Characterisation of Genome-Wide PLZF/RARA Target Genes

The PLZF/RARA fusion protein generated by the t(11;17)(q23;q21) translocation in acute promyelocytic leukaemia (APL) is believed to act as an oncogenic transcriptional regulator recruiting epigenetic factors to genes important for its transforming potential. However, molecular mechanisms associated with PLZF/RARA-dependent leukaemogenesis still remain unclear. We searched for specific PLZF/RARA target genes by ChIP-on-chip in the haematopoietic cell line U937 conditionally expressing PLZF/RARA. By comparing bound regions found in U937 cells expressing endogenous PLZF with PLZF/RARA-induced U937 cells, we isolated specific PLZF/RARA target gene promoters. We next analysed gene expression profiles of our identified target genes in PLZF/RARA APL patients and analysed DNA sequences and epigenetic modification at PLZF/RARA binding sites. We identify 413 specific PLZF/RARA target genes including a number encoding transcription factors involved in the regulation of haematopoiesis. Among these genes, 22 were significantly down regulated in primary PLZF/RARA APL cells. In addition, repressed PLZF/RARA target genes were associated with increased levels of H3K27me3 and decreased levels of H3K9K14ac. Finally, sequence analysis of PLZF/RARA bound sequences reveals the presence of both consensus and degenerated RAREs as well as enrichment for tissue-specific transcription factor motifs, highlighting the complexity of targeting fusion protein to chromatin. Our study suggests that PLZF/RARA directly targets genes important for haematopoietic development and supports the notion that PLZF/RARA acts mainly as an epigenetic regulator of its direct target genes.

MarA-mediated transcriptional repression of the rob promoter

The Escherichia coli transcriptional regulator MarA affects functions that include antibiotic resistance, persistence, and survival. MarA functions as an activator or repressor of transcription utilizing similar degenerate DNA sequences (marboxes) with three different binding site configurations with respect to the RNA polymerase-binding sites. We demonstrate that MarA down-regulates rob transcripts both in vivo and in vitro via a MarA-binding site within the rob promoter that is positioned between the -10 and -35 hexamers. As for the hdeA and purA promoters, which are repressed by MarA, the rob marbox is also in the "backward" orientation. Protein-DNA interactions show that SoxS and Rob, like MarA, bind the same marbox in the rob promoter. Electrophoretic mobility shift analyses with a MarA-specific antibody demonstrate that MarA and RNA polymerase form a ternary complex with the rob promoter DNA. Transcription experiments in vitro and potassium permanganate footprinting analysis show that MarA affects the RNA polymerase-mediated closed to open complex formation at the rob promoter.

Polymerisable squaramide receptors for anion binding and sensing

A novel series of polymerisable squaramides has been synthesised in high yields using simple chemical reactions, and evaluated in the binding of anionic species. These vinyl monomers can be used as functional building blocks in co-polymerisations with a plethora of co-monomers or cross-linkers, grace to their compatibility with free-radical polymerisation reactions. Aromatic substituted squaramides were found to be the strongest receptors, while binding of certain anions was accompanied by a strong colour change, attributed to the de-protonation of the squaramide. The best performing squaramide monomers were incorporated in molecularly imprinted polymers (MIPs) targeting a model anion and their capacities and selectivity were evaluated by rebinding experiments. Polymers prepared using the new squaramide monomers were compared to urea based co-polymers, and were found to contain up to 80% of the theoretical maximum number of binding sites, an exceptionally high value compared to previous reports. Strong polymer colour changes were observed upon rebinding of certain anions, equivalent to those witnessed in solution, paving the way for application of such materials in anion sensing devices.



Graphical abstract: Polymerisable squaramide receptors for anion binding and sensing

Putting chromatin immunoprecipitation into context

Chromatin immunoprecipitation (ChIP), when paired with sequencing or arrays, has become a method of choice for the unbiased identification of genomic-binding sites for transcription factors and epigenetic marks in various model systems. The data generated is often then interpreted by groups seeking to link these binding sites to the expression of adjacent or distal genes, and more broadly to the evolution of species, cell fate/differentiation or even cancer development. Against this backdrop is an ongoing debate over the relative importance DNA sequence versus chromatin structure and modification in the regulation of gene expression (Anon. 2008a Nature 454: 795; Anon. 2008b Nature 454: 711-715; Henikoff et al. 2008 Science 322: 853; Madhani et al. 2008 Science 322: 43-44). Rationally there is a synergy between the two and the goal of a biologist is to characterise both comprehensively enough to explain a cellular phenotype or a developmental process. If this is truly our goal then the critical factor in good science is an awareness of the constraints and potential of the biological models used. The reality however is often that this discussion is polarised by funding imperatives and the need to align to a transcription factor or epigenetic camp. This article will discuss the extrapolations involved in using ChIP data to draw conclusions about these themes and the discoveries that have resulted.

