O gene supressor tumoral RASSF1A tem sido associado com redução da proliferação celular em diversos tumores. Sua expressão é regulada por eventos epigenéticos que envolvem o complexo repressivo polycomb (PRC2), no entanto os mecanismos moleculares da modulação do recrutamento deste modificador epigenético para este locus ainda são desconhecidos. Neste trabalho identificamos e caracterizamos ANRASSF1, um RNA não codificador longo (lncRNA) intrônico unspliced, que é transcrito na fita oposta do gene RASSF1A, em várias linhagem celulares e tecidos, e se liga a PRC2. ANRASSF1 é transcrito pela RNAPII, possui cap-5´ e cauda poli-A, além de localizar-se no núcleo e possuir uma meia-vida em média quatro vezes menor comparada com outros lncRNAs ligados à PRC2. A super-expressão ectópica de ANRASSF1 reduziu os níveis de RASSF1A e aumentou a taxa de proliferação em células HeLa, enquanto seu silenciamento provocou efeito oposto. Essas mudanças nos níveis de ANRASSF1 não afetaram a abundância da isoforma RASSF1C em nenhuma das condições. A super-expressão de ANRASSF1 provocou um grande aumento tanto da ocupação de PRC2 como da marca de histona repressiva H3K27me3 especificamente na região promotora RASSF1A. Nenhum efeito da super-expressão de ANRASSF1 foi detectado na ocupação de PRC2 e na histona H3K27me3 nas regiões promotoras de RASSF1C e de outros quatro genes vizinhos, incluindo dois genes supressores tumorais bem caracterizados. Além disso, foi demonstrado que ANRASSF1 forma um híbrido de RNA/DNA e recruta SUZ12, um componente do PRC2, para o promotor de RASSF1A. Notavelmente, foi detectado pelo ensaio de RNase-ChIP que a degradação de ANRASSF1 diminui a ocupação de PRC2 neste promotor. Esses resultados demonstram um novo mecanismo de repressão epigenética do supressor tumoral RASSF1A, envolvendo um lncRNA unspliced antissenso, onde ANRASSF1 reprime seletivamente a expressão da isoforma de RASSF1 que sobrepõe o transcrito antissenso de modo local e específico. Considerando uma perspectiva mais ampla, nossos resultados sugerem que outros lncRNAs intrônicos unspliced não caracterizados no genoma humano podem contribuir para uma modulação epigenética local e específica de cada região em que os lncRNAs são transcritos. / Tumor-suppressor RASSF1A gene down-regulation has been implicated in increasing cell proliferation in several tumors. Its expression is regulated by epigenetic events involving polycomb repressive complex 2 (PRC2), however the molecular mechanisms modulating recruitment of this epigenetic modifier to the locus remain largely unknown. Here, we identify and characterize ANRASSF1, an endogenous unspliced long noncoding RNA (lncRNA) that is transcribed from the opposite strand of RASSF1 gene in several cell lines and tissues, and binds to PRC2. ANRASSF1 is transcribed by RNA Polymerase II, 5\'-capped, polyadenylated, displays nuclear localization, and has on average a four-fold shorter half-life compared to other lncRNAs that bind PRC2. ANRASSF1 ectopic overexpression decreases RASSF1A abundance and increases the proliferation rate of HeLa cells, whereas its silencing causes opposite effects. These changes in NRASSF1 levels do not affect RASSF1C isoform abundance. ANRASSF1 overexpression causes a marked increase both in PRC2 occupancy and in histone H3K27me3 repressive mark specifically at the RASSF1A promoter region. No effect of ANRASSF1 overexpression is detected on PRC2 occupancy and on histone H3K27me3 at the promoter regions of RASSF1C and of four other neighbor genes, including two well-characterized tumor suppressor genes. Additionally, we demonstrate that ANRASSF1 forms an RNA/DNA hybrid, and recruits SUZ12, a PRC2 component, to the RASSF1A promoter. Notably, depletion of ANRASSF1 disrupts SUZ12 occupancy on RASSF1A promoter as measured by RNAse-ChIP assay. Together, these results show a new mechanism of epigenetic repression of RASSF1A tumor suppressor gene involving an antisense unspliced lncRNA, in which ANRASSF1 selectively represses expression of the RASSF1 isoform overlapping the antisense transcript in a location-specific manner. In a broader perspective, our findings suggest that other non-characterized unspliced intronic lncRNAs transcribed in the human genome may contribute to a location-specific epigenetic modulation of genes.

