Contrôle génétique et épigénétique des transitions du cycle de vie chez l'algue brune Ectocarpus sp.

The brown alga Ectocarpus exhibits a haploid-diploid life cycle with an alternation between two multicellular generations : a haploid gametophyte and a diploid sporophyte. Two mutants exhibit homeotic switching between the sporophyte and gametophyte programs, reiterating the gametophyte program instead of switching to the sporophyte. These mutants, called ouroboros (oro) et samsara (sam), carry mutations into two different genes that code for TALE homeodomain transcription factors. This thesis aimed to characterize these two transcription factors and the chromatin dynamics associated with the alternation of generation in Ectocarpus. This thesis presents the characterisation of the oro and sam mutants and a transcriptomic comparison of the mutants with the sporophyte and gametophyte. DNA-binding preferences of the two transcription factors were evaluated using in vitro methods. ORO and SAM are able to heterodimerise via their respective homeodomains and a yeast two-hybrid screen showed that two C subunits of the Nuclear Factor Y family are able to interacting with ORO. This thesis also presents major advances in the study of chromatin regulation in the brown alga. A chromatin immunoprecipitation protocol was established and used to obtain genome-wide profiles for six histone modifications. Taken together, the data presented here suggests that ORO and SAM may be involved directly in chromatin reprogramming at generation-biased genes via an association with the NF-Y complex. The work presented represents a pioneer analysis of brown algal transcription factors and chromatin reprogramming events involved in the regulation of developmental pathways.L’algue brune Ectocarpus présente un cycle de vie haplo-diploïde avec l’alternance de deux générations multicellulaires : un gamétophyte haploïde et un sporophyte diploïde. Deux mutants présentent un changement homéotique entre les programmes de développement des générations sporophyte et gamétophyte. Les mutants réitèrent le programme de développement du gamétophyte à la place du sporophyte. Ces mutants, appelés ouroboros (oro) et samsara (sam), sont affectés dans deux gènes différents codant pour des facteurs de transcription à homéodomaine de classe TALE. Ma thèse porte sur la caractérisation des deux facteurs de transcription ORO et SAM ainsi que sur les dynamiques chromatiniennes sous-jacentes. Cette thèse présente les phénotypes des deux mutants oro et sam ainsi qu’une comparaison du transcriptome des mutants avec celui du gamétophyte et sporophyte. L’interaction entre ORO et SAM a été également testée et a lieu au niveau de chaque homéodomaine. Les préférences de liaison à l’ADN des deux facteurs de transcription ont été évaluées in vitro. Un criblage par double-hybride de levure a permis d’identifier deux sous-unités C de la famille de facteurs de transcription Nuclear Factor Y interagissant avec ORO. Cette thèse a également permis des avancées importantes dans l’étude de la régulation de la chromatine notamment en mettant au point un protocole d’immunoprécipitation de la chromatine. Ainsi, les profils de six modifications post-traductionnelles d’histones sur l’ensemble du génome ont été établis. Ce travail est pionnier dans la compréhension de la reprogrammation de la chromatine et la régulation de voies de développement majeures chez les algues brunes

Genetic and epigenetic control of life cycle transitions in the brown alga Ectocarpus sp.

L’algue brune Ectocarpus présente un cycle de vie haplo-diploïde avec l’alternance de deux générations multicellulaires : un gamétophyte haploïde et un sporophyte diploïde. Deux mutants présentent un changement homéotique entre les programmes de développement des générations sporophyte et gamétophyte. Les mutants réitèrent le programme de développement du gamétophyte à la place du sporophyte. Ces mutants, appelés ouroboros (oro) et samsara (sam), sont affectés dans deux gènes différents codant pour des facteurs de transcription à homéodomaine de classe TALE. Ma thèse porte sur la caractérisation des deux facteurs de transcription ORO et SAM ainsi que sur les dynamiques chromatiniennes sous-jacentes. Cette thèse présente les phénotypes des deux mutants oro et sam ainsi qu’une comparaison du transcriptome des mutants avec celui du gamétophyte et sporophyte. L’interaction entre ORO et SAM a été également testée et a lieu au niveau de chaque homéodomaine. Les préférences de liaison à l’ADN des deux facteurs de transcription ont été évaluées in vitro. Un criblage par double-hybride de levure a permis d’identifier deux sous-unités C de la famille de facteurs de transcription Nuclear Factor Y interagissant avec ORO. Cette thèse a également permis des avancées importantes dans l’étude de la régulation de la chromatine notamment en mettant au point un protocole d’immunoprécipitation de la chromatine. Ainsi, les profils de six modifications post-traductionnelles d’histones sur l’ensemble du génome ont été établis. Ce travail est pionnier dans la compréhension de la reprogrammation de la chromatine et la régulation de voies de développement majeures chez les algues brunes.The brown alga Ectocarpus exhibits a haploid-diploid life cycle with an alternation between two multicellular generations : a haploid gametophyte and a diploid sporophyte. Two mutants exhibit homeotic switching between the sporophyte and gametophyte programs, reiterating the gametophyte program instead of switching to the sporophyte. These mutants, called ouroboros (oro) et samsara (sam), carry mutations into two different genes that code for TALE homeodomain transcription factors. This thesis aimed to characterize these two transcription factors and the chromatin dynamics associated with the alternation of generation in Ectocarpus. This thesis presents the characterisation of the oro and sam mutants and a transcriptomic comparison of the mutants with the sporophyte and gametophyte. DNA-binding preferences of the two transcription factors were evaluated using in vitro methods. ORO and SAM are able to heterodimerise via their respective homeodomains and a yeast two-hybrid screen showed that two C subunits of the Nuclear Factor Y family are able to interacting with ORO. This thesis also presents major advances in the study of chromatin regulation in the brown alga. A chromatin immunoprecipitation protocol was established and used to obtain genome-wide profiles for six histone modifications. Taken together, the data presented here suggests that ORO and SAM may be involved directly in chromatin reprogramming at generation-biased genes via an association with the NF-Y complex. The work presented represents a pioneer analysis of brown algal transcription factors and chromatin reprogramming events involved in the regulation of developmental pathways

