PLZF and Polycomb group proteins : interaction and implication in normal and malignant hematopoiesis

Les protéines du groupe Polycomb (PcG) sont des facteurs épigénétiques dont le rôle est de maintenir la répression de leurs gènes cibles au niveau de la chromatine via la modification des protéines histones. EZH2 est une protéine clé dans les mécanismes de régulation puisqu’elle catalyse la mise en place de la marque répressive H3K27me3. Dans le cadre de ma thèse, je me suis intéressée au modèle des leucémies aiguës myéloïdes (LAM) dans lesquelles, contrairement à d’autres pathologies myéloïdes, des mutations affectant EZH2 ou des membres Polycomb ne sont retrouvées que très rarement (˂1%). Des études ont montré que dans ce type de leucémies, de nombreux gènes cibles d’EZH2 sont dérégulés bien que son activité répressive soit toujours présente, mettant en évidence d’éventuels défauts de recrutement de cette protéine. Parmi les facteurs de transcription susceptibles de réguler l’association des protéines PcG à la chromatine, se trouve PLZF qui est un candidat intéressant. En effet, le laboratoire a mis en évidence une interaction entre PLZF et la protéine Polycomb BMI-1 et a montré que la distribution génomique de PLZF concorde avec celle de certains composants des Polycomb. L’objectif de mon travail de thèse a donc été de déterminer dans quelle mesure PLZF intervient dans le recrutement ou l’activité d’EZH2.Polycomb group (PcG) proteins are epigenetic factors which play a major role in maintaining epigenetic silencing via histone modifications at the chromatin level. EZH2 is a key regulator that catalyzes the trimethylation of H3K27, which is a repressive mark. During my PhD, I was interested in the acute myeloid leukemia (AML) model in which, unlike other myeloid malignancies, EZH2 or other PcG protein mutations are very rare (˂1%). Studies have shown that in this type of leukemia, many of EZH2 target genes are deregulated although its repressive activity is still present highlighting possible EZH2 recruitment defects. Among the transcription factors that regulate the association of PcG proteins to chromatin, the transcription factor PLZF is an interesting candidate. Indeed, the laboratory has demonstrated an interaction between PLZF and the Polycomb protein BMI -1 and showed that the genomic distribution of PLZF is consistent with that of some Polycomb components. The aim of my thesis was therefore to determine in which extent PLZF is involved in the recruitment or activity of EZH2

Au cœur d’une complexité biologique EZH2, une protéine du groupe Polycomb.

International audience> Les protéines du groupe Polycomb (PcG) sont des facteurs épigénétiques répresseurs dont les modes de recrutement et d'action sont l'objet de nombreuses études qui viennent renverser les modèles établis. De nouvelles données montrent en effet que le recrutement de ces protéines a un caractère dynamique qui n'apparaissait pas dans le modèle hiérarchique initial. Des études dévoilent également qu'EZH2, une protéine clé des PcG, peut être associée à une chromatine permissive à la transcription, défiant la fonction de répresseur transcriptionnel des protéines PcG. Le double rôle d'EZH2, qui se comporte comme un oncogène ou un suppresseur de tumeur en fonc-tion du type cellulaire, illustre ainsi la complexité des fonctions de ces protéines. < À ce jour, trois complexes PcG associant différentes protéines ont été identifiés : PhoRC (Pho [pleiohomeotic] repressive complex), PRC1 et PRC2 (polycomb repressive complex 1 and 2) (Figure 1). On distingue plusieurs complexes PRC1 constitués de différentes sous-unités, chacune pouvant elle-même avoir plusieurs paralogues. Quel que soit le complexe, l'unité catalytique de PRC1 est portée par l'une ou l'autre des protéines RING1A ou RING1B (really interesting new gene) qui catalysent l'ajout d'une ubiquitine sur la lysine située en position 119 de l'histone H2A (H2AK119ub1) [2]. Les principales protéines composant le complexe PRC2 sont, quant à elles, au nombre de trois : EZH2 (enhancer of zeste homologue 2), EED (embryo-nic ectoderm development) et SUZ12 (suppressor of zeste 12). L'associa-tion de ces trois sous-unités est nécessaire à l'activité catalytique d'EZH2 qui est responsable de la mono-, di-ou triméthylation de la lysine située en position 27 de l'histone H3 (H3K27me1, 2 ou 3) [3]. EZH2 est une pro-téine clé du complexe PRC2 : elle porte l'activité catalytique du complexe et joue un rôle majeur dans la répression transcriptionnelle. EZH2 est la seule protéine du complexe PRC2 à posséder un paralogue connu, appelé EZH1, qui présente des fonctions partiellement redondantes, notamment dans le maintien de l'identité des cellules souches [4, 45] (➜). D'autres protéines peuvent s'associer au com-plexe PRC2. Elles augmentent son activité cata-lytique ou modifient son adressage et sa liaison à la chromatine. C'est, par exemple, le cas du facteur chromatinien JARID2 (Jumonji and AT-rich interaction domain containing 2) capable de se lier à l'ADN et d'induire l'adressage du complexe PRC2 à la chromatine [5]

CD146 Expression in Human Breast Cancer Cell Lines Induces Phenotypic and Functional Changes Observed in Epithelial to Mesenchymal Transition

