archives@tulane.edu / 1 / Anna Smith

TBX2 and TBX3, which function as repressors, are members of the T-Box transcription factor family which are conserved throughout the metazoan lineage. TBX2 is highly expressed in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, and many other cancers. Previously, our lab dissected the oncogenic properties of TBX2 and its regulation of p14, p21 and PTEN. TBX3 is also expressed in some cancer types, however, its expression profile in RMS is severely down-regulated. TBX3 is shown to repress TBX2 in chondrocytes, but the characterization and regulation of TBX3 is poorly understood in the muscle lineage. Polycomb Repressive Complex 2 (PRC2), a gene silencing complex, acts to methylate histone H3 lysine 27 (H3K27me) of target gene promoters. The catalytic subunit of PRC2, EZH2, is up-regulated in RMS and data from our lab has shown that depletion of EZH2 up-regulated TBX3 and down-regulated TBX2 in C2C12 cells, an immortalized murine cell line. The hypothesis of this project was that there would be a PRC2-TBX3-TBX2 axis in RMS cells. To examine if TBX3 represses TBX2, TBX3 was transiently expressed in RMS cells representing both subtypes of RMS and we found that TBX2 was downregulated in each cell line. In a stable RH30 cell line with ectopic TBX3, TBX2 was down-regulated and PTEN expression was up-regulated. To determine if TBX2 repression by TBX3 was direct, a TBX3 ChIP assay was performed on the TBX2 promoter as well as the PTEN promoter. We found a strong enrichment of TBX3 on the TBX2 promoter but not on the PTEN promoter. Accordingly, we also observed that TBX3 over-expression impaired tumorigenesis through reduced cell proliferation, migration, and anchorage dependent growth. Also, we found that a stable RD cell line with ectopic TBX3 could promote differentiation, strongly suggesting that these results could have therapeutic value. Next, a shEZH2 plasmid was transfected into RMS cell lines ask if TBX3 was regulated by the PRC2 complex as we had observed in C2C12 cells. Just as we hypothesized, TBX3 was up-regulated and TBX2 was down-regulated. Similar to the previous TBX3 overexpression experiments, the EZH2 depleted RMS cell lines also showed decreased cell proliferation and migration rate. Also, an EZH2 knock down treatment induced differentiation in RMS cell lines. Therefore, understanding this potent regulation axis could provide an excellent opportunity for treatment of RMS cancer in the future.

Living cells maintain stable transcriptional programs while exhibiting plasticity that allows them to respond to environmental stimuli. The Polycomb repressive complex 2 (PRC2) is a key regulator of chromatin structure that maintains gene silencing through the methylation of histone H3 on lysine 27 (H3K27me), establishing chromatin-based memory. Two variants of PRC2 are present in mammalian cells, PRC2-EZH2 which is predominantly present in differentiating cells, and PRC2-EZH1 that predominates in post-mitotic tissues. PRC2-EZH1α/β pathway is involved in the response of muscle cells to oxidative stress. Atrophied muscle cells respond to oxidative stress by enabling the nuclear translocation of EED and its assembly with EZH1α and SUZ12. Here we prove that the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) long noncoding RNA (lncRNA) is required for the assembly of PRC2-EZH1 components. The absence of MALAT1 significantly decreased the association between EED and EZH1α proteins. Biochemical analysis shows that the presence of MALAT1 increases the enzymatic activity of PRC2-EZH1 in vitro. In addition, we show that the simultaneous expression of PRC2 core components is necessary for their solubility. The successful expression of PRC2 proteins enables the execution of several downstream experiments, which will further explain the nature of the interplay between MALAT1 and PRC2.

No description available.

