Essential and recurrent roles for hairpin RNAs in silencing \u3ci\u3ede novo sex\u3c/i\u3e chromosome conflict in \u3ci\u3eDrosophila simulans\u3c/i\u3e

Meiotic drive loci distort the normally equal segregation of alleles, which benefits their own transmission even in the face of severe fitness costs to their host organism. However, relatively little is known about the molecular identity of meiotic drivers, their strategies of action, and mechanisms that can suppress their activity. Here, we present data from the fruitfly Drosophila simulans that address these questions. We show that a family of de novo, protamine- derived X-linked selfish genes (the Dox gene family) is silenced by a pair of newly emerged hairpin RNA (hpRNA) small interfering RNA (siRNA)-class loci, Nmy and Tmy. In the w[XD1] genetic background, knockout of nmy derepresses Dox and MDox in testes and depletes male progeny, whereas knockout of tmy causes misexpression of PDox genes and renders males sterile. Importantly, genetic interactions between nmy and tmy mutant alleles reveal that Tmy also specifically maintains male progeny for normal sex ratio. We show the Dox loci are functionally polymorphic within D. simulans, such that both nmy-associated sex ratio bias and tmy-associated sterility can be rescued by wild-type X chromosomes bearing natural deletions in different Dox family genes. Finally, using tagged transgenes of Dox and PDox2, we provide the first experimental evidence Dox family genes encode proteins that are strongly derepressed in cognate hpRNA mutants. Altogether, these studies support a model in which protamine-derived drivers and hpRNA suppressors drive repeated cycles of sex chromosome conflict and resolution that shape genome evolution and the genetic control of male gametogenesis

Physical and functional interaction between SET1/COMPASS complex component CFP-1 and a Sin3S HDAC complex in C. elegans.

The CFP1 CXXC zinc finger protein targets the SET1/COMPASS complex to non-methylated CpG rich promoters to implement tri-methylation of histone H3 Lys4 (H3K4me3). Although H3K4me3 is widely associated with gene expression, the effects of CFP1 loss vary, suggesting additional chromatin factors contribute to context dependent effects. Using a proteomics approach, we identified CFP1 associated proteins and an unexpected direct link between Caenorhabditis elegans CFP-1 and an Rpd3/Sin3 small (SIN3S) histone deacetylase complex. Supporting a functional connection, we find that mutants of COMPASS and SIN3 complex components genetically interact and have similar phenotypic defects including misregulation of common genes. CFP-1 directly binds SIN-3 through a region including the conserved PAH1 domain and recruits SIN-3 and the HDA-1/HDAC subunit to H3K4me3 enriched promoters. Our results reveal a novel role for CFP-1 in mediating interaction between SET1/COMPASS and a Sin3S HDAC complex at promoters

Study of the Caenorhabditis elegans SET-2 histone methyltransferase and its interactors in germline maintenance

