (i) The FANCM helicase and its cofactors (ii) The AAA-ATPase FIDGETIN-LIKE-1 (FIGL1) (iii) The RECQ4 -Topoisomerase 3α-RMI1 complex. The first objective was to understand the functional relationship between these three pathways and to address following questions: (1) how far can we increase recombination when combining mutations in FANCM, FIGL1 and RECQ4? We show that the highest increase in recombination was obtained in figl1 recq4, reaching to 7.5 fold the wild type level, on average genome wide. (2) How is the distribution of recombination events genome wide in mutants? The increased CO frequency in the mutants was not uniform throughout the genome. CO frequency rises from the centromere to telomeres, with distal intervals having highest COs (3) is the recombination frequency increase same in both male and female? In Arabidopsis wild type, male has higher recombination than female meiosis. In contrast, in recq4 and recq4 figl1, female recombination was higher than male. This suggests that certain constraints that apply to CO formation in wild type females are relieved in the mutant. By continuing the same genetic screen, a novel anti-CO mutant was identified. The second objective was to identify and functionally characterize the corresponding gene. Genetic mapping and protein interaction studies led to the identification of a factor that directly interacts with FIGL1 and appears to form a conserved complex both in Arabidopsis and humans. Hence, the factor was named FLIP (Fidgetin-like-1 interacting protein). Recombination frequency is increased in flip, confirming that FLIP limit COs. Epistasis studies showed that FLIP and FIGL1 act in same pathway. Further, FIGL1/FLIP proteins of Arabidopsis and humans directly interact with the recombinases RAD51 and DMC1 which catalyze a crucial step of homologous recombination, the inter homolog strand invasion. In addition flip like figl1 modifies dynamics of DMC1. We thus propose a model wherein the FLIP-FIGL1 complex negatively regulates RAD51/DMC1 to limit CO formation. Studying the conserved FIGL1-FLIP complex led to the identification of a novel mode of regulation of recombination, that likely acts at the key step of homologous strand invasion. Further the unprecedented level of CO increase in recq4figl1 in hybrids could be of great interest for crop improvement, allowing the production of novel allele combinations

Identification et caractérisation fonctionnelle de gènes contrôlant la fréquence de crossovers méiotiques.

