“Transcriptional and Epigenetic regulation in the marine diatom Phaeodactylum tricornutum”

Abstract : 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 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. Other experiments enabled to establish that most TEs are marked by DNA methylation in P. tricornutum. An experiment aiming to obtain genome-wide DNA methylation pattern in this species has been designed and launched and will allow to whether some genes are also methylated. Finally, small RNAs (sRNAs) constitute an additional mode of regulation of gene expression, acting at the interface between transcriptional regulation, post-transcriptional regulation, and epigenetic phenomena. I have studied sRNA-based mechanisms in diatoms by performing an in silico search that showed that proteins putatively involved in the generation of sRNAs are only poorly conserved in diatoms. I was also able to determine experimentally a link between sRNA and DNA methylation in P. tricornutum.
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Biomolecules. Ecole Normale Supérieure de Paris - ENS Paris, 2009. English


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Submitted on : Thursday, April 22, 2010 - 11:37:08 AM
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Florian Maumus. “Transcriptional and Epigenetic regulation in the marine diatom Phaeodactylum tricornutum”. Biomolecules. Ecole Normale Supérieure de Paris - ENS Paris, 2009. English. <tel-00475588>

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