Rôles fonctionnels des Poly(ADP-ribose)polymérases -1, -2 et de leur activité dans le contrôle épigénétique de la prolifération et de la différenciation cellulaire

La poly(ADP-ribosyl)ation est une modification post-traductionnelle des protéines catalysée par les Poly(ADPribose)polymérases (Parps) qui constituent une famille de 17 membres. Afin de caractériser les rôles in vivo de Parp1 et de Parp2, des souris déficientes en chacune de ces protéines ont été générées et révèlent des fonctions à la fois redondantes et spécifiques de ces deux Parps dans la maintenance de la stabilité génomique. Les souris mâles déficientes en Parp2 présentent un phénotype spécifique d'hypofertilité associée à une dérégulation du processus de spermatogenèse dans les étapes de méiose et de différenciation des spermatides en spermatozoïdes (spermiogenèse). Afin de caractériser la fonction de Parp2 au cours de la spermatogenèse, des études de microscopie électronique à transmission ont été réalisées et montrent un délai de condensation de la chromatine dans les spermatides issus de souris Parp2-/- qui peut s'expliquer par un défaut de remplacement des histones par les protéines de transition (TP1 et TP2). Des études d'interaction in vitro et ex vivo mettent en évidence un complexe protéique composé de Parp2, Parp1, TP2 et sa protéine chaperonne HSPA2 régulé par l'activité de poly(ADP-ribosyl)ation de Parp1. Nos données suggèrent un rôle de ce complexe et de l'activité Parp dans la structure de la chromatine au cours de la spermiogenèse. La recherche de partenaires potentiels de Parp2 par spectrométrie de masse a permis l'identification du régulateur transcriptionnel TIF1! (Facteur de Transcription Intermédiaire 1!) qui présente un grand nombre de similitudes fonctionnelles avec Parp2. En combinant des approches biochimique, moléculaire et cellulaire, nous avons mis en évidence une interaction physique et fonctionnelle de Parp1 et Parp2 avec TIF1! et les protéines de structure de l'hétérochromatine HP1s, qui intervient dans la différenciation des cellules de carcinome embryonnaire F9 en cellules type endoderme primitif et pariétal. Tandis que Parp2 et son activité catalytique sont requises pour la relocalisation de TIF1! au niveau de l'hétérochromatine péricentrique lors de la différenciation en cellules type endoderme primitif, Parp1 et la poly(ADPribosyl) ation modulent l'interaction entre TIF1! et HP1", indispensable pour la différenciation en cellules type endoderme pariétal. De plus, ces travaux suggèrent un rôle de ce complexe et de l'activité Parp dans la modulation de la structure de l'hétérochromatine péricentrique au travers du sous-code histone médié par les protéines HP1s. De façon à éclaircir le rôle des deux Parps dans la structure de l'hétérochromatine péricentrique, nous nous sommes intéressées à sa dynamique au cours de la réplication. Nous montrons un impact de l'absence des Parps et de leur activité dans la progression de la phase S au moment de la réplication de l'hétérochromatine péricentrique. Des études biochimiques d'interaction protéique ont été initiées avec les protéines majeures de son reformation : la protéine chaperonne des HP1s, CAF-1 et le facteur recruteur de l'ADN-méthyltransférase 1 (Dnmt1), Np95. Nos résultats préliminaires identifient un rôle de Parp2 et de son activité dans le recrutement de CAF-1 sur les foyers de réplication de l'hétérochromatine péricentrique tandis que la localisation de Np95 n est pas affectée. Nous montrons aussi que Parp1 interagit et poly(ADP-ribosyl)e Np95 sur son domaine SRA, nécessaire à la liaison avec l'ADN hémi-méthylé,Poly(ADP-ribosyl)ation is a post-translational modification of proteins catalyzed by Poly(ADP-ribose)polymerases (Parps), a large family of 17 proteins. To determine the in vivo functions of Parp1 and Parp2, Parp1-/- and Parp2-/- mice were generated and revealed both redundant and more specific functions of both Parps in genome integrity. More recently, the lab identified male hypofertility in Parp2 deficient mice associated with impaired spermatogenesis and defects in both meiosis and spermiogenesis (differentiation of spermatids to spermatozoa). To further investigate the function of Parp2 in germ cell differentiation, we performed electronic microscopy studies in Parp2-/-spermatids and identified a delay in chromatin condensation that can be explained by an impaired replacement of histones by transition proteins (TP1 and TP2). Using in vitro and ex vivo binding assays, we identified a protein complex containing Parp2, Parp1, TP2 and its chaperon HSPA2 that is regulated by Parp1 activity. Our work suggests a role of this complex and Parp activity in chromatin structure during spermiogenesis. The identification of Parp2 specific partners by mass spectrometry revealed an interaction of Parp2 with the transcriptional intermediary factor TIF1! which presents functional similarities with Parp2. Using biochemical, molecular and cellular approaches, we identified a physical and functional interaction between Parp1 and Parp2 with TIF1! and HP1 heterochromatic structural proteins which is required for the differentiation of F9 cells into primitive and parietal endoderm like cells. Whereas Parp2 and its activity are necessary for the differentiation of F9 cells into primitive endoderm like cells by targeting TIF1! to heterochromatic foci, Parp1 and its activity regulate TIF1! HP1" interaction and control the terminal F9 differentiation into parietal endoderm like cells. Altogether our results describe the involvement of Parp2, Parp1 and Parp activity in the control of pericentric heterochromatin structure through the regulation of the HP1 mediated silencing subcode. In order to highlight the role of both Parps in the pericentric heterochromatin structure, we currently analyze its dynamic during DNA replication. We identify an involvement of Parp1, Parp2 and their activity in S phase progression, when pericentric heterochromatin is replicated. Protein-protein interaction studies with two major chromatin binding proteins : HP1s chaperon CAF-1 and Dnmt1 (DNA methyl-transferase 1) partner Np95 were also initiated. Our first results show the requirement of Parp2 and its activity in CAF-1 recruitment onto pericentric heterochromatin replication foci. In contrast, the relocation of Np95 to these foci is not affected by the absence of Parps or their activities. Finally, we show that Parp1 interacts and poly(ADP-ribosyl)ates Np95 on its SRA domain, which is required for hemi-methylated DNA binding