Meiotic crossovers (CO) are formed by reciprocal exchange of genetic material between the homologous chromosomes. CO generate genetic diversity and are essential for the proper segregation of chromosomes during meiosis in most eukaryotes. Despite their significance and a large excess of CO precursors, CO number is very low in vast majority of species (typically one to three per chromosome pair). This indicates that COs are tightly regulated but the underlying mechanisms of this limit remain elusive. In order to identify genes that limit COs, a genetic screen was performed in Arabidopsis thaliana. This led to the identification and characterization of several anti-CO factors belonging to three different pathways: (i) The FANCM helicase and its cofactors (ii) The AAA-ATPase FIDGETIN-LIKE-1 (FIGL1) (iii) The RECQ4 -Topoisomerase 3α-RMI1 complex. The first objective was to understand the functional relationship between these three pathways and to address following questions: (1) how far can we increase recombination when combining mutations in FANCM, FIGL1 and RECQ4? We show that the highest increase in recombination was obtained in figl1 recq4, reaching to 7.5 fold the wild type level, on average genome wide. (2) How is the distribution of recombination events genome wide in mutants? The increased CO frequency in the mutants was not uniform throughout the genome. CO frequency rises from the centromere to telomeres, with distal intervals having highest COs (3) is the recombination frequency increase same in both male and female? In Arabidopsis wild type, male has higher recombination than female meiosis. In contrast, in recq4 and recq4 figl1, female recombination was higher than male. This suggests that certain constraints that apply to CO formation in wild type females are relieved in the mutant. By continuing the same genetic screen, a novel anti-CO mutant was identified. The second objective was to identify and functionally characterize the corresponding gene. Genetic mapping and protein interaction studies led to the identification of a factor that directly interacts with FIGL1 and appears to form a conserved complex both in Arabidopsis and humans. Hence, the factor was named FLIP (Fidgetin-like-1 interacting protein). Recombination frequency is increased in flip, confirming that FLIP limit COs. Epistasis studies showed that FLIP and FIGL1 act in same pathway. Further, FIGL1/FLIP proteins of Arabidopsis and humans directly interact with the recombinases RAD51 and DMC1 which catalyze a crucial step of homologous recombination, the inter homolog strand invasion. In addition flip like figl1 modifies dynamics of DMC1. We thus propose a model wherein the FLIP-FIGL1 complex negatively regulates RAD51/DMC1 to limit CO formation. Studying the conserved FIGL1-FLIP complex led to the identification of a novel mode of regulation of recombination, that likely acts at the key step of homologous strand invasion. Further the unprecedented level of CO increase in recq4figl1 in hybrids could be of great interest for crop improvement, allowing the production of novel allele combinations.Les crossing-overs (CO) sont issus d’échange réciproque de matériel génétique entre les chromosomes homologues. Les COs produisent de la diversité génétique et sont essentiels chez la plupart des eucaryotes, pour la distribution équilibrée des chromosomes lors de la méiose. Malgré leur importance, et un large excès de précurseurs moléculaires, le nombre de CO est très limité dans la grande majorité des espèces (Typiquement 1 à 4 par paire de chromosomes). Cela suggère que les COs sont étroitement régulés, mais les mécanismes sous-jacents sont mal connus. Pour identifier les gènes qui limitent la formation des CO, l’équipe a mené un crible génétique chez Arabidopsis thaliana. Ces travaux ont mené à l’identification de plusieurs facteurs anti-CO, définissant trois voies : (i) L’hélicase FANCM et ses co-facteurs ; (ii) L’AAA-ATPase FIDGETIN-LIKE-1 (FIGL1) ; (iii) Le complexe RECQ4-Topoisomerase 3α-RMI1.Le premier objectif de ma thèse est d’explorer les relations entre ces trois voies en s’attachant aux questions suivantes ; (1) Jusqu’où peut-on augmenter la recombinaison en combinant les mutations dans FANCM, FIGL1 et RECQ4 ? Nous avons montré que la plus forte augmentation de recombinaison était obtenue dans recq4 figl1, atteignant 7,5 fois la fréquence du sauvage en moyenne sur le génome. (2) Quel est la distribution de ces extra-COs ? L’augmentation de recombinaison n’est pas homogène le long du génome : Les fréquences de CO augmente fortement des centromères vers les télomères, avec les plus hautes fréquences observées dans les régions distales. (3) La modification des fréquences de recombinaison est-elle identique lors des méioses mâles et femelle ? Chez le sauvage, la fréquence de recombinaison est plus élevée lors de la méiose mâle que femelle. Au contraire, la recombinaison femelle devient plus élevée que la recombinaison mâle chez les mutants recq4 et recq4 figl1. Ceci suggère que des contraintes qui s’appliquent sur la formation des CO lors de la méiose femelle sont relâchées chez ces mutants. En poursuivant le crible génétique, un nouveau mutant hyper-recombinant a été identifié. Le second objectif de ma thèse fut d’identifier et de caractériser fonctionnellement le gène correspondant. Une cartographie génétique et des études d’interactions protéine-protéine, ont mené à l’identification d’un facteur qui interagit directement avec FIGL1 et semble former un complexe conservé depuis les plantes jusqu’au mammifères. Nous avons baptisé cette protéine FLIP (Fidgetin-like-1 interacting protein). Les fréquences de recombinaisons sont augmentées dans flip-1, confirmant que FLIP1 limite la formation des COs. Des études d’épistasie ont montré que FLIP et FIGL1 agissent dans la même voie. De plus les protéines FIGL1/FLIP d’Arabidopsis ou humaine, interagissent avec RAD51 et DMC1, les deux protéines qui catalyse une étape clef de la recombinaison, l’invasion d’un ADN homologue. Finalement, dans flip comme dans figl1, la dynamique de DMC1 est modifiée. Nous proposons donc un modèle dans lequel le complexe FLIP-FIGL1 régule négativement l’activité de RAD51/DMC1 pour limiter la formation des COs. L’étude du complexe conservé FLIP-FIGL1 a mis en évidence un nouveau mode de régulation de la recombinaison, qui agit vraisemblablement à l’étape clé de l’échange de brin homologue. De plus, l’augmentation des CO sans précédent obtenues chez recq4 figl1 peut être d’un grand intérêt pour l’amélioration des plantes en permettant de diversité de nouvelles combinaisons alléliques

FIGL1 and its novel partner FLIP form a conserved complex that regulates homologous recombination.

