Les océans couvrent plus de 70% de la surface de la Terre (planète bleue) et la productivité primaire nette (PPN) marine est équivalente à celle terrestre. Alors qu‟il ne représente que 1% de la biomasse totale d‟organismes photosynthétiques de la planète, le phytoplancton est responsable d‟environ 45% de la PPN globale. Le terme phytoplancton décrit un assemblage polyphylétique comprenant des eucaryotes et procaryotes photosynthétiques dérivant avec les courants. Dans les océans contemporains, les diatomées constituent un groupe d‟eucaryotes unicellulaires autotrophes très abondant, responsable de 40% de la PPN marine. Les diatomées appartiennent à la lignée des straménopiles qui sont issus d‟un évènement d‟endosymbiose entre une algue rouge et un hôte hétérotrophe. Elles sont classifiées en deux groupes majeurs : les centriques qui son apparues il y a environ 200 millions d‟années (Ma), et les pennées qui ont évolué il y a environ 90 Ma. Deux génomes de diatomées ont récemment été séquencés : celui de la diatomée centrique Thalassiosira pseudonana (32 Mb), et celui de la diatomée pennée Phaeodactylum tricornutum (27 Mb). Mon sujet de doctorat s‟est focalisé sur l‟étude de différents aspects de la régulation de l‟expression génique ainsi que sur la dynamique et l‟évolution de ces génomes. L‟expression des gènes est régulée à différents niveaux: trancriptionel, post-transcriptionel, et épigénétique. Dans le cadre de mon doctorat, une étude de la régulation transcriptionelle chez les diatomées a été effectuée et comprend l‟identification et l‟analyse in silico des facteurs de transcription (FT). Cela a permis par exemple d‟établir qu‟une classe spécifique de FT, les Heat Shock Factors, sont particulièrement abondants chez les diatomées par rapport aux autres eucaryotes. L‟analyse de la représentation des FT identifiés dans différentes librairies d‟EST élaborées à partir de cultures ayant subi divers stress a permis de détecter certaines spécificités d‟expression. L‟évolution des génomes eucaryotes est largement impactée par les effets directs et secondaires des éléments transposables (ET) qui sont des éléments génétiques mobiles se trouvant dans le génome de la plupart des organismes. Dans le but d‟étudier la dynamique des génomes de diatomées, la recherche de différents types d‟ET a permis d‟établir qu‟une certaine classe, les rétrotransposons de type Copia, est la plus abondante dans ces génomes et constitue un part significativement plus importante du génome de P. tricornutum (5,8%) par rapport à T. pseudonana (1%). D‟autre part, des analyses phylogénitiques ont montré que les rétrotransposons de type copia forment deux classes distinctes et éloignées de la lignée Copia. L‟analyse de leurs niveaux d‟expression a montré que la transcription de deux éléments s‟active en réponse à des stress spécifiques comme la limitation en nitrate dans le milieu de culture. Cette activation est accompagnée par un hypométhylation de l‟ADN et l‟analyse de profils d‟insertions chez différents écotypes de P. tricornutum ainsi que l‟étude d‟autres phénomènes suggèrent que les rétrotransposons de type Copia ont joué un rôle important dans l‟évolution des diatomées. Mon grand intérêt pour les ET m‟a ensuite amené à chercher à les caractériser dans d‟autres génomes récemment séquencés tels celui de l‟algue brune Ectocarpus siliculosus. La recherche in silico de différents gènes codant des protéines capables d‟introduire ou de stabiliser des états épigénétiques telle que la modification des histones et la méthylation de l‟ADN a montré leur présence chez P. tricornutum ainsi que leurs particularités. La présence de certaines modifications d‟histones spécifiques d‟une conformation compacte ou ouverte de la chromatine dans le proteome de P. tricornutum a été montrée. De plus, la mise au point de la technique d‟immunoprécipitation de la chromatine chez P. tricornutum a permis d‟établir que les nucléosomes enrobés d‟éléments transposables étaient marqués par des modifications spécifiques. D‟autres expériences ont permis d‟établir que l‟ADN de différents types d‟éléments transposables est marqué par la méthylation de cytosines chez P. tricornutum. Une expérience permettant l‟analyse du profil de méthylation à l‟échelle de génome en utilisant une puce à ADN a été lancée et permettra de découvrir si certains gènes portent aussi des traces de méthylation. Enfin, les ARN interférents constituent un troisième mode de régulation de l‟expression se situant à l‟interface de la régulation transcriptionelle, post-transcriptionelle et épigénétique. Les mécanismes d‟interférences chez les diatomées ont été étudié par la recherche in silico d‟enzymes clés impliquées dans ce processus ainsi qu‟en établissant expérimentalement un lien direct avec la méthylation de l‟ADN.

