Genetic and epigenetic regulation of life cycle progression in the model brown alga Ectocarpus – Epicycle

• Bioinformatic analysis of histones and histone modifiers: Blast, literature surveys, comparative genomics
• Identification of histone PTMs: histone extraction, mass spectrometry, western blotting
• Genome-wide analysis of histone PTMs: ChIP-seq, MN-seq, FAIRE-seq, RNA-seq, bioinformatic transcriptomic analyses
• Identification of ORO and SAM interactors: Y2H, in vitro pull-downs
• Development of a CRISPR-Cas9-based method to generate gene knockouts: gamete release, bioinformatic design of CRISPR-Cas9 complexes, in vitro testing of CRISPR-Cas9 complexes, biolistic delivery of CRISPR-Cas9 complexes, phenotypic and genotypic analysisof putative mutants
• General methodologies: Ectocarpus culture

We have completed an extensive characterisation of chromatin features in Ectocarpus that included: 1) detailed annotation of the genes encoding both histone proteins and histone modifiers (i.e. writers and erasers of post-translational modifications; PTMs), 2) an exhaustive mass spectrometry and western blotting analysis of histone PTMs in Ectocarpus (47 different PTMs identified) and 3) ChIP-seq analysis of the genome-wide distributions of 11 selected PTMs during the Ectocarpus life cycle. Genome-wide distributions of PTMs indicated that most of the marks analysed have similar functions in brown algae to those observed in animals and/or in land plants (as well as in micro-algae such as the model diatom Phaeodactylum tricornutum). For example, H3K4me2, H3K4me3, H3K9ac, H3K14ac and H3K27ac were associated with transcription start sites (TSSs) of active genes, H3K36me3 with the gene bodies of transcribed genes and H4K20me3 with repeated sequences and transposons. In contrast, H3K79me2 exhibited a highly novel distribution, marking extensive regions of the genome, often spanning multiple transcription units, a pattern that has not been observed in any other organism to date. Comparison of histone PTM patterns during the gametophyte and sporophyte generations of the life cycle showed that generation-biased expression patterns correlated approximately with changes in the presence of TSS-localised PTMs but these marks alone were not sufficient to entirely explain changes in patterns of gene expression during the life cycle.
A major advance in relation to Task 1 was the development of a CRISPR-Cas9-based method to generate gene knockouts (Badis et al., 2021). A paper describing the method has been published and the technique is currently being applied to life-cycle-related genes in the context of Task 1, notably the bZIP transcription factor Ec-08_005050.

1) Our comprehensive analysis of histone PTMs in the model brown alga Ectocarpus (Bourdareau et al., 2021) was the first study to analyse histone modifications in this lineage. The study will therefore represent a key reference for future studies, providing both essential methodologies (such as the adapted ChIP-seq method) and a reference inventory of brown algal histone PTMs. In addition to addressing life-cycle-related questions, this study provided several new insights into chromatin function in the brown algae.
2) The development of CRISPR-Cas9-based methodology to generate gene knockouts in the model brown alga Ectocarpus (Badis et al., 2021) also represents an important advance because, previously, no method existed to carry out reverse genetics in the brown algae. These seaweeds are an ecologically and economically important group that independently evolved complex multicellularity so this new tool is expected to be of broad interest.