Solitary and repetitive binding motifs for the AP2 complex alpha-appendage in amphiphysin and other accessory proteins

Adaptor protein (AP) complexes bind to transmembrane proteins destined for internalization and to membrane lipids, so linking cargo to the accessory internalization machinery. This machinery interacts with the appendage domains of APs, which have platform and beta-sandwich subdomains, forming the binding surfaces for interacting proteins. Proteins that interact with the subdomains do so via short motifs, usually found in regions of low structural complexity of the interacting proteins. So far, up to four motifs have been identified that bind to and partially compete for at least two sites on each of the appendage domains of the AP2 complex. Motifs in individual accessory proteins, their sequential arrangement into motif domains, and partial competition for binding sites on the appendage domains coordinate the formation of endocytic complexes in a temporal and spatial manner. In this work, we examine the dominant interaction sequence in amphiphysin, a synapse-enriched accessory protein, which generates membrane curvature and recruits the scission protein dynamin to the necks of coated pits, for the platform subdomain of the alpha-appendage. The motif domain of amphiphysin1 contains one copy of each of a DX(F/W) and FXDXF motif. We find that the FXDXF motif is the main determinant for the high affinity interaction with the alpha-adaptin appendage. We describe the optimal sequence of the FXDXF motif using thermodynamic and structural data and show how sequence variation controls the affinities of these motifs for the alpha-appendage.

A let-7 microRNA-binding site polymorphism in 3'-untranslated region of KRAS gene predicts response in wild-type KRAS patients with metastatic colorectal cancer treated with cetuximab monotherapy

PURPOSE: recent studies have found that KRAS mutations predict resistance to monoclonal antibodies targeting the epidermal growth factor receptor in metastatic colorectal cancer (mCRC). A polymorphism in a let-7 microRNA complementary site (lcs6) in the KRAS 3' untranslated region (UTR) is associated with an increased cancer risk in non-small-cell lung cancer and reduced overall survival (OS) in oral cancers. We tested the hypothesis whether this polymorphism may be associated with clinical outcome in KRAS wild-type (KRASwt) mCRC patients treated with cetuximab monotherapy.

PATIENTS AND METHODS: the presence of KRAS let-7 lcs6 polymorphism was evaluated in 130 mCRC patients who were enrolled in a phase II study of cetuximab monotherapy (IMCL-0144). Genomic DNA was extracted from dissected formalin-fixed paraffin-embedded tumor tissue, KRAS mutation status and polymorphism were assessed using direct sequencing and PCR restriction fragment length polymorphism technique.

RESULTS: KRAS let-7 lcs6 polymorphism was found to be related to object response rate (ORR) in mCRC patients whose tumors had KRASwt. The 12 KRASwt patients harboring at least a variant G allele (TG or GG) had a 42% ORR compared with a 9% ORR in 55 KRASwt patients with let-7 lcs6 TT genotype (P = 0.02, Fisher's exact test). KRASwt patients with TG/GG genotypes had trend of longer median progression-free survival (3.9 versus 1.3 months) and OS (10.7 versus 6.4 months) compared to those with TT genotypes.

CONCLUSIONS: these results are the first to indicate that the KRAS 3'UTR polymorphism may predict for cetuximab responsiveness in KRASwt mCRC patients, which warrants validation in other clinical trials.

Metal ions and protein folding: conformational and functional interplay

Dissertation presented to obtain a PhD degree in Biochemistry at Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa

CD8 kinetically promotes ligand binding to the T-cell antigen receptor.

The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate constant ((1.7 +/- 0.2) x 10(5) M(-1) s(-1)) was independent of examined cell type or MHC-bound peptides' structure. Its value was much faster than that of the association of soluble pMHC and TCR ((7.0 +/- 0.3) x 10(3) M(-1) s(-1)), and close to that of the association of soluble pMHC with CD8 ((1-2) x 10(5) M(-1) s(-1)). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate constant was slowed down approximately 10-fold after cells treatment with methyl-beta-cyclodextrin. These results suggest that the most efficient pMHC-cell association route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC association with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC association with CD8-proximal TCR. The slow binding phase is assigned to pMHC association with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, our data suggest that the colocalized, raft-associated CD8-TCR subpopulation is the one capable of inducing T-cell activation.

Monoclonal antibodies to murine lipopolysaccharide (LPS)-binding protein (LBP) protect mice from lethal endotoxemia by blocking either the binding of LPS to LBP or the presentation of LPS/LBP complexes to CD14.

