“Transcriptional and Epigenetic regulation in the marine diatom Phaeodactylum tricornutum” Florian Maumus To cite this version: Florian Maumus. “Transcriptional and Epigenetic regulation in the marine diatom Phaeodactylum tricornutum”. Biochemistry [q-bio.BM]. Ecole Normale Supérieure de Paris - ENS Paris, 2009. English. tel-00475588 HAL Id: tel-00475588 https://tel.archives-ouvertes.fr/tel-00475588 Submitted on 22 Apr 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Thèse de Doctorat “Transcriptional and Epigenetic regulation in the marine diatom Phaeodactylum tricornutum” Présentée par: Florian Maumus Soutenance le 6 juillet 2009 devant les membres du jury: Prof. Martine Boccara Dr. Chris Bowler Dr. Pascale Lesage Prof. Olivier Panaud Jury présidé par Prof. Pierre Capy Thesis director: Chris Bowler CNRS UMR 8186 Département de Biologie Ecole Normale Supérieure 46 rue d’Ulm, Paris, France External supervisors: Vincent Colot CNRS UMR 8186 Département de Biologie Ecole Normale Supérieure 46 rue d’Ulm, Paris, France David Moreira CNRS UMR 8079 Unité d'Ecologie, Systématique et Evolution Université Paris-Sud, bâtiment 360 91405 Orsay Cedex, France. I would like to dedicate this work to my parents Chantal and Olivier, my sister Laure, and my little princess Diana for their love, comprehension, and support. I also think about my dear friend Bertrand who was the most brilliant person I ever met and was very interested in physics and biology but accidentally lost the ability to go to the University ten years ago. Bertrand, I know you would have had unlimited pioneering ideas about all the topics I will talk about. Contents Summary p. 1 Résumé en français p. 4 Aims of the thesis p. 7 Acknowledgments p. 8 Chapter I: Introduction p. 10 1.1 Diatoms, marine genomics, and the marine ecosystem p. 11 1.1.1 First glance p. 11 1.1.2 Diatom biology p. 12 1.1.3 Classification and habitats p. 19 1.1.4 Role of diatoms in biogeochemical cycles p. 23 1.1.5 Evolutionary history p. 24 1.1.6 Diatom and (algal) genomics p. 31 1.1.7 The diatom Phaeodactylum tricornutum p. 37 1.2 Transposable elements p. 38 1.2.1 Introduction p. 38 1.2.2 Diversity p. 41 1.2.2.1 Class I elements p. 43 1.2.2.2 Class II elements p. 48 1.2.3 Impact of TEs on genome evolution p. 51 1.2.3.1 Domestication p. 51 1.2.3.2 The generation of genetic variability in response to stress p. 52 1.2.3.3 TE-mediated recombination p. 53 1.2.3.4 Gene duplication p. 55 1.3References p. 57 Chapter II: Transcription factors in diatom genomes p. 67 2.1 Introduction p. 68 2.2 Results and Discussion p. 71 2.2.1 Transcription factor content in stramenopiles p. 71 2.2.2 Transcription factor complement among stramenopiles p. 71 2.2.3 Heat Shock Factors (HSFs) p. 76 2.2.4 Myb factors p. 79 2.2.5 bZIP domain factors p. 85 2.2.6 bHLH factors p. 89 2.2.7 Expression analysis p. 91 2.3 Material and Methods p. 94 2.4 References p. 95 Chapter III: Potential Impact of Stress Activated Retrotransposons on Genome Evolution in a Marine Diatom p. 101 3.1 Introduction p. 102 3.2 Results p. 105 3.2.1 Expansion of LTR Retrotransposons in the P. tricornutum genome p. 105 3.2.2 Classification of LTR retrotransposon sequences p. 108 3.2.3 Phylogenetic analysis p. 108 3.2.4 Expression of LTR retrotransposons in diatoms p. 113 3.2.5 Regulation of Blackbeard p. 115 3.2.6 Insertion polymorphism between P. tricornutum accessions p. 118 3.2.7 Two distinct haplotypes at loci containing TEs p. 120 3.2.8 TE-mediated recombination in the P. tricornutum genome p. 121 3.2.9 A high diversity of RT domains from micro-planktonic organisms p. 125 3.3 Discussion p. 130 3.4 Material and Methods p. 134 3.5 References p. 142 Chapter IV: Epigenetics in P. tricornutum p. 150 4.1 Introduction p. 151 4.2 Results and Discussion p. 154 4.2.1 Histone modifications p. 154 4.2.1.1 Histone modifiers p. 154 4.2.1.2 Effectors p. 158 4.2.1.3 Chromatin Extraction and Immunoprecipitation p. 159 4.2.2 DNA methylation p. 162 4.2.2.1 DNA methyltransferases in diatoms p. 164 4.2.2.2 DNA methylation in P. tricornutum p. 170 4.2.3 RNA silencing machinery in P. tricornutum p. 179 4.2.3.1 Diatom genes putatively involved in RNAi-related processes p. 182 4.2.3.2 RNA-directed DNA methylation (RdDM) in P. tricornutum p. 190 4.3 Material and Methods p. 192 4.4 References p. 198 Chapter V: Identification and analysis of transposable elements in the genome of the brown alga Ectocarpus siliculosus p. 205 5.1 Introduction p. 206 5.2 Results and discussion p. 209 