• Bourdareau, S., Tirichine, L., Lombard, B., Loew, D., Wu, Y., Coelho, S.M. and Cock, J.M. (2021). Genome Biology, 22, 12.
• Badis, Y., Scornet, D., Harada, M., Caillard, C., Raphalen, M., Gachon, C.M.M., Coelho, S.M., Motomure, T., Nagasato, C. and Cock, J.M. (2021). New Phytologist, in press.
• Coelho, S.M., Cock, J.M. (2020). Annual Review of Genetics, 54, 71–92
• Coelho, S.M., Peters, A.F., Müller, D. and Cock, J.M. (2020). EvoDevo, 11, 19.
• Bringloe, T.T., Starko, S., Wade, R.M., Vieira, C., Kawai, H., De Clerck, O., Cock, J.M., Coelho, S.M., Destombe, C., Valero, M., Neiva, J., Pearson, G.A., Faugeron, S., Serrão, E.A. and Verbruggen, H. (2020). Critical Reviews in Plant Sciences, 39, 281-321.
• Jueterbock, A., Minne, A.J.P., Cock, J.M., Coleman, M.A., Wernberg, T., Scheschonk, L., Rautenberger, R., and Zhang, J. and Zi-Min (2021). Frontiers in Marine Science, 8, 658485.
• Yao, H., Scornet, D., Peters, A.F., Jam, M., Hervé, C., Potin, P., Coelho, S.M. and Cock, J.M. (2020). Bio-protocol, 10, e3753
• Zhao X, Rastogi A, Deton Cabanillas AF, Ait Mohamed O, Cantrel C, Lombard B, Murik O, Genovesio A, Bowler C, Bouyer D, Loew D, Lin X, Veluchamy A, Vieira FRJ, Tirichine L. (2021) New Phytol. 229, 3208-3220.
• Hoguin A, Rastogi A, Bowler C, Tirichine L. (2021) Scientific Reports, 11, 2954.
• Xue Zhao, Anne Flore Deton Cabanillas, Alaguraj Veluchamy, Chris Bowler, Fabio Rocha Jimenez Vieira and Leila Tirichine (2020). Frontiers in Marine Science, 7, 189.

To function coherently, the pathways that control development in any multicellular organism must be initiated at the appropriate stage of the life cycle, but very little is known about how these two processes - life cycle progression and multicellular development - are integrated and coordinated. Epigenetic regulatory mechanisms are expected to play an important part in this process, providing a means to ensure that only the relevant genetic components are expressed at each stage of the life cycle.

Recent genetic analysis of mutants of the brown alga Ectocarpus have identified a major life cycle transition regulator, encoded by the OUROBOROS (ORO) and SAMSARA (SAM) genes, that links life cycle progression to multicellular development. Both ORO and SAM are predicted to encode TALE-class homeodomain transcription factors (TALE HD TFs). Interestingly, TALE HD TFs have also been implicated in life cycle regulation in mosses, indicating that an extremely ancient life cycle regulatory system has been convergently recruited to control sporophyte development in the land plant and brown algal lineages.

The objective of this project is to elucidate the genetic and epigenetic processes associated with life cycle progression and the deployment of the sporophyte developmental program in the brown alga Ectocarpus. The project will combine approaches designed to specifically dissect the regulatory pathway that includes the ORO and SAM proteins with approaches aimed at monitoring genome-wide epigenetic modifications (histone post-translational modifications and topologically associating domains detected using Hi-C) during life cycle progression. The subcellular location of the ORO and SAM proteins will be determined at different stages of the life cycle and interacting proteins will be identified and characterised. A broad range of approaches including ChIP-nexus, DAP-seq, protein-binding arrays and RNA-seq will be used to identify both the direct target genes of ORO and SAM and downstream effector genes. Additional components of this regulatory network will be detected using various genetic screens, including the isolation of oro and sam suppressor mutations. Data on the ORO/SAM regulatory pathway will be integrated with genome-wide analysis of transcriptome changes and chromatin modifications and during the life cycle to provide a comprehensive view of the events associated with the alternation of life cycle generations in this brown alga. The project will also explore the role of ORO and SAM in two situations where the sporophyte developmental program is initiated in a haploid context without gamete fusion: parthenogenetic development of gametes and meio-spore heteroblasty

Epicycle will associate two highly complementary partners. The Roscoff group has a long history of brown algal research and is currently leading efforts to develop genetic and genomic approaches for this group of organisms, notably through the development of Ectocarpus as a model system and through the Phaeoexplorer project (France Genomique) which is generating complete genome sequences for 45 brown algal species. The Nantes group has extensive experience in the domain of epigenetics and has pioneered the application of epigenetic techniques to stramenopiles (diatoms). Collaboration between these two groups within the context of Epicycle will allow an innovative, interdisciplinary approach to be employed to further our understanding of life cycle regulation and progression, integrating both genetic and epigenetic factors.

The results generated by Epicycle are expected to considerably improve our understanding of how developmental programs have been integrated, during evolution, into underlying life cycle processes. In a broader sense, this information is expected to contribute significantly to our understanding of the emergence of complex multicellular development, a major and highly important evolutionary transition.