Study of the role of neddylation in the regulation of meiotic recombination

La recombinaison homologue est essentielle à la réparation des lésions de l’ADN ainsi qu’à la ségrégation correcte des chromosomes en méiose. Une étape importante de la recombinaison méiotique est la formation des crossovers (CO). Au cours de ma thèse, j’ai mis en évidence un nouveau mécanisme de régulation de la recombinaison méiotique. J'ai montré que les cycles d'activation et de désactivation des cullin-RING ligases (CRL) sont absolument nécessaires à la recombinaison méiotique. Les CRL sont activées par neddylation et désactivées par la deneddylation. De plus, elles peuvent aussi être inhibées par la séquestration via la protéine CAND1. Mon travail a démontré que ces trois niveaux de régulation des CRL jouent des rôles cruciaux dans la recombinaison homologue méiotique chez A. thaliana. J’ai montré qu'AXR1, un composant clé de la machinerie de neddylation, est nécessaire à la localisation correcte des CO méiotiques et à la recombinaison homologue somatique. J’ai aussi prouvé que le processus de deneddylation médié par CSN5A est nécessaire à la formation des CO. J'ai obtenu des données montrant que cette régulation de la localisation des CO agit à travers la régulation d’un complexe CRL4. Enfin, j’ai pu montrer que l'inhibiteur des CRL, CAND1, est requis pour la formation de plus de 90 % des CO. En utilisant des outils génétiques et cytologiques, j'ai montré que CAND1 agit probablement sur la régulation du biais inter-homologue. L’ensemble de ces données, met l’accent sur un nouveau mécanisme de la régulation de la recombinaison homologue, connectant pour la première fois la méiose et l’ubiquitination via les cullin-RING Ligases.Homologous recombination is essential to all living organisms in order to repair DNA damages. In addition, a large majority of organisms use homologous recombination in meiosis to ensure proper chromosome segregation. A main step of meiotic recombination is crossover (CO) formation. During my PhD, I was able to highlight a new pathway controlling meiotic recombination. I showed that cycles of activation and deactivation of cullin-RING ligases (CRLs) are absolutely required for correct meiosis. CRLs are activated by neddylation, and deactivated by deneddylation. In addition, they can also be inhibited by sequestration by the CAND1 protein. My work demonstrated that these three levels of CRL regulation play crucial roles in meiotic homologous recombination in A. thaliana. First, I showed that AXR1, a key component of the neddylation machinery, is required for the correct localisation of meiotic COs and for somatic homologous recombination. Second, I showed that the deneddylation process mediated by CSN5A is also necessary for normal CO formation. I obtained evidence that this regulation of CO position is likely to be mediated by a CRL4 complex. Last, I could show that the CRL inhibitor, CAND1, is required for the formation of up to 90% of the COs. Using genetic and cytological tools, I showed that CAND1 probably acts on the regulation of the inter-homolog bias. Considering all these data, my work draws the attention to a new mechanism regulating meiotic homologous recombination, connecting for the first time meiosis to CRL-mediated ubiquitylation

Avaliação da expressão gênica diferencial entre folhas e tecidos vasculares de Eucalyptus grandis