The developing brain is highly sensitive to environmental influences. Unfavorable nutrition is one kind of stress that can cause acute metabolic disorders during the neonatal period [1,2,3] and severe diseases in later life [4,5]. These early life experiences occurring during heightened periods of brain plasticity help determine the lifelong structural and functional aspects of brain and behavior. In humans, for example, weight gain during the first week of life increased the propensity for developing obesity several decades later [5]. This susceptibility is, if not all, related to the dynamic reversible epigenetic imprints left on the histones [6,7,8], especially during the prenatal and postpartum period [9]. Histones are highly dynamic and responsive towards environmental stress [10,11]. Through covalent modification of the histone tail, histones are able to direct DNA scaffolding and regulate gene expression [10,12]. Thus far, various types of post translational modifications have been identified on various histones tails [12]. Among them, the methylation and acetylation on lysine residue (K) 27 on histone 3 (H3) has been tightly linked to gene repression [13,14] and activation [15], respectively. EZh2 (enhancer of zeste 2) in the polycomb repressive complex 2 (PRC2) is the only methyltransferase that has been linked to catalyze this methylation reaction. In addition, SUZ (suppressor of zeste) and EED (embryonic ectoderm development) are two other key proteins in PRC2 function core that help EZH2. As previous reported, increased H3K27 methylation was monitored after fasting stress during neonatal period in chicks' paraventricular nucleus (PVN). In this study, we investigated the detailed mechanism behind changes in H3K27 methylation following fasting stress. After 24 hours fasting on 3 days-of-age (D3), chicks exhibited elevated mRNA levels of PRC2 key components, including EZH2, SUZ and EED, in the PVN on D4. Western blots confirmed this finding by showing increased global methylation status at the H3K27 site in the PVN on D4. In addition, until 38 days post fasting, SUZ and EZH2 remained inhibited. A newly identified anorexigenic factor, Brain-derived neurotrophic factor (BDNF), was used as an example of multiple hormones expressed in PVN to verify this finding. Both BDNF protein and mRNA exhibited compatible changes to global changes of tri- (me3) and di-methylated (me2) H327. Furthermore, by using chromatin immunoprecipitation assays (ChIP), we were able to monitor the changes of H3K27me2/me3 deposition along the Bdnf gene. Fasting significantly increased H3K27me2/me3 as well as EZH2 at the Bdnf's promoter, transcription start site and 3'-untranslated region. These data show that fasting stress during the early life period could leave epigenetic imprinting in PVN for a long time. Next, we tried to understand the function of this epigenetic imprinting in the chicks' PVN. Thus, we compared naive chicks (never fasted) to chicks that received either a single 24 hour fast on D3 or two 24 hour fast on both D3 and 10 days-of-age (D10). We found that the D3 fasted group significantly increased the level of PRC2 key components and its product H3K27me2/me3 compared to the naive group. However, D3 fasting and D10 fasting together decreased the surges of H3K27me2/me3, SUZ and EED (not EZH2) compared to the naive group. We called this phenomenon "epigenetic memory". The Western blot, qPCR and CHIP assay results from BDNF all confirmed the existence of "epigenetic memory" for PRC2. These data suggested that fasting stress during the early period of brain development could leave long term epigenetic modifications in neurons. These changes could be beneficial to the body, which keeps homeostasis of inner environment and prevent massive response to future same stress. The EZH2 protein was knocked down and the H3K27 methylation status changes were monitored after applying the same treatment. We first confirmed that EZH2 antisense oligonucleotides (5.5 ug), but not EZH2 siRNA and artificial cerebrospinal fluid (ACSF), inhibit EZH2 protein by 86 % in the PVN. Then, on D3, chicks were subjected to a 24 hour fasting stress (D3-fasting) post either EZH2 antisense or ACSF injection. The EZH2 antisense blocked the surge of both EZH2 mRNA and H3K27 methylation after D3-fasting. At the same time, BDNF exhibited elevated expression levels and less methylated H3K27 deposition along the Bdnf gene. In addition, we were also interested in the changes of "epigenetic memory" post EZH2 antisense injection. We found that after EZH2 antisense injection, chicks' PVN no longer exhibited any "epigenetic memory" to repetitive fasting stress. While EZH2 mRNA was constantly inhibited, SUZ, EED and H3K27me2/3 levels were unpredictable. These findings suggested that neurons in the PVN utilized PRC2 as a major H3K27 methylation tool. Knockdown of EZH2 in the PRC2 impaired the proper response in PVN to fasting stress and PVN's ability to acclimate to repetitive fasting stresses. Thus, EZH2 is an important H3K27 methyltransferase inside chicken hypothalamus to maintain homeostasis. In conclusion, fasting stress during the early life period could leave epigenetic markers on chromosomes of neurons in the feeding regulation center. These epigenetic markers will be left on chromosomes for a long period of time and have a beneficial role in keeping homeostasis when individuals face future fasting stress again. H3K27 methylation is one of these epigenetic markers and inhibits expression of various genes inside neurons. EZH2 is so far the only detected methyltransferases for H3K27 that form the PRC2. Thus EZH2 plays a key function in the body's response to fasting. / Ph. D.

