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

Mining old transcriptomes to predict HSC age

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Rôle des protéines du groupe polycomb dans la leucémie aiguë promyélocytaire associée à PLZF/RARA

Les leucémies aiguës promyélocytaires (APLs) sont caractérisées par un blocage de la différenciation des granulocytes au stade promyélocytaire et par une augmentation de la prolifération des cellules leucémiques dans la moelle osseuse. Ce phénotype est induit par des translocations chromosomiques spécifiques qui impliquent dans tous les cas le gène RARA générant ainsi des protéines de fusions X/RARAs. L expression de ces dernières induit le blocage de la transcription des gènes cibles du RARA qui sont importants pour la différenciation granulocytaire. Les APLs associées à PML/RARA répondent d une façon favorable à la thérapie différenciatrice de l acide rétinoïque (AR) alors que les APLs associées à PLZF/RARA se révèlent résistantes à ce traitement. Cette différence de réponse a été expliquée par un modèle décrivant un recrutement de corépresseurs insensible au traitement par l AR au niveau de PLZF/RARA. Cependant, l existence de mutants de PLZF/RARA avoir perdu leurs propriétés oncogéniques sans pour autant perdu leur capacité à recruter ces complexes corépresseurs questionne ce modèle. De plus, l observation que le traitement à l acide rétinoïque dégrade la protéine PLZF/RARA explique mal l absence de réponse et suggère l intervention d autres facteurs probablement de nature épigénétique capables de maintenir la répression induite par PLZF/RARA même après sa dégradation. Comme des résultats antérieurs suggéraient une interaction fonctionnelle entre PLZF et Bmi1, un membre du complexe PRC1 du groupe Polycomb, nous avons recherché à établir un lien entre les activités répressives des protéines PRC1 et les propriétés oncogéniques de PLZF/RARA. Dans cette optique, nous avons tout d abord validé l interaction entre PLZF/RARA et Bmi1 dans différents modèles cellulaires in vitro comme in vivo. De plus, nous avons montré que PLZF/RARA était capable de former un complexe stable avec les protéines du complexe PRC1 et de les recruter au niveau de ses gènes cibles in vitro comme in vivo. Le recrutement de ce nouveau complexe répresseur est spécifique à PLZF/RARA par rapport au RARA ou PML/RARA et n est pas sensible au traitement AR. Finalement, nous avons testé l importance de ce gain de fonction sur les propriétés de transformation de PLZF/RARA et nous avons montré que l absence de Bmi1 réduit les propriétés oncogéniques de PLZF/RARA. L ensemble de nos résultats montre l implication directe du complexe PRC1 dans la transformation induite par PLZF/RARA et probablement le phénomène de la résistance qui l accompagne. Mettre en évidence de nouveaux partenaires aide à mieux cerner les mécanismes impliqués dans la pathogenèse de cette leucémie et à améliorer les stratégiesthérapeutiques.Acute promyelocytic leukemia (APL) is characterized by a differentiation block of granulocytes at the promyelocytic stage and an increase self renewal capacity by leukemic cells in the bone marrow. This phenotype is induced by specific chromosomal translocation always involving the RARA gene and leading to the generation of X/RARAs fusion proteins with gain of function and dominant negative effects on the original proteins. The expression of these fusions is sufficient to block the transcription of RARA s target genes and induce leukemic phenotype in mice similar to those observed in human cases. PML/RARA-APL responds well to retinoic acid treatment and chemotherapy, whereas PLZF/RARA-APL responds poorly to both treatments, thus defining a resistant APL. The model that has been proposed to explain the distinct response describes a retinoic acidresistant binding site in PLZF/RARA for co-repressor recruitment. However, this model has been challenged with the description of PLZF/RARA s mutants, which loose their oncogenic properties, but still interact with the co-repressors. Furthermore, PLZF/RARA s degradation induced by retinoic acid treatment does not explain the lack of response and suggests the implication of others mechanisms mostly epigenetic, which maintain the repression induced by PLZF/RARA even after its degradation. As previous studies showed a functional interaction between PLZF and Bmi1, a member of the PRC1 Polycomb group, we wanted to assess the link between PRC1 repressive activities and PLZF/RARA oncogenic properties. For this purpose, we first validated the interaction between PLZF/RARA and Bmi1 in several cellular models in vitro and in vivo. Then, we showed that PLZF/RARA is able to form a stable complex with others PRC1 components and to recruit them to RARA s target genes in vitro and in vivo. This recruitment was specific to PLZF/RARA when compared to RARA or PML/RARA and was unaffected after retinoic acid treatment. Finally, we attempted to evaluate the importance of this gain of function on the transformation mediated by PLZF/RARA. The loss of the leukemic phenotype from hematopoietic progenitors transformed with PLZF/RARA ex vivo led us to conclude that Bmi1 is necessary for this transformation. Thus, our findings demonstrate the involvement of an epigenetic mechanism in the PLZF/RARA mediated transformation and this could provide an interpretation of its related resistance phenomenon. The evidence presented here could help in better understanding of the mechanisms implicated in the pathogenesis of this type of leukemia and improve therapeutic strategies to overcome the disease.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

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]

Single Cell Transcriptomics to Understand HSC Heterogeneity and Its Evolution upon Aging