Les modifications post-traductionelles des histones contribuent à l’expression génique et à la stabilité du génome. La méthylation de la lysine 4 de l’histone H3 (H3K4me), une marque associée aux promoteurs de gènes transcrits, est déposé par les methyltransferases hautement conservées de la famille SET1, dans le contexte du complexe COMPASS. SET-2, l’homologue de SET1 chez Caenorhabditis elegans, est responsable de la déposition de H3K4me dans la lignée germinale, et son inactivation provoque une perte progressive de la fertilité. Le but de mon travail de thèse a été d’étudier comment SET-2 et la méthylation de H3K4 contribuent au maintien de la lignée germinale. J’ai montré que l’absence de SET-2 provoque une sensibilité accrue aux dommages à l’ADN. Cependant, les voies de signalisation et de réparation de ces dommages sont fonctionnelles dans le mutant set-2. Par séquençage de l’ADN, j’ai par ailleurs montré que la stérilité progressive observée en l’absence de set-2 n’est pas due à une capacité de réparation réduite. L’ensemble de mes résultats suggère que H3K4me pourrait agir en aval de la signalisation de dommages à l’ADN, en influençant l’organisation de la chromatine aux sites des cassures double brin. J’ai d’autre part mis en évidence une nouvelle fonction pour la méthylation de H3K4 dans l’organisation de la chromatine en montrant que set-2 interagit génétiquement avec le complexe Condensine II et la Topoisomérase II, facteurs clefs de l’organisation mitotique des chromosomes. Des expériences de microscopie par FLIM-FRET ont d’ailleurs validé une fonction de H3K4 méthylée dans l’organisation de la chromatine dans la lignée germinale. Enfin, j’ai montré par analyses transcriptomiques que la protéine CFP-1 du complexe COMPASS est impliquée dans la régulation du programme transcriptionnel de la lignée germinale et que cette fonction est indépendante de SET-2. L’ensemble de mes résultats montre comment la régulation chromatinienne impacte le maintien d’une lignée germinale fonctionnelle à plusieurs niveaux.Post-translational modifications of histones contribute to gene expression and genome stability. Methylation of lysine 4 of histone H3 (H3K4me), a mark associated with actively transcribed genes, is deposited by the highly conserved SET1 family methyltransferases acting in COMPASS related complexes. SET-2, the SET1 homologue in Caenorhabditis elegans, is responsible for the deposition of H3K4me in the germ line, and its inactivation causes progressive loss of fertility. The purpose of my PhD work was to study how SET-2 and the methylation of H3K4 contribute to the maintenance of the germ line. I have shown that the absence of SET-2 causes increased sensitivity to DNA damage. However, the DNA damage-induced signaling and repair pathways are functional in the set-2 mutant. By DNA sequencing, I have also shown that the progressive sterility observed in the absence of set-2 is not due to a reduced repair capacity. Together, my results suggest that H3K4 methylation may act downstream of DNA damage signaling, potentially by influencing the organization of chromatin at the sites of double-strand breaks. I have also described a new function for H3K4 methylation in the organization of chromatin by showing that set-2 genetically interacts with the Condensitin II complex and Topoisomerase II, key factors in mitotic chromosome organization. Moreover, FLIM-FRET microscopy experiments have validated a role for H3K4 methylation in germline chromatin organization. Finally, using transcriptomic analyses, I have described a function for CFP-1, a component of the COMPASS complex, in the regulation of the germline transcriptional program independent of SET-2. Altogether, my results show how chromatin regulation affects the maintenance of a functional germline through multiple mechanisms

Étude de la fonction de l’histone méthyltransférase SET-2 et de ses interacteurs dans le maintien de la lignée germinale de Caenorhabditis elegans

Post-translational modifications of histones contribute to gene expression and genome stability. Methylation of lysine 4 of histone H3 (H3K4me), a mark associated with actively transcribed genes, is deposited by the highly conserved SET1 family methyltransferases acting in COMPASS related complexes. SET-2, the SET1 homologue in Caenorhabditis elegans, is responsible for the deposition of H3K4me in the germ line, and its inactivation causes progressive loss of fertility. The purpose of my PhD work was to study how SET-2 and the methylation of H3K4 contribute to the maintenance of the germ line. I have shown that the absence of SET-2 causes increased sensitivity to DNA damage. However, the DNA damage-induced signaling and repair pathways are functional in the set-2 mutant. By DNA sequencing, I have also shown that the progressive sterility observed in the absence of set-2 is not due to a reduced repair capacity. Together, my results suggest that H3K4 methylation may act downstream of DNA damage signaling, potentially by influencing the organization of chromatin at the sites of double-strand breaks. I have also described a new function for H3K4 methylation in the organization of chromatin by showing that set-2 genetically interacts with the Condensitin II complex and Topoisomerase II, key factors in mitotic chromosome organization. Moreover, FLIM-FRET microscopy experiments have validated a role for H3K4 methylation in germline chromatin organization. Finally, using transcriptomic analyses, I have described a function for CFP-1, a component of the COMPASS complex, in the regulation of the germline transcriptional program independent of SET-2. Altogether, my results show how chromatin regulation affects the maintenance of a functional germline through multiple mechanisms.Les modifications post-traductionelles des histones contribuent à l’expression génique et à la stabilité du génome. La méthylation de la lysine 4 de l’histone H3 (H3K4me), une marque associée aux promoteurs de gènes transcrits, est déposé par les methyltransferases hautement conservées de la famille SET1, dans le contexte du complexe COMPASS. SET-2, l’homologue de SET1 chez Caenorhabditis elegans, est responsable de la déposition de H3K4me dans la lignée germinale, et son inactivation provoque une perte progressive de la fertilité. Le but de mon travail de thèse a été d’étudier comment SET-2 et la méthylation de H3K4 contribuent au maintien de la lignée germinale. J’ai montré que l’absence de SET-2 provoque une sensibilité accrue aux dommages à l’ADN. Cependant, les voies de signalisation et de réparation de ces dommages sont fonctionnelles dans le mutant set-2. Par séquençage de l’ADN, j’ai par ailleurs montré que la stérilité progressive observée en l’absence de set-2 n’est pas due à une capacité de réparation réduite. L’ensemble de mes résultats suggère que H3K4me pourrait agir en aval de la signalisation de dommages à l’ADN, en influençant l’organisation de la chromatine aux sites des cassures double brin. J’ai d’autre part mis en évidence une nouvelle fonction pour la méthylation de H3K4 dans l’organisation de la chromatine en montrant que set-2 interagit génétiquement avec le complexe Condensine II et la Topoisomérase II, facteurs clefs de l’organisation mitotique des chromosomes. Des expériences de microscopie par FLIM-FRET ont d’ailleurs validé une fonction de H3K4 méthylée dans l’organisation de la chromatine dans la lignée germinale. Enfin, j’ai montré par analyses transcriptomiques que la protéine CFP-1 du complexe COMPASS est impliquée dans la régulation du programme transcriptionnel de la lignée germinale et que cette fonction est indépendante de SET-2. L’ensemble de mes résultats montre comment la régulation chromatinienne impacte le maintien d’une lignée germinale fonctionnelle à plusieurs niveaux