La neurofibromatose de type 1 (NF1) est un syndrome de prédisposition tumorale causée par une mutation perte-de-fonction du gène suppresseur de tumeurs NF1. Près de la moitié des patients atteints de NF1 développent un type de tumeurs bénignes des gaines des nerfs périphériques appelés neurofibromes plexiformes. Ces tumeurs sont majoritairement constituées de cellules de Schwann présentant une inactivation somatique du deuxième allèle NF1. Les neurofibromes plexiformes peuvent se transformer en tumeurs malignes dénommées MPNST (Malignant Peripheral Nerve Sheath Tumor) qui sont des sarcomes extrêmement agressifs, résistants aux thérapies actuelles et représentant la première cause de mortalité des patients NF1. A ce jour, les acteurs à l’origine de cette transformation maligne ne sont pas clairement établis. Leur identification représente donc un enjeu majeur pour une prise en charge appropriée des patients et le développement de nouvelles molécules thérapeutiques. Dans ce contexte, le travail mené au cours de ma thèse a eu pour objectifs la recherche et la caractérisation de nouvelles voies de signalisations impliquées dans la tumorigenèse NF1. D’une part, une approche orientée par les travaux antérieurs au laboratoire a permis de montrer l’implication de la voie WNT dans la tumorigenèse NF1. D’autre part, une approche génomique plus large a conduit à la mise en évidence de l’inactivation du répresseur transcriptionnel PRC2 (Polycomb Repressive Complex 2) dans près de la moitié des MPNST. La génération de modèles cellulaires in vitro a facilité l’exploration des gènes surexprimés lors de la perte de fonction du PRC2. Elle a également permis d’entreprendre un crible lentiCRISPR pan-génomique à la recherche des gènes essentiels à la survie des cellules tumorales mutées pour le PRC2. / Neurofibromatosis type 1 (NF1) is a tumor predisposition syndrome caused by loss-offunction mutations in the NF1 tumor suppressor gene. Almost half of NF1 patients develop a specific type of benign peripheral nerve sheath tumor called plexiform neurofibromas. These tumors are mainly composed of Schwann cells in which the second NF1 allele is inactivated. Plexiform neurofibromas can give rise to malignant tumors called MPNST that are extremely aggressive sarcomas, resistant to therapy and which represents the first cause of early demise of NF1 patients. The molecular mechanisms underlying this malignant transformation remain enigmatic. Their identification is crucial for appropriate management of NF1 patients and development of new therapies. The goal of my PhD was to identify and characterize new signaling pathways involved in NF1 tumorigenesis. On the one hand, we highlighted the involvement of WNT pathway in NF1 tumorigenesis. On the other hand, a larger genomic approach led to the identification of the transcriptional repressor PRC2 (Polycomb Repressive Complex 2) inactivation in almost half of MPNST. We have generated various cell models, which facilitated the exploration of genes aberrantly expressed consequently to PRC2 loss-offunction. These models also allowed performing a pan-genomic lentiCRISPR screen searching for essential genes for PRC2-mutated tumor cells survival.

Polycomb Proteins (PcG) are chromatin proteins that control the maintenance of “transcriptional memory” and cell identity by fixing the repressed state of developmentally regulated genes. This function has been linked to interaction with RNA moieties, in particular long non-coding RNAs (lncRNAs). However, specificity of PcG-RNA interactions has been controversial (Beltran et al., 2016; Chen Davidovich, Leon Zheng, Karen J. Goodrich, & Thomas R. Cech, 2013). In this study we took advantage of recent work published from our lab reporting about a novel and reversible mechanism regulating genome wide Ezh1-PRC2 activation in mouse skeletal muscle cells in response to atrophic stress (Bodega et al., 2017). Using this physiological, in vivo tool we could identify a functional dynamic crosstalk between Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) and PRC2-Ezh1 complex. By combining immuno-fluorescence, biochemistry, epigenomics, ChIRP, DNA and RNA immunoprecipitation we identified a novel pathway in which Malat1 plays a role in compartmentalization, assembly and activity of PRC2 in chromatin, allowing epigenetic plastic response to atrophic stress and recovery. We conclude that Malat1 is an essential partner for PRC2-Ezh1 adaptive function in skeletal muscle cells.