Dans ce travail, j'ai utilisé les trichomes (poils foliaires) d'Arabidopsis comme modèle pour étudier la différenciation cellulaire et l'endoréplication. Mon travail a révélé que les cycles d’endoréplication chez Arabidopsis étaient contrôlés par les protéines inhibitrices CYCLIN DEPENDENT KINASE (CDK), elles-mêmes contrôlées par dégradation via l'action de complexes SKP-CULLIN-F-BOX (SCF). Ceci crée vraisemblablement des niveaux variables d'activité de CDK, qui sont nécessaires pour la progression répétée au travers des phases de synthèse d'ADN dans les cellules entrées en endoréplication. Cependant, la sur-expression des inhibiteurs des CDK ne bloque pas seulement l'endoréplication mais résulte aussi dans la dédifférenciation des cellules précurseurs des trichomes. Des résultats similaires ont été obtenus en utilisant des allèles faibles de perte de fonction pour CDKA;1, la principale CDK chez Arabidopsis, laissant émerger la notion que l'endoréplication est nécessaire à la maintenance du devenir des cellules. De manière surprenante, la dédifférenciation peut être au moins partiellement réprimée quand RBR1, l'homologue chez Arabidopsis de la protéine animale suppresseur de tumeur RETINOBLASTOMA (Rb), est mutée de manière concomitante. De même, une mutation de la methyltransferase CURLY LEAF, composante du complexe PRC2, rétablit le défaut de maintenance des trichomes chez les mutants faibles pour CDKA;1. Pris dans leur ensemble, ces résultats suggèrent que le complexe PRC2 et la protéine RBR1 établissent, au niveau tissulaire, un seuil pour la différenciation cellulaire au cours du développement de l'épiderme chez Arabidopsis. / Cell differentiation is often linked with a switch from a mitotic to an endoreplication cycle, in which cells re-replicate their DNA without cell division. The molecular regulation of endoreplication and its biological fonction are only poorly understood. Here, I have used trichomes (leaf hairs) of Arabidopsis as a model to study cell differentiation and endoreplication. My work revealed that endoreplication cycles in Arabidopsis are controlled by cyclin dependent kinase (CDK) inhibitor proteins, which in turn are subject to protein degradation mediated by the action of SKP-CULLIN-F-BOX (SCF) complexes. This presumably creates oscillating levels of CDK activity, which are needed for repeated progression through DNA synthesis phases in endoreplicating cells. However, overexpression of CDK inhibitors did not only block endoreplication but also resulted in the dedifferentiation of trichome precursor cells. Similar observations were made with weak- loss-of-function alleles for the major CDK in Arabidopsis, CDKA;1, giving rise to the notion that endoreplication is required for cell fate maintenance. Trichome dedifferentiation was enhanced when trichome fate regulators were mutated. Surprisingly, the dedifferentiation could be at least partially repressed when RBR1, the Arabidopsis homolog of the animal tumor suppressor protein Retinoblastoma (Rb), was concomitantly mutated. Similarly, a mutation in PRCZ-methyltransfcrase CURLY LEAF (CLF) rescued the trichome maintenance defect of weak CDKA;1 mutants. Taken together, this suggests that PRC2 and RBR1 set a dynamic tissue threshold for cell differentiation during epidermis development in Arabidopsis.