Étude de la fonction de l’histone méthyltransférase SET-2 et de ses interacteurs dans le maintien de la lignée germinale de Caenorhabditis elegans

Post-translational modifications of histones contribute to gene expression and genome stability. Methylation of lysine 4 of histone H3 (H3K4me), a mark associated with actively transcribed genes, is deposited by the highly conserved SET1 family methyltransferases acting in COMPASS related complexes. SET-2, the SET1 homologue in Caenorhabditis elegans, is responsible for the deposition of H3K4me in the germ line, and its inactivation causes progressive loss of fertility. The purpose of my PhD work was to study how SET-2 and the methylation of H3K4 contribute to the maintenance of the germ line. I have shown that the absence of SET-2 causes increased sensitivity to DNA damage. However, the DNA damage-induced signaling and repair pathways are functional in the set-2 mutant. By DNA sequencing, I have also shown that the progressive sterility observed in the absence of set-2 is not due to a reduced repair capacity. Together, my results suggest that H3K4 methylation may act downstream of DNA damage signaling, potentially by influencing the organization of chromatin at the sites of double-strand breaks. I have also described a new function for H3K4 methylation in the organization of chromatin by showing that set-2 genetically interacts with the Condensitin II complex and Topoisomerase II, key factors in mitotic chromosome organization. Moreover, FLIM-FRET microscopy experiments have validated a role for H3K4 methylation in germline chromatin organization. Finally, using transcriptomic analyses, I have described a function for CFP-1, a component of the COMPASS complex, in the regulation of the germline transcriptional program independent of SET-2. Altogether, my results show how chromatin regulation affects the maintenance of a functional germline through multiple mechanisms.Les modifications post-traductionelles des histones contribuent à l’expression génique et à la stabilité du génome. La méthylation de la lysine 4 de l’histone H3 (H3K4me), une marque associée aux promoteurs de gènes transcrits, est déposé par les methyltransferases hautement conservées de la famille SET1, dans le contexte du complexe COMPASS. SET-2, l’homologue de SET1 chez Caenorhabditis elegans, est responsable de la déposition de H3K4me dans la lignée germinale, et son inactivation provoque une perte progressive de la fertilité. Le but de mon travail de thèse a été d’étudier comment SET-2 et la méthylation de H3K4 contribuent au maintien de la lignée germinale. J’ai montré que l’absence de SET-2 provoque une sensibilité accrue aux dommages à l’ADN. Cependant, les voies de signalisation et de réparation de ces dommages sont fonctionnelles dans le mutant set-2. Par séquençage de l’ADN, j’ai par ailleurs montré que la stérilité progressive observée en l’absence de set-2 n’est pas due à une capacité de réparation réduite. L’ensemble de mes résultats suggère que H3K4me pourrait agir en aval de la signalisation de dommages à l’ADN, en influençant l’organisation de la chromatine aux sites des cassures double brin. J’ai d’autre part mis en évidence une nouvelle fonction pour la méthylation de H3K4 dans l’organisation de la chromatine en montrant que set-2 interagit génétiquement avec le complexe Condensine II et la Topoisomérase II, facteurs clefs de l’organisation mitotique des chromosomes. Des expériences de microscopie par FLIM-FRET ont d’ailleurs validé une fonction de H3K4 méthylée dans l’organisation de la chromatine dans la lignée germinale. Enfin, j’ai montré par analyses transcriptomiques que la protéine CFP-1 du complexe COMPASS est impliquée dans la régulation du programme transcriptionnel de la lignée germinale et que cette fonction est indépendante de SET-2. L’ensemble de mes résultats montre comment la régulation chromatinienne impacte le maintien d’une lignée germinale fonctionnelle à plusieurs niveaux