Cellular responses to LPS, the major lipid component of the outer membrane of Gram-negative bacteria, are enhanced markedly by the LPS-binding protein (LBP), a plasma protein that transfers LPS to the cell surface CD14 present on cells of the myeloid lineage. LBP has been shown previously to potentiate the host response to LPS. However, experiments performed in mice with a disruption of the LBP gene have yielded discordant results. Whereas one study showed that LBP knockout mice were resistant to endotoxemia, another study did not confirm an important role for LBP in the response of mice challenged in vivo with low doses of LPS. Consequently, we generated rat mAbs to murine LBP to investigate further the contribution of LBP in experimental endotoxemia. Three classes of mAbs were obtained. Class 1 mAbs blocked the binding of LPS to LBP; class 2 mAbs blocked the binding of LPS/LBP complexes to CD14; class 3 mAbs bound LBP but did not suppress LBP activity. In vivo, class 1 and class 2 mAbs suppressed LPS-induced TNF production and protected mice from lethal endotoxemia. These results show that the neutralization of LBP accomplished by blocking either the binding of LPS to LBP or the binding of LPS/LBP complexes to CD14 protects the host from LPS-induced toxicity, confirming that LBP is a critical component of innate immunity.

Differential peptide binding to CD40 evokes counteractive responses.

The antigen-presenting cell-expressed CD40 is implied in the regulation of counteractive immune responses such as induction of pro-inflammatory and anti-inflammatory cytokines interleukin (IL)-12 and IL-10, respectively. The mechanism of this duality in CD40 function remains unknown. Here, we investigated whether such duality depends on ligand binding. Based on CD40 binding, we identifed two dodecameric peptides, peptide-7 and peptide-19, from the phage peptide library. Peptide-7 induces IL-10 and increases Leishmania donovani infection in macrophages, whereas peptide-19 induces IL-12 and reduces L. donovani infection. CD40-peptide interaction analyses by surface plasmon resonance and atomic force microscopy suggest that the functional differences are not associated with the studied interaction parameters. The molecular dynamic simulation of the CD40-peptides interaction suggests that these two peptides bind to two different places on CD40. Thus, we suggest for the first time that differential binding of the ligands imparts functional duality to CD40.

Études Structurales par Résonance Magnétique Nucléaire (RMN) du Site Actif du Ribozyme VS de Neurospora.

Nous étudions le ribozyme VS de Neurospora, en tant que système modèle, pour augmenter nos connaissances sur la relation entre la structure et la fonction chez les ARNs, ainsi que pour mieux comprendre le mécanisme de clivage de ce ribozyme. Il a été proposé précédemment que la boucle interne A730 dans la tige-boucle VI (SLVI) contient le site actif du ribozyme et lie un ou plusieurs ions métalliques qui pourraient participer au mécanisme réactionnel. Nous avons déterminé par spectroscopie RMN la structure de la tige-boucle SLVI contenant la boucle A730 afin d’éclaircir ce mécanisme. La structure obtenue est en accord avec les études biochimiques antérieures et présente un ou plusieurs sites de liaison au magnésium associé à la boucle interne. Suite à des études de cinétique et de mutagenèse, il a été proposé qu’une adénine localisée dans le site actif, A756, participe à la catalyse par acide/base générale. Des études de pH effectuées précédemment ont identifié un pKa catalytique (5.2-5.8) qui correspond probablement à l’équilibre de protonation du A756. À l’aide de méthodes utilisant le carbone-13, nous avons identifié un pKa modifié appartenant au A756, ce qui supporte le rôle de ce résidu dans la catalyse par acide/base générale. Les études structurales présentées ici aident donc à augmenter notre compréhension du mécanisme de clivage chez le ribozyme VS.

Impacts fonctionnels des polymorphismes dans les promoteurs des gènes de l’apoptose

La susceptibilité ou la résistance aux cancers peuvent impliquer plusieurs mécanismes, incluant l’apoptose, la croissance cellulaire et la différenciation, la réplication et la réparation de l’ADN. Mon projet porte plus particulièrement sur l’apoptose. Une dérégulation dans les voies d’activation de l’apoptose entraîne une accumulation de cellules déréglées, créant ainsi un environnement propice à l’instabilité génétique et au développement du cancer. Comme l’apoptose est une voie biologique hautement régulée, nous proposons l’hypothèse que des polymorphismes « fonctionnels » dans les régions de régulations des gènes (rSNPs) perturberaient cette voie à cause de taux variables de transcrits et des protéines correspondantes dû à la modification des sites de reconnaissances des facteurs de transcription. Les principaux objectifs de mon projet sont : (i) identifier les SNPs présents dans la région promotrice des gènes d’apoptose; (ii) déterminer les haplotypes de promoteurs les plus fréquents présents dans la population générale; (rHaps) (iii) vérifier leurs impacts fonctionnels sur l’expression génique par des essais in vitro (gène rapporteur et retard sur gel). Cette étude permettra d’identifier des rSNPs et rHaps ayant un impact sur le niveau d’expression des gènes d’apoptose, au moins dans un contexte in vitro. Ces différences alléliques au niveau de l’expression de ces gènes d’apoptose pourraient contribuer à la susceptibilité interindividuelle de développer un cancer.