O gene supressor tumoral RASSF1A tem sido associado com redução da proliferação celular em diversos tumores. Sua expressão é regulada por eventos epigenéticos que envolvem o complexo repressivo polycomb (PRC2), no entanto os mecanismos moleculares da modulação do recrutamento deste modificador epigenético para este locus ainda são desconhecidos. Neste trabalho identificamos e caracterizamos ANRASSF1, um RNA não codificador longo (lncRNA) intrônico unspliced, que é transcrito na fita oposta do gene RASSF1A, em várias linhagem celulares e tecidos, e se liga a PRC2. ANRASSF1 é transcrito pela RNAPII, possui cap-5´ e cauda poli-A, além de localizar-se no núcleo e possuir uma meia-vida em média quatro vezes menor comparada com outros lncRNAs ligados à PRC2. A super-expressão ectópica de ANRASSF1 reduziu os níveis de RASSF1A e aumentou a taxa de proliferação em células HeLa, enquanto seu silenciamento provocou efeito oposto. Essas mudanças nos níveis de ANRASSF1 não afetaram a abundância da isoforma RASSF1C em nenhuma das condições. A super-expressão de ANRASSF1 provocou um grande aumento tanto da ocupação de PRC2 como da marca de histona repressiva H3K27me3 especificamente na região promotora RASSF1A. Nenhum efeito da super-expressão de ANRASSF1 foi detectado na ocupação de PRC2 e na histona H3K27me3 nas regiões promotoras de RASSF1C e de outros quatro genes vizinhos, incluindo dois genes supressores tumorais bem caracterizados. Além disso, foi demonstrado que ANRASSF1 forma um híbrido de RNA/DNA e recruta SUZ12, um componente do PRC2, para o promotor de RASSF1A. Notavelmente, foi detectado pelo ensaio de RNase-ChIP que a degradação de ANRASSF1 diminui a ocupação de PRC2 neste promotor. Esses resultados demonstram um novo mecanismo de repressão epigenética do supressor tumoral RASSF1A, envolvendo um lncRNA unspliced antissenso, onde ANRASSF1 reprime seletivamente a expressão da isoforma de RASSF1 que sobrepõe o transcrito antissenso de modo local e específico. Considerando uma perspectiva mais ampla, nossos resultados sugerem que outros lncRNAs intrônicos unspliced não caracterizados no genoma humano podem contribuir para uma modulação epigenética local e específica de cada região em que os lncRNAs são transcritos. / Tumor-suppressor RASSF1A gene down-regulation has been implicated in increasing cell proliferation in several tumors. Its expression is regulated by epigenetic events involving polycomb repressive complex 2 (PRC2), however the molecular mechanisms modulating recruitment of this epigenetic modifier to the locus remain largely unknown. Here, we identify and characterize ANRASSF1, an endogenous unspliced long noncoding RNA (lncRNA) that is transcribed from the opposite strand of RASSF1 gene in several cell lines and tissues, and binds to PRC2. ANRASSF1 is transcribed by RNA Polymerase II, 5\'-capped, polyadenylated, displays nuclear localization, and has on average a four-fold shorter half-life compared to other lncRNAs that bind PRC2. ANRASSF1 ectopic overexpression decreases RASSF1A abundance and increases the proliferation rate of HeLa cells, whereas its silencing causes opposite effects. These changes in NRASSF1 levels do not affect RASSF1C isoform abundance. ANRASSF1 overexpression causes a marked increase both in PRC2 occupancy and in histone H3K27me3 repressive mark specifically at the RASSF1A promoter region. No effect of ANRASSF1 overexpression is detected on PRC2 occupancy and on histone H3K27me3 at the promoter regions of RASSF1C and of four other neighbor genes, including two well-characterized tumor suppressor genes. Additionally, we demonstrate that ANRASSF1 forms an RNA/DNA hybrid, and recruits SUZ12, a PRC2 component, to the RASSF1A promoter. Notably, depletion of ANRASSF1 disrupts SUZ12 occupancy on RASSF1A promoter as measured by RNAse-ChIP assay. Together, these results show a new mechanism of epigenetic repression of RASSF1A tumor suppressor gene involving an antisense unspliced lncRNA, in which ANRASSF1 selectively represses expression of the RASSF1 isoform overlapping the antisense transcript in a location-specific manner. In a broader perspective, our findings suggest that other non-characterized unspliced intronic lncRNAs transcribed in the human genome may contribute to a location-specific epigenetic modulation of genes.