No presente trabalho está descrita a análise de experimentos de microarranjos de DNA realizados pelos pesquisadores do Projeto GENOLYPTUS em conjunto com a empresa NimbleGen Systems Inc. (Reykjavik, Iceland), assim como a validação dos resultados gerados por essa análise, com a finalidade de encontrar genes diferencialmente expressos entre folhas e xilema de E. grandis para futuro estudo. O processo de análise dos microarranjos de DNA envolveu a busca por um software com programas estatísticos adequados ao estudo de grande quantidade de dados com mínima probabilidade de erro. A comprovação dos resultados gerados pelo software escolhido foi realizada com o uso da técnica da reação em cadeia da DNA-polimerase quantitativa (em tempo real) precedida de transcrição reversa (qRT-PCR). Alguns dos genes mais diferencialmente expressos foram selecionados para esta etapa, juntamente com alguns genes com expressão não tão alta. Os genes mais expressos nas folhas que foram escolhidos codificam para; (i) uma proteína semelhante à metalotioneína do tipo 3 (MT-3); (ii) uma glicolato-oxidase; (iii) uma catalase; (iv) uma fosforribulocinase precursora de cloroplasto (PRK); (v) um fator de transcrição MYB do tipo MYB142 e, (vi) uma proteína tipo “dedo-de-zinco”. Os genes mais expressos no xilema escolhidos foram os que codificam (i e ii) duas proteínas expressas em Arabidopsis thaliana; (iii) uma proteína hipotética expressa em Nicotiana benthamiana; (iv) uma descarboxilase de UDPglicuronato do tipo 2, (v) uma quitinase do tipo ELP; (vi) uma celulosesintase tipo 3; (vii) um fator de transcrição MYB; (viii) uma cafeoil-CoA-3- O-metiltransferase (CCoAOMT) e; (ix) uma proteína rica em prolina híbrida do tipo 2 (HyPRP2). Os resultados das qRT-PCRs demonstraram que o experimento de microarranjo e seus resultados foram consistentes e válidos, embora os primeiros tenham demonstrado valores de expressão freqüentemente mais altos. Acreditamos que tanto a análise dos microarranjos de DNA quanto a equivalência das amostras (duplicatas) biológicas estudadas foram totalmente sustentadas pelos resultados da qRT-PCR, pois foram robustas o suficiente para estimar um grande número de genes simultaneamente e indicar aqueles mais importantes como candidatos para futuras análises.In the present work it is described the analysis of DNA microarray experiments developed by GENOLYPTUS researchers together with Nimblegen Systems Inc. (Reykjavik, Iceland), as well as the validation of the generated results with the aim to find differentially expressed genes between leaves and xylem tissue of E. grandis for further study. The DNA microarray analysis process comprised the search for software containing statistical programs satisfactory for studying an enormous amount of data with a very low false discovery rate. The confirmation of the generated data by the chosen software was made with the use of quantitative (real-time) reverse-transcription followed by polymerase chain reaction (qRT-PCR). Some of the most differentially expressed genes were selected for this step, along with some genes with average expression. Leaf chosen genes were those that codify (i) a metallothionein-like protein type 3 (MT-3), (ii) a glycolate oxidase, (iii) a catalase, (iv) a precursor phosphoribulokinase (PRK) from chloroplast, (v) a MYB transcription factor (MYB142), and (vi) a CONSTANS-LIKE 16 zincfinger protein. Xylem chosen genes were those codifying (i and ii) two expressed proteins from Arabidopsis thaliana, (iii) a hypothetic protein expressed in Nicotiana benthamiana, (iv) a putative UDP-glucuronate decarboxylase type 2, (v) a chitinase type ELP (ectopic deposition of lignin in pith), (vi) a cellulose synthase type 3, (vii) a MYB transcription factor, (viii) a caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), and (ix) a hybrid proline-rich protein type 2 (HyPRP2). Our qRT-PCR results proved the full consistency and validation of the microarray experiments, although the relative expression ratios of the first were often higher. We believe that our microarray analysis as well as the equivalence of biological samples (duplicates) were fully supported by the qRT-PCR findings since they were robust enough to evaluate a massive number of genes and able to point out important candidate genes

Étude du rôle de la neddylation dans la régulation de la recombinaison méiotique

Homologous recombination is essential to all living organisms in order to repair DNA damages. In addition, a large majority of organisms use homologous recombination in meiosis to ensure proper chromosome segregation. A main step of meiotic recombination is crossover (CO) formation. During my PhD, I was able to highlight a new pathway controlling meiotic recombination. I showed that cycles of activation and deactivation of cullin-RING ligases (CRLs) are absolutely required for correct meiosis. CRLs are activated by neddylation, and deactivated by deneddylation. In addition, they can also be inhibited by sequestration by the CAND1 protein. My work demonstrated that these three levels of CRL regulation play crucial roles in meiotic homologous recombination in A. thaliana. First, I showed that AXR1, a key component of the neddylation machinery, is required for the correct localisation of meiotic COs and for somatic homologous recombination. Second, I showed that the deneddylation process mediated by CSN5A is also necessary for normal CO formation. I obtained evidence that this regulation of CO position is likely to be mediated by a CRL4 complex. Last, I could show that the CRL inhibitor, CAND1, is required for the formation of up to 90% of the COs. Using genetic and cytological tools, I showed that CAND1 probably acts on the regulation of the inter-homolog bias. Considering all these data, my work draws the attention to a new mechanism regulating meiotic homologous recombination, connecting for the first time meiosis to CRL-mediated ubiquitylation.La recombinaison homologue est essentielle à la réparation des lésions de l’ADN ainsi qu’à la ségrégation correcte des chromosomes en méiose. Une étape importante de la recombinaison méiotique est la formation des crossovers (CO). Au cours de ma thèse, j’ai mis en évidence un nouveau mécanisme de régulation de la recombinaison méiotique. J'ai montré que les cycles d'activation et de désactivation des cullin-RING ligases (CRL) sont absolument nécessaires à la recombinaison méiotique. Les CRL sont activées par neddylation et désactivées par la deneddylation. De plus, elles peuvent aussi être inhibées par la séquestration via la protéine CAND1. Mon travail a démontré que ces trois niveaux de régulation des CRL jouent des rôles cruciaux dans la recombinaison homologue méiotique chez A. thaliana. J’ai montré qu'AXR1, un composant clé de la machinerie de neddylation, est nécessaire à la localisation correcte des CO méiotiques et à la recombinaison homologue somatique. J’ai aussi prouvé que le processus de deneddylation médié par CSN5A est nécessaire à la formation des CO. J'ai obtenu des données montrant que cette régulation de la localisation des CO agit à travers la régulation d’un complexe CRL4. Enfin, j’ai pu montrer que l'inhibiteur des CRL, CAND1, est requis pour la formation de plus de 90 % des CO. En utilisant des outils génétiques et cytologiques, j'ai montré que CAND1 agit probablement sur la régulation du biais inter-homologue. L’ensemble de ces données, met l’accent sur un nouveau mécanisme de la régulation de la recombinaison homologue, connectant pour la première fois la méiose et l’ubiquitination via les cullin-RING Ligases