Down syndrome (DS) is caused by the triplication of chromosome 21, but science is still investigating the precise mechanisms by which this results in the various phenotypes, such as anatomical abnormalities, intellectual deficits, and early development of Alzheimer’s disease (AD). The global changes in transcriptional activity and the altered expression of genes not transcribed from chromosome 21 point to changes to the epigenetic landscape. One of the candidate genes for this global gene dysregulation is High Mobility Group Nucleosome Binding Domain 1 (HMGN1) which is triplicated in DS. While investigating DS-associated B-cell acute lymphoblastic leukemia (B-ALL), researchers found the triplication of HMGN1 alone led to many of the same transcriptional and phenotypic changes that marked DS-associated B-cells from a mouse model with all 31 genes orthologous to human chromosome 21 genes triplicated. Amongst the pathways most affected by triplication, enrichment was greatest for targets of the Polycomb Repressive Complex 2 (PRC2) and sites of the transcriptionally repressive mark it catalyzes, H3K27me3. HMGN1 instead, promotes transcriptional activation and its overexpression leads to a global increase in RNA transcript levels. Therefore, overexpression of HMGN1 in DS may cause an increase in transcriptional activity and prevent the silencing of genes normally silenced by PRC2, with downstream effects on neurogenesis and gliogenesis, abnormal cellular migration, and deviant developmental timing that result in known DS phenotypes. With this hypothesis, we first wanted to quantify the levels of acetylation versus methylation at H3K27 in trisomy 21 induced pluripotent stem (iPS) cell-derived cellular models: neural progenitor cells (NPCs) and cortical organoids and to determine if there are measurable differences between the genotypes. We found a decrease in H3K27me3 in 130-day-old organoids, but not in NPCs. No changes were detected in the levels of H3K27ac. With the high comorbidity between DS and AD, and changes to the epigenome found in both diseases, we wondered whether there were specific alterations at H3K27 in DS-AD. To determine this, we performed an analysis of human postmortem brain tissue from individuals with DS-AD, AD, and control and quantified H3K27me3 and H3K27ac marks. Our data indicated that there are marginally significant changes in H3K27me3 that are unique to DS-AD as compared to control and AD samples. Encouraged by this data, we next measured gene expression levels of specific PRC2 target genes increased in trisomy. Our goal was to identify the causative relationship between the increased expression of HMGN1 in trisomy and upregulation of specific PRC2 target genes with known brain-related functions. We found that enhanced expression of particular PRC2 target genes in trisomic cells could be normalized with the short-hairpin RNA (shRNA)-induced knockdown of HMGN1 expression in trisomic NPCs. This implicates HMGN1 overexpression in DS in the dysregulation and overexpression of particular genes involved in morphogenesis, neurogenesis, neuronal migration, apoptosis, and cell viability through the antagonism of the PRC2 activity. We provided novel evidence for a possible mechanism for the cellular, molecular, and transcriptomic changes originating from the triplication of HMGN1 that can potentially lead to DS-related phenotypes such as intellectual disability and AD-related pathology. Furthermore, our findings suggest a possible therapeutic avenue to mitigate these phenotypes by regulating HMGN1 expression. Taken together, our work is the first to causatively connect HMGN1-induced epigenetic changes to DS-related brain cell phenotypes and to point out to a potential approach for correcting them. .

No description available.