Multiple myeloma (MM) is a hematological malignancy of antibody producing plasmablasts/plasma cells. MM is characterized by extensive genetic and clonal heterogeneity, which have hampered the attempts to identify a common underlying mechanism for disease establishment and development of appropriate treatment regimes. This thesis is focused on understanding the role of epigenetic regulation of gene expression mediated by the polycomb repressive complexes 1 and 2 (PRC1 and 2) in MM and their impact on disease biology and therapy. In paper I the genome-wide distribution of two histone methylation marks; H3K27me3 and H3K4me3 were studied in plasma cells isolated from newly diagnosed MM patients or age-matched normal donors. We were able to define targets of H3K27me3, H3K4me3 and bivalent (carry both marks) which are, when compared to normal individuals, unique to MM patients. The presence of H3K27me3 correlated with silencing of MM unique H3K27me3 targets in MM patients at advanced stages of the disease. Notably, the expression pattern of H3K27me3-marked genes correlated with poor patient survival. We also showed that inhibition of the PRC2 enzymatic subunit EZH2 using highly selective inhibitors (GSK343 and UNC1999) demonstrated anti-myeloma activity using relevant in vitro models of MM. These data suggest an important role for gene repression mediated by PRC2 in MM, and highlights the PRC2 component EZH2 as a potential therapeutic target in MM. In paper II we further explored the therapeutic potential of UNC1999, a highly selective inhibitor of EZH2 in MM. We showed that EZH2 inhibition by UNC1999 downregulated important MM oncogenes; IRF-4, XBP-1, BLIMP-1and c-MYC. These oncogenes have been previously shown to be crucial for disease establishment, growth and progression. We found that EZH2 inhibition reactivated the expression of microRNAs genes previously found to be underexpressed in MM and which possess potential tumor suppressor functions. Among the reactivated microRNAs we identified miR-125a-3p and miR-320c as predicted negative regulators of the MM-associated oncogenes. Notably, we defined miR-125a-3p and miR-320c as targets of EZH2 and H3K27me3 in MM cell lines and patients samples.  These findings described for the first time PRC2/EZH2/H3K27me3 as regulators of microRNA with tumor suppressor functions in MM. This further strengthens the oncogenic features of EZH2 and its potential as a therapeutic target in MM. In paper III we evaluated the therapeutic potential of targeting PRC1 in MM using the recently developed chemical PTC-209; an inhibitor targeting the BMI-1 subunit of PRC1. Using MM cell lines and primary cells isolated from newly diagnosed or relapsed MM patients, we found that PTC-209 has a potent anti-MM activity. We showed, for the first time in MM, that PTC-209 anti-MM effects were mediated by on-target effects i.e. downregulation of BMI-1 protein and the associated repressive histone mark H2AK119ub, but that other subunits of the PRC1 complex were not affected. We showed that PTC-209 reduced MM cell viability via significant induction of apoptosis. More importantly, we demonstrated that PTC-209 shows synergistic anti-MM activity with other epigenetic inhibitors targeting EZH2 (UNC1999) and BET-bromodomains (JQ1). This work highlights the potential use of BMI-1 and PRC1 as potential therapeutic targets in MM alone or in combination with other anti-MM agents including epigenetic inhibitors.

L’inactivation d’un des deux chromosomes X dans les cellules d’organismes femelles permet d’assurer un taux similaire des transcrits des gènes liés aux chromosomes X entre les deux sexes. L’ARN non codant Xist d’environ 17000 nts joue un rôle central dans ce processus. Il habille le futur chromosome X inactivé et induit la mise en place de modifications épigénétiques qui permettent d’éteindre l’expression des gènes. Une région d’approximativement 500 nts située à l’extrémité 5’ de l’ARN Xist est nécessaire à l’initiation de l’inactivation. Cette région appelée region des A-repeats contient 8 répétitions d’une séquence de 24 nucléotides. La délétion de cette région provoque un défaut d’inactivation, ce qui souligne son importance dans le processus. Etant donné que la fonction d’un ARN est bien souvent conditionnée par sa structure 2D, mon travail de thèse a consisté à réaliser l’étude expérimentale de la structure 2D de la région des A-repeats, ceci en utilisant des sondes de la structure secondaire des ARN en solution et une méthode de FRET. Nous avons montré que la région des A-repeats se structure selon 2 grandes