Rôles fonctionnels des Poly(ADP-ribose)polymérases -1, -2 et de leur activité dans le contrôle épigénétique de la prolifération et de la différenciation cellulaire

La poly(ADP-ribosyl)ation est une modification post-traductionnelle des protéines catalysée par les Poly(ADPribose)polymérases (Parps) qui constituent une famille de 17 membres. Afin de caractériser les rôles in vivo de Parp1 et de Parp2, des souris déficPoly(ADP-ribosyl)ation is a post-translational modification of proteins catalyzed by Poly(ADP-ribose)polymerases (Parps), a large family of 17 proteins. To determine the in vivo functions of Parp1 and Parp2, Parp1-/- and Parp2-/- mice were generated and

The role of poly(ADP-ribosyl)ation in epigenetic events

Epigenetic refers to a range of heritable chromatin modifications including DNA methylation, histone modifications, remodeling of nucleosomes and higher order chromatin modifications. In the framework of chromatin remodeling activities, the poly(ADP-ribosyl)ation of nuclear proteins catalyzed by PARPs, particularly PARP-1 and PARP-2, plays a fundamental role and as such have the potential to orchestrate various chromatin-based biological tasks including transcription, DNA repair and differentiation. In this review, we propose a short overview of the more recent experimental data that shed light on the role of poly(ADP-ribosyl)ation in the translation of the histone code. We will essentially focus on the different mechanisms by which PARP activity regulates the global chromatin environment and how this affects cellular pathways

The histone subcode: poly(ADP-ribose) polymerase-1 (Parp-1) and Parp-2 control cell differentiation by regulating the transcriptional intermediary factor TIF1beta and the heterochromatin protein HP1alpha.