Study of the role of neddylation in the regulation of meiotic recombination

La recombinaison homologue est essentielle à la réparation des lésions de l ADN ainsi qu à la ségrégation correcte des chromosomes en méiose. Une étape importante de la recombinaison méiotique est la formation des crossovers (CO). Au cours de ma thèse, j ai mis en évidence un nouveau mécanisme de régulation de la recombinaison méiotique. J'ai montré que les cycles d'activation et de désactivation des cullin-RING ligases (CRL) sont absolument nécessaires à la recombinaison méiotique. Les CRL sont activées par neddylation et désactivées par la deneddylation. De plus, elles peuvent aussi être inhibées par la séquestration via la protéine CAND1. Mon travail a démontré que ces trois niveaux de régulation des CRL jouent des rôles cruciaux dans la recombinaison homologue méiotique chez A. thaliana. J ai montré qu'AXR1, un composant clé de la machinerie de neddylation, est nécessaire à la localisation correcte des CO méiotiques et à la recombinaison homologue somatique. J ai aussi prouvé que le processus de deneddylation médié par CSN5A est nécessaire à la formation des CO. J'ai obtenu des données montrant que cette régulation de la localisation des CO agit à travers la régulation d un complexe CRL4. Enfin, j ai pu montrer que l'inhibiteur des CRL, CAND1, est requis pour la formation de plus de 90 % des CO. En utilisant des outils génétiques et cytologiques, j'ai montré que CAND1 agit probablement sur la régulation du biais inter-homologue. L ensemble de ces données, met l accent sur un nouveau mécanisme de la régulation de la recombinaison homologue, connectant pour la première fois la méiose et l ubiquitination via les cullin-RING Ligases.Homologous recombination is essential to all living organisms in order to repair DNA damages. In addition, a large majority of organisms use homologous recombination in meiosis to ensure proper chromosome segregation. A main step of meiotic recombination is crossover (CO) formation. During my PhD, I was able to highlight a new pathway controlling meiotic recombination. I showed that cycles of activation and deactivation of cullin-RING ligases (CRLs) are absolutely required for correct meiosis. CRLs are activated by neddylation, and deactivated by deneddylation. In addition, they can also be inhibited by sequestration by the CAND1 protein. My work demonstrated that these three levels of CRL regulation play crucial roles in meiotic homologous recombination in A. thaliana. First, I showed that AXR1, a key component of the neddylation machinery, is required for the correct localisation of meiotic COs and for somatic homologous recombination. Second, I showed that the deneddylation process mediated by CSN5A is also necessary for normal CO formation. I obtained evidence that this regulation of CO position is likely to be mediated by a CRL4 complex. Last, I could show that the CRL inhibitor, CAND1, is required for the formation of up to 90% of the COs. Using genetic and cytological tools, I showed that CAND1 probably acts on the regulation of the inter-homolog bias. Considering all these data, my work draws the attention to a new mechanism regulating meiotic homologous recombination, connecting for the first time meiosis to CRL-mediated ubiquitylation.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Ssb2/Nabp1 is dispensable for thymic maturation, male fertility, and DNA repair in mice

SSB1 and SSB2 are newly identified singlestranded (ss)DNAbinding proteins that play a crucial role in genome maintenance in humans. We recently generated a knockout mouse model of Ssb1 and revealed its essential role for neonatal survival. Notably, we found compensatory up-regulation of Ssb2 protein levels in multiple tissues of conditional Ssb1-/- mice, suggesting functional compensation between these 2 proteins. We report here the first description of Ssb2-/- knockout mice. Surprisingly, unlike Ssb1 knockout mice, Ssb2-/- mice are viable and fertile and do not exhibit marked phenotypic changes when compared with their Ssb2+/+ and Ssb2+/- littermates. Notably, we did not detect any pathologic changes in the thymus, spleen, or testes, tissues with the most abundant expression of Ssb2. Moreover, Ssb2-/- mouse embryonic fibroblasts (MEFs) did not show any sensitivity to DNA-damaging agents, or defects in DNA repair capacity. However, we observed modest up-regulation of Ssb1 levels in Ssb2-/- MEFs as well as in Ssb2-/- thymus and spleen, suggesting that Ssb1 is likely able to compensate for the loss of Ssb2 in mice. Altogether, our results show that Ssb2 is dispensable for embryogenesis and adult tissue homeostasis, including thymopoiesis, splenic development, male fertility, and DNA repair in mice. -Boucher, D., Vu, T., Bain, A. L., Tagliaro-Jahns,M., Shi, W., Lane, S. W., Khanna, K. K. Ssb2/Nabp1 is dispensable for thymic maturation, male fertility, and DNA repair in mice