structures tige-boucle irrégulières formées par l’appariement 2 à 2 des éléments répétés. Par purification des RNP et identification de leurs protéines, nous avons démontré que le complexe PRC2, impliqué dans la mise en place des marques épigénétiques du Xi, se lie à la région des A-repeats. Nous avons également identifié un grand nombre d’autres protéines pouvant avoir un rôle dans l’activité de la région des A-repeats (PTB, KSRP, Sam68, Vigiline, RHA, TIAR, DEK, H1, BRML1, Rod1, Lin28). Leurs implications dans l’inactivation du chromosome X est en cours de vérification. / Silencing of one X chromosome (XCI) in cells of mammalian female ensures sex chromosome dosage compensation between male and female. The 17kb Xist ncRNA plays an essential role in XCI. Its spread along the future inactivated X chromosome is associated with major modifications of the epigenetic status of this chromosome, including histone H3K27 methylations mediated by PRC2 complex. One key part of Xist necessary for XCI initiation is the phylogenetically conserved A region. It lies at the 5’ end of the Xist molecule and contains 8 of a 24-nucleotides motif. Female mouse embryos carrying a mutated Xist deleted for the A region are selectively lost during embryogenesis, which underlines the importance of this element. We performed the first experimental analysis of the structure of the entire A region in solution. By the use of chemical and enzymatic probes and FRET experiments, using oligonucleotides carrying fluorescent dyes, we established a 2D structure for the A region that contains two long stem-loop structures each including 4 repeats which interact together two by two. By immunoprecipitation assays and mass spectrometry analysis, we identified the protein partners of the A region. We demonstrated that the A region associate with PRC2 components which is responsible for the apposition of epigenetic modifications of X inactive chromosome. Others proteins which would have a role in A region function were also identified (PTB, KSRP, Sam68, Vigiline, RHA, TIAR, DEK, H1, BRML1, Rod1, Lin28).

EZH2 is a SET domain-containing methyltransferase and the catalytic component of the multimeric Polycomb- group (PcG) protein complex, PRC2. When in complex with other PRC2 members (EED, SUZ12, AEBP2, and RBBP4), EZH2 catalyzes methylation of H3K27, a histone modification associated with transcriptional repression and developmental regulation. As several PRC2 components are upregulated or mutated in a variety of human cancers, efforts to discover small-molecule modulators of PRC2 and understand its regulation may yield therapeutic insights. Identification of small-molecule probes with distinct chemotypes, MOAs, and selectivity profiles are not only of great value, but necessary in establishing comprehensive probe sets capable of illuminating the various roles of EZH2 in oncogenesis. Here we describe efforts to identify and characterize small-molecule modulators of PRC2 and further understand its regulation. Chapter II outlines the expression and purification of 5-component PRC2 (EZH2-EED-SUZ12-AEBP2-RBBP4) and the establishment of biochemical and cellular HTS assays. These assays were used to screen a diverse set of small molecules (>120,000), identifying biochemical PRC2 inhibitors and activators (described in Chapter III). One biochemical PRC2 inhibitor, BRD1835, appeared to inhibit PRC2 activity through a novel artifactual mechanism involving interaction with peptide substrate, leading to apparent peptide-competitive behavior and putative cellular activity (described in Chapter IV). The characterization of novel biochemical PRC2 activators, BRD3934 and BRD8284, is discussed in Chapter V. Chapter VI describes the use of an HCS assay to identify known bioactive compounds that alter intracellular levels of H3K27me3 through modulating H3K27me3-connected regulatory nodes or by targeting PRC2 directly. These efforts led to the discovery that an antifungal agent, miconazole, is capable of activating PRC2 activity in vitro, while a mucolytic agent, bromhexine, selectively ablates cellular H3K27me3 levels through targeting an activity distinct from PRC2. Finally, Chapter VII discusses novel PRC2-connected crosstalk mechanisms identified through screening libraries of uniquely modified histone peptides for their ability to bind or support methylation by PRC2. These studies enhance our understanding of PRC2 regulation by revealing the effects of H3R26 and H3K23me1 modifications on enzymatic activity, implicating their respective methyltransferases in PRC2 regulation.