International audienceRecent advances reveal emerging unique functions of poly(ADP-ribose) polymerase-1 (Parp-1) and Parp-2 in heterochromatin integrity and cell differentiation. However, the chromatin-mediated molecular and cellular events involved remain elusive. Here we describe specific physical and functional interactions of Parp-1 and Parp-2 with the transcriptional intermediary factor (TIF1beta) and the heterochromatin proteins (HP1) that affect endodermal differentiation. We show that Parp-2 binds to TIF1beta with high affinity both directly and through HP1alpha. Both partners colocalize at pericentric heterochromatin in primitive endoderm-like cells. Parp-2 also binds to HP1beta but not to HP1gamma. In contrast Parp-1 binds weakly to TIF1beta and HP1beta only. Both Parps selectively poly(ADP-ribosyl)ate HP1alpha. Using shRNA approaches, we provide evidence for distinct participation of both Parps in endodermal differentiation. Whereas Parp-2 and its activity are required for the relocation of TIF1beta to heterochromatic foci during primitive endodermal differentiation, Parp-1 and its activity modulate TIF1beta-HP1alpha association with consequences on parietal endodermal differentiation. Both Parps control TIF1beta transcriptional activity. In addition, this work identifies both Parps as new modulators of the HP1-mediated subcode histone.-Qu?t, D., Gasser, V., Fouillen, L., Cammas, F., Sanglier-Cianferani, S., Losson, R., Dantzer, F. The histone subcode: poly(ADP-ribose) polymerase-1 (Parp-1) and Parp-2 control cell differentiation by regulating the transcriptional intermediary factor TIF1beta and the heterochromatin protein HP1alpha

Parp2 is required for the differentiation of post-meiotic germ cells: identification of a spermatid-specific complex containing Parp1, Parp2, TP2 and HSPA2.

International audienceSpermiogenesis is a complex male germ cell post-meiotic differentiation process characterized by dramatic changes in chromatin structure and function, including chromatin condensation, transcriptional inhibition and the sequential replacement of histones by transition proteins and protamines. Recent advances, in mammalian cells, suggest a possible role of poly(ADP-ribosyl)ation catalyzed by Parp1 and/or Parp2 in this process. We have recently reported severely compromised spermiogenesis in Parp2-deficient mice characterized by a marked delay in nuclear elongation whose molecular mechanisms remain however unknown. Here, using in vitro protein-protein interaction assays, we show that Parp2 interacts significantly with both the transition protein TP2 and the transition chaperone HSPA2, whereas Parp1 binds weakly to HSPA2. Parp2-TP2 interaction is partly mediated by poly(ADP-ribosyl)ation. Only Parp1 poly(ADP-ribosyl)ates HSPA2. In addition, a detailed analysis of spermatid maturation in Parp2-deficient mice, combining immunohistochemistry and electron microscopic approaches, reveals a loss of spermatids expressing TP2, a defect in chromatin condensation and abnormal formation of the manchette microtubules that, together, contribute to spermatid-specific cell death. In conclusion, we propose both Parps as new participants of a spermatid-specific protein complex involved in genome reorganization throughout spermiogenesis

Poly(ADP-ribose) polymerase 1 (PARP1) associates with E3 ubiquitin-protein ligase UHRF1 and modulates UHRF1 biological functions

Poly(ADP-ribose) polymerase 1 (PARP1, also known as ARTD1) is an abundant nuclear enzyme that plays important roles in DNA repair, gene transcription, and differentiation through the modulation of chromatin structure and function. In this work we identify a physical and functional poly(ADP-ribose)-mediated interaction of PARP1 with the E3 ubiquitin ligase UHRF1 (also known as NP95, ICBP90) that influences two UHRF1-regulated cellular processes. On the one hand, we uncovered a cooperative interplay between PARP1 and UHRF1 in the accumulation of the heterochromatin repressive mark H4K20me3. The absence of PARP1 led to reduced accumulation of H4K20me3 onto pericentric heterochromatin that coincided with abnormally enhanced transcription. The loss of H4K20me3 was rescued by the additional depletion of UHRF1. In contrast, although PARP1 also seemed to facilitate the association of UHRF1 with DNMT1, its absence did not impair the loading of DNMT1 onto heterochromatin or the methylation of pericentric regions, possibly owing to a compensating interaction of DNMT1 with PCNA. On the other hand, we showed that PARP1 controls the UHRF1-mediated ubiquitination of DNMT1 to timely regulate its abundance during S and G2 phase. Together, this report identifies PARP1 as a novel modulator of two UHRF1-regulated heterochromatin-associated events: the accumulation of H4K20me3 and the clearance of DNMT1