Loss of noradrenaline transporter sites in frontal cortex of rats with acute (ischemic) liver failure.

There is increasing evidence that central noradrenaline (NA) transport mechanisms are implicated in the central nervous system complications of acute liver failure. In order to assess this possibility, binding sites for the high affinity NA transporter ligand [3H]-nisoxetine were measured by quantitative receptor autoradiography in the brains of rats with acute liver failure resulting from hepatic devascularization and in appropriate controls. In vivo microdialysis was used to measure extracellular brain concentrations of NA. Severe encephalopathy resulted in a significant loss of [3H]-nisoxetine sites in frontal cortex and a concomitant increase in extracellular brain concentrations of NA in rats with acute liver failure. A loss of transporter sites was also observed in thalamus of rats with acute liver failure. This loss of NA transporter sites could result from depletion of central NA stores due to a reserpine-like effect of ammonia which is known to accumulate to millimolar concentrations in brain in ischemic liver failure. Impaired NA transport and the consequent increase in synaptic concentrations and increased stimulation of neuronal and astrocytic noradrenergic receptors could be implicated in the pathogenesis of the encephalopathy and brain edema characteristic of acute liver failure.

Une nouvelle approche computationnelle pour la découverte des sites de fixation de facteurs de transcription à l’ADN, adaptée aux données de ChIP-chip et de ChIP-séquençage

Les facteurs de transcription sont des protéines spécialisées qui jouent un rôle important dans différents processus biologiques tel que la différenciation, le cycle cellulaire et la tumorigenèse. Ils régulent la transcription des gènes en se fixant sur des séquences d’ADN spécifiques (éléments cis-régulateurs). L’identification de ces éléments est une étape cruciale dans la compréhension des réseaux de régulation des gènes. Avec l’avènement des technologies de séquençage à haut débit, l’identification de tout les éléments fonctionnels dans les génomes, incluant gènes et éléments cis-régulateurs a connu une avancée considérable. Alors qu’on est arrivé à estimer le nombre de gènes chez différentes espèces, l’information sur les éléments qui contrôlent et orchestrent la régulation de ces gènes est encore mal définie. Grace aux techniques de ChIP-chip et de ChIP-séquençage il est possible d’identifier toutes les régions du génome qui sont liées par un facteur de transcription d’intérêt. Plusieurs approches computationnelles ont été développées pour prédire les sites fixés par les facteurs de transcription. Ces approches sont classées en deux catégories principales: les algorithmes énumératifs et probabilistes. Toutefois, plusieurs études ont montré que ces approches génèrent des taux élevés de faux négatifs et de faux positifs ce qui rend difficile l’interprétation des résultats et par conséquent leur validation expérimentale. Dans cette thèse, nous avons ciblé deux objectifs. Le premier objectif a été de développer une nouvelle approche pour la découverte des sites de fixation des facteurs de transcription à l’ADN (SAMD-ChIP) adaptée aux données de ChIP-chip et de ChIP-séquençage. Notre approche implémente un algorithme hybride qui combine les deux stratégies énumérative et probabiliste, afin d’exploiter les performances de chacune d’entre elles. Notre approche a montré ses performances, comparée aux outils de découvertes de motifs existants sur des jeux de données simulées et des jeux de données de ChIP-chip et de ChIP-séquençage. SAMD-ChIP présente aussi l’avantage d’exploiter les propriétés de distributions des sites liés par les facteurs de transcription autour du centre des régions liées afin de limiter la prédiction aux motifs qui sont enrichis dans une fenêtre de longueur fixe autour du centre de ces régions. Les facteurs de transcription agissent rarement seuls. Ils forment souvent des complexes pour interagir avec l’ADN pour réguler leurs gènes cibles. Ces interactions impliquent des facteurs de transcription dont les sites de fixation à l’ADN sont localisés proches les uns des autres ou bien médier par des boucles de chromatine. Notre deuxième objectif a été d’exploiter la proximité spatiale des sites liés par les facteurs de transcription dans les régions de ChIP-chip et de ChIP-séquençage pour développer une approche pour la prédiction des motifs composites (motifs composés par deux sites et séparés par un espacement de taille fixe). Nous avons testé ce module pour prédire la co-localisation entre les deux demi-sites ERE qui forment le site ERE, lié par le récepteur des œstrogènes ERα. Ce module a été incorporé à notre outil de découverte de motifs SAMD-ChIP.