Homologous recombination is central to repair DNA double-strand breaks, either accidently arising in mitotic cells or in a programed manner at meiosis. Crossovers resulting from the repair of meiotic breaks are essential for proper chromosome segregation and increase genetic diversity of the progeny. However, mechanisms regulating crossover formation remain elusive. Here, we identified through genetic and protein-protein interaction screens FIDGETIN-LIKE-1 INTERACTING PROTEIN (FLIP) as a new partner of the previously characterized anti-crossover factor FIDGETIN-LIKE-1 (FIGL1) in Arabidopsis thaliana. We showed that FLIP limits meiotic crossover together with FIGL1. Further, FLIP and FIGL1 form a protein complex conserved from Arabidopsis to human. FIGL1 interacts with the recombinases RAD51 and DMC1, the enzymes that catalyze the DNA strand exchange step of homologous recombination. Arabidopsis flip mutants recapitulate the figl1 phenotype, with enhanced meiotic recombination associated with change in counts of DMC1 and RAD51 foci. Our data thus suggests that FLIP and FIGL1 form a conserved complex that regulates the crucial step of strand invasion in homologous recombination

FIGL1 and its novel partner FLIP form a conserved complex that regulates homologous recombination - Fig 4

<p>Mutation in <i>FLIP</i> restores crossover formation in <i>zmm</i> mutants: A. Schematic representation of the <i>FLIP</i> gene (Fidgetin-Like-1 Interacting Protein). Exons appear as blue boxes. The red line and red triangle indicate the missense mutation in <i>flip-1</i> and the <i>flip-2</i> T-DNA insertion, respectively. B. Average number of bivalents (blue) and pairs of univalents (red) per male meiocyte at metaphase I (Fig 4C). Light blue represents rod shaped bivalents indicating that one chromosome arm has at least one CO, and one arm has no CO. Dark blue represents ring shaped bivalent indicating the presence of at least one CO on both chromosome arms. The number of cells analyzed for each genotype is indicated in brackets. C. DAPI staining of Chromosome spreads of male meiocytes at metaphase I. Scale bars 10μm. D. Fertility measured as number of seeds per fruit. Each dot represents a plant; at least 10 fruits per plant were analyzed.</p

Yeast-two-hybrid experiments testing interactions between Arabidopsis FIGL1, FLIP, RAD51 and DMC1 proteins.

<p>Proteins of interest were fused with Gal4 DNA binding domain (BD, left) and with Gal4 activation domain (AD, top), respectively, and co-expressed in yeast cells. Full-length and truncated protein are schematically represented. For each combination, serial dilutions of yeast cells were spotted on non-selective medium (-LW), moderately selective media (-LWH) and more selective media (-LWHA). ++: Growth on both LWH and LWHA, interpreted as strong interaction. +: Growth on LWH and not on LWHA, interpreted as weak interaction. +*: Growth on LWH but cannot be interpreted as positive interaction because of auto-activation of one of the construct.—: Growth on neither LWH nor LWHA. n.d. Not determined. Pictures of yeasts are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007317#pgen.1007317.s001" target="_blank">S1 Fig</a>.</p

<i>FLIP</i> genetically interacts with <i>SDS</i>.

<p>A. Immunostaining of DMC1 (green) and the chromosome axis protein ASY1 (red) on leptotene/zygotene meiotic chromosome spreads. B. Quantification of DMC1 foci at leptotene/zygotene in <i>sds</i>, <i>sds figl1</i> and <i>sds flip</i>. Each dot represents an individual cell and bars indicate the mean. C. Co-immunolocalization of ASY1 (red) and ZYP1 (green), which mark respectively chromosome axes and synapsed regions. Synapsis was partially restored in <i>sds flip</i> compared to single mutant <i>sds</i>. Scale bars 10μm. D. DAPI staining of chromosome spreads of male meiocytes at metaphase I and anaphase I. Scale bars 10μm. E. Fertility measured as number of seeds per fruit. Each dot represents a plant; at least 12 fruits per plant were analyzed. P values are the results of Fisher's LSD tests.</p

Phylogenetic tree depicting the evolutionary conservation of FLIP, FIGL1, RAD51 and DMC1 orthologs in a range of eukaryotic species.

<p>FLIP, FIGL1, DMC1 and RAD51 are presented as dots in green, red, blue and turquoise color, respectively. Gene accession numbers are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007317#pgen.1007317.s005" target="_blank">S2 Table</a>. A version of this figure with a larger number of species can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007317#pgen.1007317.s006" target="_blank">S3 Fig</a> and as an interactive tree at <a href="http://itol.embl.de/tree/132166555992271498216301" target="_blank">http://itol.embl.de/tree/132166555992271498216301</a>.</p

DMC1 foci in wild type <i>figl1</i>, <i>flip</i> and <i>figl1 flip</i>.

<p>A. Triple immunolocalization of ASY1 (red), ZYP1 (blue) and DMC1 (green) on meiotic chromosome spreads. Merged pictures are shown. Partial and full ZYP1 polymerization defines the zygotene and pachytene stages, respectively. Scale bars 10μm. B. Quantification of DMC1 foci at leptotene, zygotene and pachytene in wild type, <i>figl1</i>, <i>flip</i> and <i>figl1 flip</i>. Each dot represents an individual cell and bars indicate the mean. P values are the results of Fisher's LSD tests.</p