Eukaryotic DNA is packaged into highly ordered structures knows as chromatin that further packs the DNA into higher-order structures, limiting the accessibility of the underlying genetic information for the processes like transcription, replication, and repair. However, eukaryotic cells have evolved proteins called chromatin regulators that regulate the accessibility of the genetic information when needed. This dissertation aims to characterize the role of two such proteins, JARID2 and the polycomb repressive complex-2 (PRC2), during skeletal muscle proliferation and differentiation.JARID2 is an inactive yet evolutionarily conserved histone demethylase that is shown to be a sub-stoichiometric component of the PRC2 complex. The PRC2 complex represses gene expression through the trimethylation of lysine 27 of histone 3 (H3K27me) tails. H3K27 methylation leads to chromatin compaction. JARID2 helps in targeting of the PRC2 complex to its target loci. JARID2 is shown to be required for the normal development of mice, as loss of Jarid2 leads to lethality in utero. We, for the first time, show that JARID2 is required for the normal skeletal muscle differentiation. We show that the JARID2 regulates the expression of myogenic regulatory factor, Myod1, both through direct repression and activation through the modulation of canonical Wnt signaling pathway. JARID2, in association with the PRC2 complex, represses Wnt antagonist Sfrp1 to modulate the activity of the canonical Wnt signaling pathway. The translocation of Wnt effector protein, b-catenin, from the cytoplasm to the nucleus modulates the activity of the canonical Wnt signaling pathway during activation. We also show that b-catenin directly regulates the expression of Myod1 gene through its direct binding in the distal regulatory region.We further extend the role of JARID2 during skeletal muscle proliferation. We show that JARID2 also plays an essential role in restraining the skeletal muscle proliferation through its direct repression of positive cell cycle regulators cyclin D1 (Ccnd1) and cyclin E1 (Ccne1). Furthermore, we show that retinoblastoma protein 1 (Rb1), a negative regulator of cell proliferation that promotes cell cycle exit and differentiation, is also directly regulated by JARID2 in PRC2 dependent manner. Together, we show that JARID2 precisely controls cell proliferation and differentiation during skeletal muscle differentiation.Further, we show that the regulation of cell proliferation by JARID2 is PRC2 complex dependent. When the PRC2 complex was depleted or inhibited to a modest level, the cells have an increased cell proliferation ability compared to severe loss or inhibition of EZH2, the catalytic subunit of the PRC2 complex, that leads to the apoptosis of the cells. It is also marked by increased expression of known PRC2 targets genes. We show that the increased proliferation upon modest inhibition or depletion of EZH2 is through direct de-repression of positive cell cycle genes, Ccnd1, and Ccne1. It is the first work that shows a context-dependent role of the PRC2 complex during skeletal muscle proliferation and differentiation.My dissertation also makes an extraordinary discovery as to why myogenin is required for the proper function of MyoD during skeletal muscle differentiation, even though both proteins share a large set of overlapping target genes. We show that myogenin is required for the nucleosome disassembly and reassembly at the target genes through recruitment of the FACT complex, a histone chaperone. We also show that myogenin is required for the assembly of the basic transcription machinery and RNA polymerase II to the target muscle genes during differentiation. Surprisingly, we also show that myogenin reinforces its own expression through the activation of Myod1 expression during skeletal muscle differentiation. Myogenin is a known target of MyoD.Taken together, this dissertation provides a molecular mechanism for the crosstalk between a signaling pathway with chromatin regulatory proteins, JARID2, and the PRC2 complex in regulating skeletal muscle differentiation. It also extends the role of JARID2 and the PRC2 complex - known oncogenes, in precise, context-dependent control of cell proliferation and differentiation in skeletal muscle.

