Par ce travail expérimental, nous essayons d’établir un lien entre les propriétés mécaniques de gels d’actine branchés de levure et la composition biochimique des réseaux. L’actine est un polymère semi-flexible qui fait partie du cytosquelette. De nombreux partenaires protéiques de l’actine (notés ABPs par la suite) se lient aux filaments d’actine et les agencent en différents types de réseaux. Arp2/3 est un complexe protéique qui génère la croissance de réseaux d’actine branchés. Les réseaux d’actine branchés en croissance intéressent tout particulièrement physiciens comme biologistes car ils sont capables de développer des forces nécessaires à de nombreux processus vitaux pour la cellule, comme l’endocytose. Nous avons ici étudié les propriétés mécaniques de gels d’actine branchés reconstitués in vitro, en nous focalisant sur le rôle d’un type d’ABPs en particulier, les protéines de réticulation. Il nous a été possible de quantifier et de comparer l’effet de trois protéines de réticulation différentes sur la mécanique des réseaux d’actine branchés de levure.Afin de mener à bien cette étude, nous avons combiné deux puissantes techniques expérimentales.Nous avons utilisé une technique de mesure des propriétés mécaniques basée sur l’utilisation de colloïdes superparamagnétiques développée au laboratoire. Cette technique permet de réaliser des mesures quantitatives et à haut débit sur des gels polymères très fins (quelques centaines de nanomètres d’épaisseur). Les réseaux ont été reconstitués in vitro grâce à la fonctionnalisation des billes superparamagnétiques avec Las17, une protéine que notre collaborateur biologiste a identifiée comme suffisant à activer Arp2/3 chez la levure. Nous avons de plus combiné deux approches complémentaires en travaillant à la fois sur des extraits cellulaires de levure contenant toutes les ABPs des réseaux Arp2/3 et à la fois sur des mélanges de quelques protéines purifiées.L’approche « top-down » est basée sur l’utilisation d’extraits cellulaires de mutants de la levure n’exprimant pas une ou des protéine(s) d’intérêt(s), et l’approche « bottom-up » sur l’addition de la protéine étudiée dans le système simplifié de quelques protéines purifiées. / In this experimental work we tried to quantify the mechanical properties of yeast branchedactin networks with regard to their biochemical composition. Actin is a semi-flexible biopolymerthat is assembled as part of the cytoskeleton. Proteins partners of actin (ABPs) shape itsfilaments into different type of networks. Arp2/3 is a protein complex that has the propertyto generate branched actin gels. Growing branched actin networks are of particular interest forboth biologists and physicists because of their ability to generate forces necessary to many vitalprocesses such as endocytosis. Here we study in vitro the mechanical properties of such networks,and we focus on the role of one type of actin binding proteins, the crosslinkers. This family ofproteins appears to play a role in both the elastic, viscous and plastic properties of the gels. Weare able to quantify and to compare the impact of three different crosslinkers on branched actinnetworks in yeast.In order to conduct said study, we combined two powerful experimental methods. We used asuperparamagnetic particle-based mechanical measurement technique that was developed in thelab and allows quantitative, high-throughput measurements on very thin gels. And the networkswere reconstituted in vitro by functionalization of the magnetic particles with Las17, which hasbeen showed to activate Arp2/3 for the yeast by our biologist collaborator. We furthermoreworked on both yeast extracts containing all the ABPs of the Arp2/3 networks, and with setsof a few purified proteins, in order to combine a « top-down » (use of mutations in yeast toprevent the expression of protein(s) of interest) and a « bottom-up » (addition of a protein ofinterest in a simplified system) approaches.

The ParaHox cluster is the evolutionary sister of the Hox cluster. Like the Hox cluster, the ParaHox cluster is subject to complex regulatory phenomena such as collinearity. Despite the breakup of the ParaHox cluster within many animals, intact and collinear clusters have now been discovered within the chordate phyla in amphioxus and the vertebrates, and more recently within the hemichordates and echinoderms. The archetypal ParaHox cluster of amphioxus places it in a unique position in which to examine the regulatory mechanisms controlling ParaHox gene expression within the last common ancestor of chordates, and perhaps even the wider Deuterostomia. In this thesis, the genomic and regulatory landscape of the amphioxus ParaHox cluster is characterised in detail. New genomic and transcriptomic resources are used to better characterise the B.floridae ParaHox cluster and surrounding genomic region, and conserved non-coding regions and regulatory motifs are identified across the ParaHox cluster of three species of amphioxus. In conjunction with this, the impact of retrotransposition upon the ParaHox cluster is examined and analyses of transposable elements and the AmphiSCP1 retrogene reveal that the ParaHox cluster may be more insulated from outside influence than previously thought. Finally, the detailed analyses of a regulatory element upstream of AmphiGsx reveals conserved mechanisms regulating Gsx CNS expression within the chordates, and TCF/Lef is likely a direct regulator of AmphiGsx within the CNS. The work in this thesis makes use of new genomic and transcriptomic resources available for amphioxus to better characterise the genomic and regulatory landscape of the amphioxus ParaHox cluster, serving as a basis for the improved identification and characterisation of functional regulatory elements and conserved regulatory mechanisms. This work also highlights the potential of Ciona intestinalis as a ‘living test tube' to allow the detailed characterisation of amphioxus ParaHox regulatory elements.

In eukaryotes, the first step of interpreting the genetic information is the transcription of DNA into RNA. For protein-coding genes, such transcription is carried out by RNA polymerase II. A special domain of RNA polymerase II, called the C-terminal domain (CTD), functions as a master controller for the transcription process by providing a platform to recruit regulatory proteins to nascent mRNA (Chapter 1-2). The modifications and conformational states of the CTD, termed the 'CTD code', represent a critical regulatory checkpoint for transcription. The CTD, found only in eukaryotes, consists of 26--52 tandem heptapeptide repeats with the consensus sequence, Tyr₁Ser₂Pro₃Thr₄Ser₅Pro₆Ser₇. Phosphorylation of the serines and prolyl isomerization of the prolines represent two major regulatory mechanisms of the CTD. Interestingly, the phosphorylation sites are typically close to prolines, thus the conformation of the adjacent proline could impact the specificity of the corresponding kinases and phosphatases. Understanding how those modifying enzymes recognize and regulate the CTD is important for expanding our knowledge on the transcription regulation and deciphering the 'CTD code'. During my PhD study, I studied the function of CTD phosphatases and prolyl isomerase in the CTD regulation using Scp1, Ssu72 and Pin1 as model regulators. Scp1 and Ssu72 are both Ser5 phosphatases. However, Ssu72 is an essential protein and regulates the global transcription while Scp1 epigenetically silences the expression of specific neuronal genes. Pin1 is a highly conserved phosphorylation-specific prolyl isomerase that recognizes the phospho-Ser/Thr-Pro motif within the CTD as one of its primary substrates in vivo. Among these enzymes, Scp1 is the focal point of this dissertation, as it was studied from different angles, such as enzymatic mechanism (Chapter 3 describes the capture of phospho-aspartyl intermediate of Scp1 as a direct evidence for the proposed two-step mechanism), specific inhibition (Chapter 4 describes the identification and characterization of the first specific inhibitor of Scp1), and its non-active-site contact with the CTD (Chapter 5 describes the structural basis of this contact). These studies are of great importance towards understanding the molecular mechanism of the dephosphorylation process of the CTD by Scp1. / text