Crossover localisation is regulated by the neddylation posttranslational regulatory pathway

Crossovers (COs) are at the origin of genetic variability, occurring across successive generations, and they are also essential for the correct segregation of chromosomes during meiosis. Their number and position are precisely controlled, however the mechanisms underlying these controls are poorly understood. Neddylation/rubylation is a regulatory pathway of posttranslational protein modification that is required for numerous cellular processes in eukaryotes, but has not yet been linked to homologous recombination. In a screen for meiotic recombination-defective mutants, we identified several axr1 alleles, disrupting the gene encoding the E1 enzyme of the neddylation complex in Arabidopsis. Using genetic and cytological approaches we found that axr1 mutants are characterised by a shortage in bivalent formation correlated with strong synapsis defects. We determined that the bivalent shortage in axr1 is not due to a general decrease in CO formation but rather due to a mislocalisation of class I COs. In axr1, as in wild type, COs are still under the control of the ZMM group of proteins. However, in contrast to wild type, they tend to cluster together and no longer follow the obligatory CO rule. Lastly, we showed that this deregulation of CO localisation is likely to be mediated by the activity of a cullin 4 RING ligase, known to be involved in DNA damage sensing during somatic DNA repair and mouse spermatogenesis. In conclusion, we provide evidence that the neddylation/rubylation pathway of protein modification is a key regulator of meiotic recombination. We propose that rather than regulating the number of recombination events, this pathway regulates their localisation, through the activation of cullin 4 RING ligase complexes. Possible targets for these ligases are discussed

Bivalent shortage has a similar effect on each pair of chromosomes.

<p>Fluorescent in situ hybridisation (FISH) on metaphase I cells was performed with probes directed against the 45S (green) and the 5S (red) rDNA, which allow the identification of chromosomes 1 (unlabelled), 2 (green labelled), and 4 (green and red labelled), whereas chromosomes 3 and 5 cannot be distinguished (red labelled). In wild type, each chromosome pair represents 20% of the total number of bivalents (A and D, centre circle, in light, <i>n</i> = 21 cells). In <i>axr1</i> (B and D, N877898 allele, external circle, <i>n</i> = 28), the proportion of each bivalent pair is the same as in wild type. Bar = 5 µm.</p

Synapsis is strongly perturbed in <i>axr1</i>, but HEI10 dynamics during early prophase are unchanged.

<p>ZYP1 and HEI10 proteins were co-immunolocalised on lipsol-spread chromosomes from wild-type (A–D) and <i>axr1</i> (N877989 allele, E–L) meiotic cells. The overlay of both signals is shown here (ZYP1 in red, HEI10 in green), but single channels can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001930#pbio.1001930.s007" target="_blank">Figures S7</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001930#pbio.1001930.s008" target="_blank">S8</a>. In wild type as in <i>axr1</i>, ZYP1 appears on chromosomes as foci that quickly elongate, yielding a mixture of foci and short stretches (A, B, E, F, and I). Synapsis then progresses until complete synapsis is reached in wild type, defining the pachytene stage (C–D). In <i>axr1</i>, ZYP1 elongation can be detected, but full synapsis was never achieved (G, H, and K). In <i>axr1</i>, the ZYP1 signal is often uneven in thickness or forms dotted lines rather than a homogeneous and continuous signal (J and G). In addition, in some cases, only short and thick ZYP1 stretches were detected which could correspond to ZYP1 polycomplexes (L). During early zygotene, in wild type as in <i>axr1</i>, HEI10 forms numerous foci of variable sizes on chromatin (A, E, and I). Then, although synapsis progresses, combinations of large and small foci are observed, forming “strings of pearls” on ZYP1 stretches (B, F, and J, arrows). As meiosis progresses, a few bigger and brighter HEI10 foci can be observed in wild type (D) and in <i>axr1</i> (G, H, K, and L), which generally co-exist with smaller and fainter HEI1O foci (C, D, G, H, and K). Whereas this latter HEI10 pattern is associated with complete synapsis in wild type (C–D), synapsis is only partial in <i>axr1</i> (G, H, K, and L). Bar = 2 µm.</p