<p>The mouse xenobiotic-inducible <i>Cyp2a5</i> gene is both transcriptionally and posttranscriptionally regulated. One of the most potent <i>Cyp2a5</i> inducers, the hepatotoxin pyrazole, increases the CYP2A5 mRNA half-life. The induction is accomplished through the interaction of a pyrazole-inducible protein with a 71 nt long, putative hairpin-loop region in the 3' UTR of the CYP2A5 mRNA.</p><p>The aims of this thesis have been to identify the pyrazole-inducible protein, to investigate its role in the <i>Cyp2a5</i> expression and the significance of the 71 nt hairpin-loop region for the <i>Cyp2a5</i> expression, and to examine a possible coupling between transcriptional and posttranscriptional processes in <i>Cyp2a5</i> expression.</p><p>The pyrazole-inducible protein was identified as the heterogeneous nuclear ribonucleoprotein (hnRNP) A1. Studies performed in mouse primary hepatocytes overexpressing hnRNP A1, and in mouse erythroleukemia derived cells lacking hnRNP A1, revealed that the 71 nt region in the 3' UTR of the CYP2A5 mRNA is essential for <i>Cyp2a5</i> expression.</p><p>The hnRNP A1 is a multifunctional nucleocytoplasmic shuttling protein, with the ability to bind both RNA and DNA. These properties make it an interesting candidate mediating a coupling between nuclear and cytoplasmic gene regulatory events, which was investigated for the <i>Cyp2a5</i>. In conditions of cellular stress hnRNP A1 translocates from the nucleus to the cytoplasm. The accumulation of cytoplasmic hnRNP A1 after RNA polymerase II transcription inhibition, resulted in an increased binding of hnRNP A1 to the CYP2A5 mRNA, parallel with a stabilization of the CYP2A5 mRNA.</p><p>Treating primary mouse hepatocytes with phenobarbital (PB), a <i>Cyp2a5</i> transcriptional inducer, resulted in a mainly nuclear localization of the hnRNP A1. Electrophoretic mobility shift assays with nuclear extracts from control or PB-treated mice, revealed that hnRNP A1 interacts with two regions in the <i>Cyp2a5</i> proximal promoter, and that the interaction to one of the regions was stimulated by PB treatment.</p><p>In conclusion, the change in hnRNP A1 subcellular localization after transcriptional inhibition or activation, together with the effects on the interaction of hnRNP A1 with the CYP2A5 mRNA and <i>Cyp2a5</i> promoter, suggest that hnRNP A1 could couple the nuclear and cytoplasmic events of the <i>Cyp2a5</i> expression.</p><p>The presented studies are the first showing involvement of an hnRNP protein in the regulation of a <i>Cyp</i> gene. Moreover, it is the first time an interconnected transcriptional and posttranscriptional regulation has been suggested for a member of the <i>Cyp</i> gene family.</p>