Rapid Evolution of microRNA Loci in the Brown Algae

International audienceStringent searches for microRNAs (miRNAs) have so far only identified these molecules in animals, land plants, chlorophyte green algae, slime molds and brown algae. The identification of miRNAs in brown algae was based on the analysis of a single species, the filamentous brown alga Ectocarpus sp. Here, we have used deep sequencing of small RNAs and a recently published genome sequence to identify miRNAs in a second brown alga, the kelp Saccharina japonica. S. japonica possesses a large number of miRNAs (117) and these miRNAs are highly diverse, falling into 98 different families. Surprisingly, none of the S. japonica miRNAs share significant sequence similarity with the Ectocarpus sp. miRNAs. However, the miRNA repertoires of the two species share a number of structural and genomic features indicating that they were generated by similar evolutionary processes and therefore probably evolved within the context of a common, ancestral miRNA system. This lack of sequence similarity suggests that miRNAs evolve rapidly in the brown algae (the two species are separated by ~95 Myr of evolution). The sets of predicted targets of miRNAs in the two species were also very different suggesting that the divergence of the miRNAs may have had significant consequences for miRNA function

Chromatin landscape associated with sexual differentiation in a UV sex determination system

International audienceAbstract In many eukaryotes, such as dioicous mosses and many algae, sex is determined by UV sex chromosomes and is expressed during the haploid phase of the life cycle. In these species, the male and female developmental programs are initiated by the presence of the U- or V-specific regions of the sex chromosomes but, as in XY and ZW systems, sexual differentiation is largely driven by autosomal sex-biased gene expression. The mechanisms underlying the regulation of sex-biased expression of genes during sexual differentiation remain elusive. Here, we investigated the extent and nature of epigenomic changes associated with UV sexual differentiation in the brown alga Ectocarpus, a model UV system. Six histone modifications were quantified in near-isogenic lines, leading to the identification of 16 chromatin signatures across the genome. Chromatin signatures correlated with levels of gene expression and histone PTMs changes in males versus females occurred preferentially at genes involved in sex-specific pathways. Despite the absence of chromosome scale dosage compensation and the fact that UV sex chromosomes recombine across most of their length, the chromatin landscape of these chromosomes was remarkably different to that of autosomes. Hotspots of evolutionary young genes in the pseudoautosomal regions appear to drive the exceptional chromatin features of UV sex chromosomes

Characterisation of brown alga mutants indicates deep evolutionary origins of life cycle regulatory pathways

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Convergent recruitment of TALE homeodomain life cycle regulators to direct sporophyte development in land plants and brown algae

International audienceThree amino acid loop extension homeodomain transcription factors (TALE HD TFs) act as life cycle regulators in green algae and land plants. In mosses these regulators are required for the deployment of the sporophyte developmental program. We demonstrate that mutations in either of two TALE HD TF genes, OUROBOROS or SAMSARA, in the brown alga Ectocarpus result in conversion of the sporophyte generation into a gametophyte. The OUROBOROS and SAMSARA proteins heterodimerise in a similar manner to TALE HD TF life cycle regulators in the green lineage. These observations demonstrate that TALE-HD-TF-based life cycle regulation systems have an extremely ancient origin, and that these systems have been independently recruited to regulate sporophyte developmental programs in at least two different complex multicellular eukaryotic supergroups, Archaeplastida and Chromalveolata