The majority of our physiological and metabolic processes are coordinated by an internal clock, which has evolved as an adaptive response to the daily light-dark cycles. Thus, several physiological and behavioral activities display an oscillatory rhythmic period of 24 hours. This highly conserved molecular mechanism is achieved through a specific program of gene expression, characterized by a complex interaction between clock-core proteins, chromatin remodelers and epigenetic events associated with the oscillatory nature of circadian transcriptional activity in the genome. Clock disruption leads to a wide spectrum of severe health problems including chronic metabolic disorders, muscle waste and cardiopathies. Previous studies revealed that each cell and organ possess an intrinsic clock and that coordination between central versus peripheral clocks is key for health. Furthermore, it has been found that under nutritional challenge such as High Fat Diet (HFD), the circadian transcriptome and metabolome are rapidly remodeled in the mouse model. Surprisingly, metabolome and gene expression analysis on various tissues revealed that skeletal muscle is the most affected under HFD. Mechanisms that regulate circadian cycle and stress induced rapid adaptation and in particular metabolic stress at the chromatin level are largely unknown. In this study, we investigated the role of Polycomb proteins group (PcG) mediate cell memory system by maintaining transcriptional gene silencing, in particular the PRC2-Ezh1. We hypothesized that Ezh1 could play an important role in circadian clock regulation in post-mitotic skeletal muscle, and this pathway has never been explored in this context. We explored the circadian role of PRC2-Ezh1 in the mouse skeletal muscle. Intriguingly, we found that the oscillatory profile of a novel isoform of Ezh1 (Ezh1beta), localized specifically in the cytoplasm and controlling stress induced nuclear PRC2 activity, was completely disrupted under HFD. More interestingly, the circadian pattern of core clock components was impaired in Ezh1 depleted cells. Our data unveils an interesting physiological role of the PcG memory system, from cytoplasm to chromatin, which could indicate a new link between the chromatin remodeler Polycomb proteins and the endogenous clock in adaptation mechanism in skeletal muscle.

Polycomb repressive complex 2 (PRC2) catalyses the methylation of ‘Lys-27’ of histone H3, leading to transcriptional repression of target genes through its catalytic subunit Enhancer of zeste homolog 1/2 (EZH1/2). PRC2 functions as a critical regulator of stem cells in mouse embryonic and adult tissues. However, the role of PRC2 in human skin remains largely unknown. This study investigated the role of PRC2 in human epidermal homeostasis and hair growth. The expression of EZH2 was elevated in differentiating suprabasal layers of the human epidermis. Consistently, EZH1/2 expression and enzymatic activity was upregulated in differentiating primary human keratinocytes (NHEKs) in vitro. Inhibition of EZH2 and Embryonic ectoderm development (EED) in NHEKs stimulated the expression of differentiation-associated genes, therefore leading to their premature differentiation; while inhibition of EZH1/2 reduced cell proliferation and promoted apoptosis. Silencing of EZH2 in NHEKs induced complex changes in gene expression programmes, including the upregulation of terminal differentiation genes, such as Filaggrin. EZH2 expression was downregulated in aged keratinocytes accompanied with upregulation of senescence-associated genes, p16INK4A and p19INK4D, suggesting EZH2 involvement in epidermal aging. In human anagen hair follicle (HF), EZH2 was detected in stem and progenitor cells; and hair matrix keratinocytes. Silencing EZH2 in HFs accelerated anagen-catagen transition and retarded hair growth accompanied by decreased proliferation and increased apoptosis. Silencing EZH2 in outer root sheath keratinocytes resulted in upregulation of p14ARF and K15, suggesting EZH2 involvement in regulating proliferation and stem cell activity. Thus, this study demonstrates that PRC2-mediated repression is crucial for epidermal homeostasis and hair growth. Modulating the activities of PRC2 in skin might offer a new therapeutic approach for disorders of epidermal differentiation and hair growth.