<div><h3>Background</h3><p>Metastasis is an important step in tumor progression leading to a disseminated and often incurable disease. First steps of metastasis include down-regulation of cell adhesion molecules, alteration of cell polarity and reorganization of cytoskeleton, modifications associated with enhanced migratory properties and resistance of tumor cells to anoikis. Such modifications resemble Epithelial to Mesenchymal Transition (EMT). In breast cancer CD146 expression is associated with poor prognosis and enhanced motility.</p> <h3>Methodology/Principal Findings</h3><p>On 4 different human breast cancer cell lines, we modified CD146 expression either with shRNA technology in CD146 positive cells or with stable transfection of CD146 in negative cells. Modifications in morphology, growth and migration were evaluated. Using Q-RT-PCR, we analyzed the expression of different EMT markers. We demonstrate that high levels of CD146 are associated with loss of cell-cell contacts, expression of EMT markers, increased cell motility and increased resistance to doxorubicin or docetaxel. Experimental modulation of CD146 expression induces changes consistent with the above described characteristics: morphology, motility, growth in anchorage independent conditions and Slug mRNA variations are strictly correlated with CD146 expression. These changes are associated with modifications of ER (estrogen receptor) and Erb receptors and are enhanced by simultaneous and opposite modulation of JAM-A, or exposure to heregulin, an erb-B4 ligand.</p> <h3>Conclusions</h3><p>CD146 expression is associated with an EMT phenotype. Several molecules are affected by CD146 expression: direct or indirect signaling contributes to EMT by increasing Slug expression. CD146 may also interact with Erb signaling by modifying cell surface expression of ErbB3 and ErbB4 and increased resistance to chemotherapy. Antagonistic effects of JAM-A, a tight junction-associated protein, on CD146 promigratory effects underline the complexity of the adhesion molecules network in tumor cell migration and metastasis.</p> </div

Changes in EMT markers levels are associated with CD146 modulation.

<p>The CD146-pCMV6-XL4 vector was transfected in MCF-7 and SKBR3 cells, CD146 expression was down-modulated (KD) in CAL51 and MDA-MB-231 cells with siRNAs. mRNAs from CD146 or siRNAs transfected cells were analyzed against mRNAs from mock transfected cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043752#s2" target="_blank">Results</a> are expressed as the mean of the fold change (± standard error of the mean) for at least six different mRNA preparations. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043752#s2" target="_blank">Results</a> were analyzed with the Wilcoxon matched pairs test and considered as significant when p<0.05 (indicated with S for significant).</p

PLZF mutation alters mouse hematopoietic stem cell function and cell cycle progression

International audienceKey Points Inactivation of PLZF promotes phenotype of HSC aging. PLZF controls HSC cell cycle

IC50 values for doxorubicin and docetaxel depending on the level of CD146 expression.

<p>The specific MFI represents the ratio of the mean fluorescence intensity for the CD146 antibody over the mean fluorescence intensity of the isotypic control (mean ± standard error of the mean of at least six independent experiments). IC50 is expressed as the mean (± standard error of the mean) for six different experiments. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043752#s2" target="_blank">Results</a> were analyzed with the Wilcoxon matched pairs test and considered as significant when p<0.05.</p

Characteristics of the breast cancer cell lines used in this study.

<p>The specific MFI represents the ratio of the mean fluorescence intensity for the CD146 antibody over the mean fluorescence intensity of the isotypic control (mean ± standard error of the mean of at least six independent experiments).</p

Functional properties of breast cancer cells depend on the level of CD146 expression.

<p>A: Anchorage-dependent growth was estimated by the cell number after four days of culture. B: Anchorage-independent growth was assessed by soft agar colony-forming assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043752#s4" target="_blank">Materials and Methods</a>. C, Chemotactic migration evaluated after 36 hours (CAL51 cells) or 96 hours (MCF-7 and SKBR3 cells) using uncoated Boyden chambers and 10% FCS as chemo-attractant. Migrating cells were stained with Crystal Violet solution, lysed and absorbance measured at 550 nm. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043752#s2" target="_blank">Results</a> represented as the mean ± standard error of seven independent experiments were analyzed with the Wilcoxon signed rank test. White bars: CD146^low cells, black bars: CD146^high cells.</p

Morphologic changes associated with the modulation of CD146 expression.

<p>A: Morphology of MCF-7 cells stably transfected with CD146 and of CAL51 cells stably transfected with a shRNA against CD146 compared to mock transfected cells. The diminution in cell-cell contacts is associated with CD146 expression (magnification×40). B: Proportion of scattered colonies in mocked-transfected and CD146-modulated MCF-7 and CAL51 cells. Cells expressing high levels of CD146 produce a higher number of scattered colonies than their CD146 low or negative counterparts. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043752#s2" target="_blank">Results</a> expressed as the mean ± standard error of the mean of at least six independent experiments were analyzed with the Wilcoxon signed rank test.</p

CD146 modulation reveals a link with ER, PR and Erb receptor expression and function.

<p>A: analysis of ER, PR and Erb receptors in CD146 transfected MCF-7 cells versus mock transfected cells. Q-RT-PCR was performed on six different RNA preparations. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043752#s2" target="_blank">Results</a> expressed as the mean ± standard error to the mean were analyzed with the one sample t-test assuming a theoretical mean equal to 1 (** = p<0.01). B: CD146 increases the response to heregulin in MCF-7 cells. Q-RT-PCR was performed on six different RNA preparations isolated from mock transfected cells (CD146 negative cells) or CD146 transfected cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043752#s2" target="_blank">Results</a> are expressed as fold change in expression comparing HRG-treated cells with untreated cells for mock transfected cells (CD146 negative) as well as CD146 transfected cells. Mean ± standard error to the mean is shown. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043752#s2" target="_blank">Results</a> were analyzed with the Wilcoxon signed rank test.</p