Cells sense the properties of the surrounding environment and convert this information into changes in gene expression. Bacteria are, in contrast to many multi-cellular eukaryotes, remarkable in their ability to cope with rapid environmental changes and to endure harsh and extreme milieus. Previously, control of gene expression was thought to be carried out exclusively by proteins. However, it is now clear that small regulatory RNAs (sRNA) also carry out gene regulatory functions. Bacteria such as E. coli harbor a large class of sRNAs that bind to mRNAs to alter translation and/or mRNA stability. By identifying mRNAs that are targeted by sRNAs, my studies have broadened the understanding of the mechanisms that underlie sRNA-dependent gene regulation, and have shed light on the impact that this type of regulation has on bacterial physiology. Control of gene expression often relies on the interplay of many regulators. This interplay is exemplified by our discovery of mutual regulation between the sRNA MicF and the globally acting transcription factor Lrp. Through double negative feedback, these two regulators respond to nutrient availability in the environment which results in reprogramming of downstream gene expression. We have also shown that both the transcription factor CsgD, and the anti-sigma factor FlgM, are repressed by the two sRNAs OmrA and OmrB, suggesting that these sRNAs are important players in the complex regulation that allow bacteria to switch between motility and sessility. Bacterial populations of genetically identical individuals show phenotypic variations when switching to the sessile state due to bistability in gene expression. While bistability has previously been demonstrated to arise from stochastic fluctuations in transcription, our results suggest that bistability possibly may arise from sRNA-dependent regulatory events also on the post-transcriptional level.

The mouse xenobiotic-inducible Cyp2a5 gene is both transcriptionally and posttranscriptionally regulated. One of the most potent Cyp2a5 inducers, the hepatotoxin pyrazole, increases the CYP2A5 mRNA half-life. The induction is accomplished through the interaction of a pyrazole-inducible protein with a 71 nt long, putative hairpin-loop region in the 3' UTR of the CYP2A5 mRNA. The aims of this thesis have been to identify the pyrazole-inducible protein, to investigate its role in the Cyp2a5 expression and the significance of the 71 nt hairpin-loop region for the Cyp2a5 expression, and to examine a possible coupling between transcriptional and posttranscriptional processes in Cyp2a5 expression. The pyrazole-inducible protein was identified as the heterogeneous nuclear ribonucleoprotein (hnRNP) A1. Studies performed in mouse primary hepatocytes overexpressing hnRNP A1, and in mouse erythroleukemia derived cells lacking hnRNP A1, revealed that the 71 nt region in the 3' UTR of the CYP2A5 mRNA is essential for Cyp2a5 expression. The hnRNP A1 is a multifunctional nucleocytoplasmic shuttling protein, with the ability to bind both RNA and DNA. These properties make it an interesting candidate mediating a coupling between nuclear and cytoplasmic gene regulatory events, which was investigated for the Cyp2a5. In conditions of cellular stress hnRNP A1 translocates from the nucleus to the cytoplasm. The accumulation of cytoplasmic hnRNP A1 after RNA polymerase II transcription inhibition, resulted in an increased binding of hnRNP A1 to the CYP2A5 mRNA, parallel with a stabilization of the CYP2A5 mRNA. Treating primary mouse hepatocytes with phenobarbital (PB), a Cyp2a5 transcriptional inducer, resulted in a mainly nuclear localization of the hnRNP A1. Electrophoretic mobility shift assays with nuclear extracts from control or PB-treated mice, revealed that hnRNP A1 interacts with two regions in the Cyp2a5 proximal promoter, and that the interaction to one of the regions was stimulated by PB treatment. In conclusion, the change in hnRNP A1 subcellular localization after transcriptional inhibition or activation, together with the effects on the interaction of hnRNP A1 with the CYP2A5 mRNA and Cyp2a5 promoter, suggest that hnRNP A1 could couple the nuclear and cytoplasmic events of the Cyp2a5 expression. The presented studies are the first showing involvement of an hnRNP protein in the regulation of a Cyp gene. Moreover, it is the first time an interconnected transcriptional and posttranscriptional regulation has been suggested for a member of the Cyp gene family.