Distinct spermiogenic phenotypes underlie sperm elimination in the Segregation Distorter meiotic drive system

International audienceSegregation Distorter (SD) is a male meiotic drive system in Drosophila melanogaster. Males heterozygous for a selfish SD chromosome rarely transmit the homologous SD + chromosome. It is well established that distortion results from an interaction between Sd, the primary distorting locus on the SD chromosome and its target, a satellite DNA called Rsp, on the SD + chromosome. However, the molecular and cellular mechanisms leading to postmeiotic SD + sperm elimination remain unclear. Here we show that SD/SD + males of different genotypes but with similarly strong degrees of distortion have distinct spermiogenic phenotypes. In some genotypes, SD + spermatids fail to fully incorporate protamines after the removal of histones, and degenerate during the individualization stage of spermiogenesis. In contrast, in other SD/SD + genotypes, protamine incorporation appears less disturbed, yet spermatid nuclei are abnormally compacted, and mature sperm nuclei are eventually released in the seminal vesicle. Our analyses of different SD + chromosomes suggest that the severity of the spermiogenic defects associates with the copy number of the Rsp satellite. We propose that when Rsp copy number is very high (> 2000), spermatid nuclear compaction defects reach a threshold that triggers a checkpoint controlling sperm chromatin quality to eliminate abnormal spermatids during individualization

Diversity of puroindolines as revealed by two-dimensional electrophoresis

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A Role for Caenorhabditis elegans COMPASS in Germline Chromatin Organization

International audienceDeposition of histone H3 lysine 4 (H3K4) methylation at promoters is catalyzed by the SET1/COMPASS complex and is associated with context-dependent effects on gene expression and local changes in chromatin organization. The role of SET1/COMPASS in shaping chromosome architecture has not been investigated. Here we used Caenorhabditis elegans to address this question through a live imaging approach and genetic analysis. Using quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) on germ cells expressing histones eGFP-H2B and mCherry-H2B, we find that SET1/COMPASS influences meiotic chromosome organization, with marked effects on the close proximity between nucleosomes. We further show that inactivation of set-2, encoding the C. elegans SET1 homologue, or CFP-1, encoding the chromatin targeting subunit of COMPASS, enhances germline chromosome organization defects and sterility of condensin-II depleted animals. set-2 loss also aggravates germline defects resulting from conditional inactivation of topoisomerase II, another structural component of chromosomes. Expression profiling of set-2 mutant germlines revealed only minor transcriptional changes, suggesting that the observed effects are at least partly independent of transcription. Altogether, our results are consistent with a role for SET1/COMPASS in shaping meiotic chromosomes in C. elegans, together with the non-histone proteins condensin-II and topoisomerase. Given the high degree of conservation, our findings expand the range of functions attributed to COMPASS and suggest a broader role in genome organization in different species

Identification of Antioxidant Metabolites from Five Plants (Calophyllum inophyllum, Gardenia taitensis, Curcuma longa, Cordia subcordata, Ficus prolixa) of the Polynesian Pharmacopoeia and Cosmetopoeia for Skin Care

Oxidative stress contributes to impairment of skin health, the wound healing process, and pathologies such as psoriasis or skin cancer. Five Polynesian medicinal plants, among the most traditionally used for skin care (pimples, wounds, burns, dermatoses) are studied herein for their antioxidant properties: Calophyllum inophyllum, Gardenia taitensis, Curcuma longa, Cordia subcordata, and Ficus prolixa. Plant extracts were submitted to in vitro bioassays related to antioxidant properties and their bioactive constituents were identified by a metabolomic analytical approach. High performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) analysis was performed leading to the characterization of 61 metabolites. Compounds annotated for F. prolixa and C. subcordata extracts were reported for the first time. Antioxidant properties were evaluated by total phenolic content (TPC), free radical scavenging DPPH (1,1-diphenyl-2-picryl-hydrazyl), and Ferric Reducing Antioxidant Power activity (FRAP) assays. F. prolixa extract was the most active one and showed antioxidant intracellular activity on keratinocytes by Anti Oxydant Power 1 assay. Online HPLC-DPPH allowed the identification of phenolic bioactive compounds such as quercetin-O-rhamnoside, rosmarinic acid, chlorogenic acid, procyanidins, epicatechin, 5-O-caffeoylshikimic acid, and curcumin as being responsible for the scavenging properties of these plant extracts. These results highlight the potential of F. prolixa aerial roots as a source of antioxidants for skin care application

Usefulness of polymerase chain reaction for diagnosing Whipple's disease in rheumatology

International audienc