International audienceSingle-cell transcriptomic technologies enable the uncovering and characterization of cellular heterogeneity and pave the way for studies aiming at understanding the origin and consequences of it. The hematopoietic system is in essence a very well adapted model system to benefit from this technological advance because it is characterized by different cellular states. Each cellular state, and its interconnection, may be defined by a specific location in the global transcriptional landscape sustained by a complex regulatory network. This transcriptomic signature is not fixed and evolved over time to give rise to less efficient hematopoietic stem cells (HSC), leading to a well-documented hematopoietic aging. Here, we review the advance of single-cell transcriptomic approaches for the understanding of HSC heterogeneity to grasp HSC deregulations upon aging. We also discuss the new bioinformatics tools developed for the analysis of the resulting large and complex datasets. Finally, since hematopoiesis is driven by fine-tuned and complex networks that must be interconnected to each other, we highlight how mathematical modeling is beneficial for doing such interconnection between multilayered information and to predict how HSC behave while aging

A novel Boolean network inference strategy to model early hematopoiesis aging

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SC-RNA-seq assisted synthesis of a Boolean network to model early haematopoiesis aging

We previously analyzed 15 000 transcriptomes of mouse hematopoietic stem and progenitor cells (HSPCs) from young and aged mice and characterized the early differentiation of the hematopoietic stem cells (HSCs) according to age, thanks to cell clustering and pseudotime analysis (Herault et al, 2021). In this study, we propose an original strategy to build a Boolean gene network explaining HSC priming and homeostasis based on our previous single cell data analysis and the actual knowledge of these biological processes (graphical abstract). We first made an exhaustive analysis of the transcriptional network on selected HSPC states in the differentiation trajectory of HSCs by identifying regulons, modules formed by a transcription factor (TFs) and its targets, from the scRNA-seq data., From this global view of transcriptional regulation in early hematopoiesis, we chose to focus on 15 components, 13 selected TFs (Tal1, Fli1, Gata2, Gata1, Zfpm1, Egr1, Junb, Ikzf1, Myc, Cebpa, Bclaf1, Klf1, Spi1) and two complexes regulating the ability of HSC to cycle (CDK4/6 - Cyclines D and CIP/KIP). We then defined the relations in the differentiation dynamics we want to model ((non) reachability, attractors) between the HSPC states that are partial observations of binarized activity levels of the 15 components. Besides, we defined an influence graph of possibly involved TF interactions in the dynamic using regulon analysis on our single cell data and interactions from the literature. Next, using Answer Set Programming (ASP) and considering these inputs, we obtained a Boolean model as a final solution of a Boolean satisfiability problem. Finally, we perturbed the model according to aging differences underlined from our regulon analysis. This led us to propose new regulatory mechanisms at the origin of the differentiation bias of aged HSCs, explaining the decrease in the transcriptional priming of HSCs toward all mature cell types except megakaryocytes

Delta 1-activated Notch inhibits muscle differentiation without affecting Myf5 and Pax3 expression in chick limb myogenesis

International audienceThe myogenic basic helix-loop-helix (bHLH) transcription factors, Myf5, MyoD, myogenin and MRF4, are unique in their ability to direct a program of specific gene transcription leading to skeletal muscle phenotype. The observation that Myf5 and MyoD can force myogenic conversion in non-muscle cells in vitro does not imply that they are equivalent. In this paper, we show that Myf5 transcripts are detected before those of MyoD during chick limb development. The Myf5 expression domain resembles that of Pax3 and is larger than that of MyoD. Moreover, Myf5 and Pax3 expression is correlated with myoblast proliferation, while MyoD is detected in post-mitotic myoblasts. These data indicate that Myf5 and MyoD are involved in different steps during chick limb bud myogenesis, Myf5 acting upstream of MyoD. The progression of myoblasts through the differentiation steps must be carefully controlled to ensure myogenesis at the right place and time during wing development. Because Notch signalling is known to prevent differentiation in different systems and species, we sought to determine whether these molecules regulate the steps occurring during chick limb myogenesis. Notch1 transcripts are associated with immature myoblasts, while cells expressing the ligands Delta1 and Serrate2 are more advanced in myogenesis. Misexpression of Delta1 using a replication-competent retrovirus activates the Notch pathway. After activation of this pathway, myoblasts still express Myf5 and Pax3 but have downregulated MyoD, resulting in inhibition of terminal muscle differentiation. We conclude that activation of Notch signalling during chick limb myogenesis prevents Myf5-expressing myoblasts from progressing to the MyoD-expressing stag

C/EBPβ: a major PML–RARA-responsive gene in retinoic acid-induced differentiation of APL cells

In acute promyelocytic leukemia (APL), the translocation t(15;17) induces a block at the promyelocytic stage of differentiation in an all-trans-retinoic acid (ATRA)-responsive manner. Here we report that upon treatment with ATRA, t(15;17) cells (NB4) reveal a very rapid increase in protein level and binding activity of C/EBPβ, a C/EBP family member, which was not observed in an ATRA-resistant NB4 cell line. We further provide evidence that ATRA mediates a direct increase of C/EBPβ, only in PML–RARA (promyelocytic leukemia–retinoic acid receptor α)-expressing cells. In addition, transactivation experiments indicate that the PML–RARA fusion protein, but not PML–RARA mutants defective in transactivation, strongly transactivates the C/EBPβ promoter. These results suggest that PML–RARA mediates ATRA-induced C/EBPβ expression. Finally, we demonstrate the importance of C/EBPβ in granulocytic differentiation. We show that not only does C/EBPβ induce granulocytic differentiation of non-APL myeloid cell lines independent of addition of ATRA or other cytokines, but also that C/EBPβ induction is required during ATRA-induced differentiation of APL cells. Taken together, C/EBPβ is an ATRA-dependent PML–RARA target gene involved in ATRA-induced differentiation of APL cells

Noggin acts downstream of Wnt and Sonic Hedgehog to antagonize BMP4 in avian somite patterning

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