In eukaryotes the genome adopts a non-random spatial organisation, which is important for gene regulation. However, very little is known about the driving forces behind nuclear organisation. In the simple model eukaryote fission yeast, Schizosaccharomyces pombe, it has been known for a long time that transcriptionally repressed heterochromatin localise to the nuclear membrane (NM); the centromeres attaches to spindle pole body (SPB), while the telomeres are positioned at the NM on the opposite side of the nucleus compared to the SPB. Studies presented in this thesis aimed at advancing our knowledge of nuclear organisation in Schizosaccharomyces pombe. We show that the heterochromatic mating-type region localises to the NM in the vicinity of the SPB. This positioning was completely dependent on Clr4, a histone methyl transferase crucial for the formation of heterochromatin. Additional factors important for localisation were also identified: the chromo domain protein Swi6, and the two boundary elements IR-L and IR-R surrounding this locus. We further identify two other chromo domain proteins; Chp1 and Chp2, as crucial factors for correct subnuclear localisation of this region. From these results we suggest that the boundary elements together with chromodomain proteins in balanced dosage and composition cooperate in organising the mating-type chromatin. Gene regulation can affect the subnuclear localisation of genes. Using nitrogen starvation in S. pombe as a model for gene induction we determined the subnuclear localisation of two gene clusters repressed by nitrogen: Chr1 and Tel1. When repressed these loci localise to the NM, and this positioning is dependent on the histone deacetylase Clr3. During induction the gene clusters moved towards the nuclear interior in a transcription dependent manner. The knowledge gained from work presented in this thesis, regarding nuclear organisation in the S. pombe model system, can hopefully aid to a better understanding of human nuclear organisation.

With accellerating global warming and pollution problems a change of energy regime is necessary. Solar energy offers a clean and unlimited energy source of enormous potential. Due to it’s intermittenet nature solar energy must be stored - ideally in the chemical bond of a carrier molecule. Hydrogen gas, H2, an energy carrier with water as only emission when used in a fuel cell, is considered to be the choise for the future. In this context cyanobacteria show promising potential as future H2 factories since they can produce H2 from solar energy and water. The main enzymes directly involved in cyanobacterial hydrogen metabolism are nitrogenases and hydrogenases. Cyanobacterial hydrogenases are either uptake hydrogenases or bidirectional hydrogenases and their maturation requires assistance of six maturation proteins and two hydrogenase specific proteases. In this thesis the transcriptional regulation, maturation and function of the cyanobacterial uptake hydrogenases were investigated in the filamentous, heterocyst forming strains Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120. Five genes, encoding proteins putatively involved in the maturation of the uptake hydrogenase were identified upstream the known maturation genes. Two transcription factors, CalA and CalB, were found interacting with the stretch of DNA forming the upstream regions of the uptake hydrogenase structural genes and the novel maturation genes. The expression of the uptake hydrogenase were  heterocysts specific and the specificity mapped to a short promoter region starting -57 bp upstream the transcription start point. In addition, the function of the uptake hydrogenase was inserted in a metabolic context. Among the proteases, a conserved region was discovered possibly involved in determining the hydrogenase specificity. This thesis has given valuable information about the transcriptional regulation, maturation and function of the uptake hydrogenase in filamentous, heterocystous cyanobacteria and identified new targets for bioengineering of mutant strains with higher H2 production rates.

It is evident that components of the 26S proteasome function beyond protein degradation in the regulation of transcription. Studies in yeast implicate the 26S proteasome, specifically the 19S cap, in the epigenetic regulation of transcription. Saccharomyces cerevisiae 19S ATPases remodel chromatin by facilitating histone acetylation and methylation. However, it is unclear if the 19S ATPases play similar roles in mammalian cells. We previously found that the 19S ATPase Sug1 positively regulates transcription of the critical inflammatory gene MHC-II and that the MHC-II promoter fails to efficiently bind transcription factors upon Sug1 knockdown. MHC-II transcription is regulated by the critical coactivator CIITA. We now find that Sug1 is crucial for regulating histone H3 acetylation at the cytokine inducible MHC-II and CIITA promoters. Histone H3 acetylation is dramatically decreased upon Sug1 knockdown with a preferential loss occurring at lysine 18. Research in yeast indicates that the ortholog of Sug1, Rpt6, acts as a mediator between the activating modifications of histone H2B ubiquitination and H3 methylation. Therefore, we characterized the role the 19S proteasome plays in regulating additional activating modifications. As with acetylation, Sug1 is necessary for proper histone H3K4 and H3R17 methylation at cytokine inducible promoters. In the absence of Sug1, histone H3K4me3 and H3R17me2 are substantially inhibited. Our observation that the loss of Sug1 has no significant effect on H3K36me3 implies that Sug1’s regulation of histone modifications is localized to promoter regions as H3K4me3 but not H3K36me3 is clustered around gene promoters. Here we show that multiple H3K4 histone methyltransferase subunits bind constitutively to the inducible MHC-II and CIITA promoters and that over-expressing one subunit significantly enhances promoter activity. Furthermore, we identified a critical subunit of the H3K4 methyltransferase complex that binds multiple histone modifying enzymes, but fails to bind the CIITA promoter in the absence of Sug1, implicating Sug1 in recruiting multi-enzyme complexes responsible for initiating transcription. Finally, Sug1 knockdown maintains gene silencing as elevated levels of H3K27 trimethylation are observed upon Sug1 knockdown. Together these studies strongly implicate the 19S proteasome in mediating the initial reorganization events to relax the repressive chromatin structure surrounding inducible genes.