5.2.1 Identification of repeated sequences in the E. siliculosus genome p. 209 5.2.2 Masking the E. siliculosus genome p. 212 5.2.3 Phylogeny of E. siliculosus LTR-retrotransposons p. 219 5.2.4 Expression analysis p. 223 5.2.5 DNA Methylation in Ectocarpus siliculosus p. 229 5.3 Material and Methods p. 232 5.4 References p. 239 Chapter VI: Conclusions and perspectives p.241 6.1 Conclusions and perspectives p.242 6.2 References p.249 Supplementary Material p. Summary The unicellular chlorophyll c-containing algal class Bacillariophyceae (diatoms) is among the most successful and diversified groups of photosynthetic eukaryotes, with possibly over 100,000 extant species (Round et al., 1990) widespread in all kinds of open water masses. The contribution of diatom photosynthesis to marine primary productivity has been estimated to be around 40% (Nelson et al., 1995; Raven and Waite, 2004). Diatoms have a peculiar genetic makeup in that they are likely to have emerged following a secondary endosymbiotic process between a photosynthetic eukaryote, most probably red algal-like, and a heterotrophic eukaryote (Falkowski et al., 2004). They are traditionally divided into two orders: the centric diatoms which are radially symmetrical and are thought to have arisen around 180 Million years ago (Mya), followed by the pennate diatoms around 90 Mya which are bilaterally symmetrical. The complete nuclear, mitochondrial, and plastid genome sequences of the centric diatom Thalassiosira pseudonana (32 Mb) and the pennate diatom Phaeodactylum tricornutum (27 Mb) have recently become available (Armbrust et al., 2004 and Bowler et al., 2008). My PhD focalized on the study of various aspects of the regulation of gene expression in diatoms as well as on the study of genome evolution and dynamics in these species. Gene expression is regulated at the transcriptional, post-transcriptional, and epigenetic (or pre- transcriptional) levels. In the framework of my PhD, I performed an in silico search in diatom genomes for transcription factors (TFs), which are master control proteins involved in transcriptional regulation, in order to get a panorama of the TF complement in these species and to identify lineage-specific peculiarities. We found, for example, that Heat Shock Factors (HSFs) have been amplified dramatically during the course of diatom evolution. Analysis of 1 the abundance of TFs in different P. tricornutum and T. pseudonana-derived EST libraries enabled us to identify some specificities of their expression. The evolution of eukaryotic genomes is impacted by the direct or secondary effects of transposable elements (TEs), which are mobile DNA sequences that inhabit the genomes of most organisms. In order to evaluate genome dynamics in diatoms, the search for transposable elements in diatoms enabled to establish that a specific class of TEs, the Copia-like retrotransposons, is the most abundant in diatom genomes and has been significantly amplified in the P. tricornutum genome with respect to T. pseudonana, constituting 5.8 and 1% of the respective genomes. Phylogenetic analysis enabled me to demonstrate that diatom genomes harbor two classes of diatom-specific Long Terminal Repeat retrotransposons (LTR- RTs), as well as another class which is widespread among eukaryotes. Analysis of their abundance in various P. tricornutum-derived EST libraries has shown that two of these elements are activated in response to stress such as nitrate starvation. This activation is accompanied by DNA hypomethylation and the analysis of insertion profiles of different P. tricornutum ecotypes from around the world as well as other clues suggest that TEs play an important role in the generation of variability in diatom genomes. My interest in TEs further led me to try to characterize them in the genome of the brown alga Ectocarpus siliculosus. In silico searches for genes encoding proteins putatively capable of introducing or stabilizing epigenetic modifications such as histone modifiers and DNA methyltransferase has shown the presence of a large set of such proteins in the P. tricornutum genome, as well as their particularities. The presence of particular histone modifications in the P. tricornutum proteome has also been assessed by western blot experiments. In addition, I adapted a chromatin immunoprecipitation protocol for P. tricornutum which was used successfully to observe that histones within nucleosomes associated with TEs were marked with specific modifications.