Le raccourcissement progressif des télomères, en grande partie dû à la réplication incomplète des télomères, est l’une des caractéristiques principales du vieillissement. De façon encore plus frappante, une attrition trop marquée ou rapide des télomères est l’une des causes majeures d’un vieillissement prématuré. Les patients diagnostiqués avec un tel syndrome présentent généralement des mutations délétères dans un gène de maintien de l’homéostasie des télomères. Parmi les symptômes physiologiques, on remarque chez ces patients, des dégénérescences dans les tissus hautement prolifératifs, dus à l’exhaustion des cellules souches. Les cellules souches adultes, spécialisées et présentes dans nos tissus, permettent normalement la régénération des organes grâce à leur potentiel prolifératif et de différenciation. Afin de maintenir leur intégrité génomique, les cellules souches expriment une enzyme, la télomérase, capable de rallonger les extrémités terminales des chromosomes, et ainsi de maintenir intègre les télomères de ces cellules subissant des divisions cellulaire et subsistant dans l’organisme durant la vie de l’individu. En revanche, l’expression de la télomérase s’amenuise au cours du temps, les télomères se raccourcissent et les fonctions des cellules souches sont altérées. Nous étudions ce phénomène en utilisant comme modèle cellulaire, les cellules souches embryonnaires de souris, dont le gène de la télomérase réverse transcriptase a été anéanti génétiquement (mESC Tert -/-). Notamment, ces cellules, qui possèdent des télomères extrêmement courts n’arrivent pas à se différencier correctement. D’une étude précédente, mon laboratoire avait montré que ces cellules ne réussissaient pas à réprimer l’expression des gènes de pluripotence, comme Pou5F1/Oct4 et Nanog, et donc montraient des difficultés à sortir de l’état indifférencié. Au cours de mes recherches, nous avons montré que ce défaut était en fait dû à une altération en profondeur de l’épigénétique de ces cellules. Après avoir observé une déméthylation globale de l’ADN, nous avons constaté une augmentation de la marque d’histone H3K27me3 à travers le génome. De plus, moduler la présence de H3K27me3 via des petits inhibiteurs du complexe PRC2, ou par une approche génétique, modifie également le potentiel de différenciation des cellules souches. À la suite de cette étude, nous avons voulu en savoir davantage sur le signal liant attrition des télomères et défaut de différenciation. Lorsque les télomères sont très courts, la voie de réparation de l’ADN les reconnait comme une cassure double-brin. Parmi les acteurs de la réparation de l’ADN, la protéine p53 joue un rôle central puisqu’elle influence le destin cellulaire. Candidat idéal, nous avons cherché à savoir si p53 influençait la différenciation des mESC Tert -/- en utilisant une approche génétique. Nous avons été surpris de constater que l’absence de p53 restaurait le potentiel de différenciation des mESC Tert -/-. Ainsi et pour la première fois, nous avons montré que le raccourcissement des télomères pouvait avoir un effet très global sur les cellules. Nos recherches permettront de mieux appréhender certaines problématiques, notamment en matière de vieillissement. / The progressive decline of telomere length, mainly due to incomplete DNA replication at telomeres, is one of the main features of aging. Even more strikingly, excessive or rapid telomere attrition is one of the major causes of premature aging. Patients diagnosed with such syndromes have deleterious mutations in genes that maintain telomere homeostasis. For these patients, physiological manifestations include degeneration in highly proliferative tissues due to stem cell exhaustion. Adult stem cells, present in our tissues, typically allow the regeneration of organs due to their proliferative and differentiation potential. To maintain their replicative potential, stem cells express an enzyme, called telomerase, that is capable of lengthening the terminal ends of chromosomes. The maintenance of telomere length and, consequently favoring genomic stability, is crucial for stem cells that undergo cell division and remain in the organism throughout the individual's life. However, in stem cells, telomerase expression decreases over time, leading to telomere attrition and defects in stem cell functions. We studied this phenomenon using mouse embryonic stem cells disrupted for telomerase reverse transcriptase (mESC Tert -/-) as a model. In particular, these cells, which have extremely short telomeres, are unable to differentiate appropriately. Previously, my colleagues had shown that these cells failed to repress the expression of pluripotency genes, such as Pou5f1/Oct4 and Nanog, and therefore were refractory to differentiation. Here, we uncovered that profound epigenetic alterations influenced mESC Tert-/- cell fate. In addition to global DNA demethylation, we found an increase in H3K27me3 throughout the genome. Furthermore, modulating the levels of H3K27me3 via small inhibitors of the PRC2 complex or by a genetic approach also altered the differentiation potential of stem cells. Following this study, we wanted to learn more about the signals linking telomere attrition and differentiation failure. When telomeres are very short, the DNA repair pathway recognizes them as a double-strand break. Among the actors in DNA repair, the p53 protein plays a central role in influencing cell fate. As an ideal candidate, we investigated whether p53 influenced the differentiation of Tert-/- mESCs using a genetic approach. We were surprised to find that the absence of p53 restored the differentiation potential of Tert-/- mESCs. Thus, for the first time, we have shown that telomere shortening can have a very global effect on stem cells. Our research will allow us to better understand specific problems, particularly in the field of aging.