The transcription in the eukaryotic cells involves epigenetic regulatory mechanisms that control local and higher-order chromatin remodelling. In the skin, keratinocyte-specific genes are organized into distinct loci including Epidermal Differentiation Complex (EDC) and Keratin type I/II loci. This thesis introduces bioinformatics approaches to analyze multi-level regulatory mechanisms that control skin development and keratinocyte-specific differentiation. Firstly, integration of gene expression data with analyses of linear genome organization showed dramatic downregulation of the genes that comprise large genomic domains in the sweat glands including EDC locus, compared to ii hair follicles, suggesting substantial differences in global genome rearrangement during development of these two distinct skin appendages. Secondly, comparative analysis of the genetic programmes regulated in keratinocytes by Lhx2 transcription factor and chromatin remodeler Satb1 revealed that significant number of their target genes is clustered in the genome. Furthermore, it was shown in this study that Satb1 target genes are lineage-specific. Thirdly, analysis of the topological interactomes of Loricrin and Keratin 5 in hair follicle steam cells revealed presence of the cis- and trans-interactions and lineage specific genes (Wnt, TGF-beta/activin, Notch, etc.). Expression levels of the genes that comprise interactomes show correlation with their histone modification status. This study demonstrates the crucial role for integration of transcription factormediated and epigenetic regulatory mechanisms in establishing a proper balance of gene expression in keratinocytes during development and differentiation into distinct cell lineages and provides an integrated bioinformatics platform for further analyses of the changes in global organization of keratinocyte-specific genomic loci in normal and diseased skin.

In times with elevated CO2 levels and global warming there is a need of finding alternatives to carbon based energy carriers. One such environmental friendly solution could be H2 produced by living organisms. Cyanobacteria are good candidates since they can produce H2 from sunlight and water through the combination of photosynthesis and H2 producing enzymes i.e. nitrogenases and/or [NiFe]-hydrogenases. This thesis investigates the maturation and transcriptional regulation of [NiFe]-hydrogenases in cyanobacteria, with a special focus on hydrogenase specific proteases. The core of all hydrogenases consists of the small and large subunit. The large subunit in which the catalytic site is located goes through an extenstive maturation process which ends with a proteolytic cleavage performed by a hydrogenase specific protease (HupW/HoxW). This thesis shows that within the maturation process of hydrogenases, the proteolytic cleavage is probably the only step that is specific with respect to different types of hydrogenases i.e. one type of protease cleaves only one type of hydrogenase. Further in-silico analysis revealed that these proteases and the hydrogenases might have co-evolved since ancient time and that the specificity observed could be the result of a conserved amino acid sequence which differs between the two types of proteases (HupW/HoxW). A number of different transcription factors were revealed and shown to interact with the promoter regions of several of the genes encoding maturation proteins. The results indicate that the hydrogenase specific proteases are regulated on a transcriptional level in a similar manner as the hydrogenases they cleave. This thesis contributes with knowledge concerning transcriptional regulation and protein regulation of hydrogenases which will be useful for designing genetically engineered cyanobacteria with an improved and adjustable H2 production. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 722

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