Les cortico-surrénalomes (CCS) sont considérés comme des tumeurs malignes endocriniennes rares, associées à un pronostic sombre. Les trois mécanismes moléculaires les plus fréquemment altérés dans les CCS comprennent les mutations inactivatrices du gène suppresseur de tumeur TP53,la surexpression de IGF-II et l'activation constitutive de la voie de signalisation Wnt/β-caténine. En utilisant des modèles de souris transgéniques, nous avons montré que ces altérations, même combinées, ne sont pas suffisantes pour permettre la progression maligne.Nous avons précédemment identifié l'histone méthyltransférase EZH2 comme le modificateur d'histone le plus dérégulé dans les CCS. Nous avons également montré que sa surexpression est associée à une progression tumorale et à un mauvais pronostic. Cependant, les mécanismes sous-jacents de cette agressivité sont largement inconnus. Dans cette étude, nous avons cherché à identifier les gènes cibles de EZH2 dans les CCS, qui sont soient activés, soient réprimés. Ainsi, nous avons effectué une analyse bio-informatique des données du transcriptome de trois cohortes de patients porteurs de CCS. L’analyse montre une forte corrélation entre la surexpression de EZH2 et les gènes régulés positivement, suggérant un rôle majeur d’inducteur transcriptionnel de EZH2 dans les CCS. Nous avons montré que cette activité positive repose sur une interaction entre EZH2 et E2F1, qui entraîne la surexpression de gènes impliqués dans la régulation du cycle cellulaire et la mitose tels que RRM2,PTTG1 et PRC1/ASE1. Nous avons montré que l'inhibition de RRM2 par ARN interférent ou traitement pharmacologique avec le GW8510 inhibe la croissance cellulaire, la capacité à combler les blessures, la croissance clonogénique, la migration et induit l'apoptose des cellules H295R en culture. En revanche, l'expression du facteur pro-apoptotique NOV/CCN3 est diminuée dans les CCS, ce qui est corrélé au développement de tumeurs agressives. Nos analyses moléculaires montrent que l'inhibition de EZH2 augmente l'expression de NOV/CCN3, suggérant que la surexpression de EZH2 pourrait favoriser la progression maligne des CCS en inhibant les stimulateurs de l'apoptose. Le facteur NOV a déjà été identifié comme cible négative du récepteur nucléaire SF1 dans les cellules du CCS, bien que les mécanismes moléculaires à l'origine de cette inhibition n'aient pas été identifiés. De manière intéressante, dans le cancer de la prostate, l'expression de NOV est inhibée par le récepteur des androgènes AR, grâce au recrutement de EZH2 qui pose la marque répressive H3K27me3. Nous avons pu identifier une coopération similaire entre SF1 et EZH2 pour réprimer l'expression de NOV et bloquer ainsi l'apoptose dans les CCS.Au total, ces résultats identifient SF1 et E2F1 comme deux partenaires indépendants de EZH2, induisant la répression de facteurs pro-apoptotiques et l'activation des gènes du cycle cellulaire respectivement, conduisant ainsi à l'agressivité des CCS. / Adrenocortical carcinomas (ACC) are regarded as rare endocrinemalignancies associated with dismal prognosis. The three common molecularmechanisms predominantly altered in ACC include inactivating mutations of theTP53 tumor suppressor gene, overexpression of IGF-II and constitutive activationof the Wnt/β-catenin signaling pathway. Using transgenic mouse models, wehave shown that these alterations, even when combined together, were notsufficient to induce malignant progression.We previously identified the histone methyltransferase EZH2 as the mostderegulated histone modifier in ACC. We have also shown that its overexpressionis associated with tumor progression and poor prognosis. Yet, the mechanismsunderlying this aggressiveness are largely unknown. Here, we aimed to identifyEZH2 target genes in ACC, which are either activated or repressed.Thus, we conducted a bio-informatics analysis of transcriptome data fromthree cohorts of ACC patients. The analysis showed a strong correlation betweenhighly expressed EZH2 and positively regulated genes suggesting a major role of‘transcriptional inducer‘ for EZH2 in ACC. We have shown that this positiveactivity relies on an interaction between EZH2 and E2F1 that results in theupregulation of genes implicated in cell cycle regulation and mitosis such asRRM2, PTTG1 and PRC1/ASE1. We showed that Inhibition of RRM2 by RNAinterference or pharmacological treatment with GW8510 inhibits cellular growth,wound healing, clonogenic growth, migration and induces apoptosis of H295Rcells in culture.In contrast, expression of the pro-apoptotic factor NOV/CCN3 is decreasedin ACC, which is correlated with development of aggressive tumours. Ourmolecular analyses show that EZH2 inhibition increases expression ofNOV/CCN3, suggesting that EZH2 overexpression may also favour malignantprogression in ACC by inhibition of apoptosis stimulators. NOV has previouslybeen identified as a negative target of the nuclear receptor SF1 in ACC cells,although the molecular mechanisms underlying this inhibition were unidentified.Interestingly, in prostate cancer, NOV expression is inhibited by the androgenreceptor, through recruitment of EZH2 and deposition of the H3K27me3 mark.We have been able to identify a similar cooperation between SF1 and EZH2 tosuppress NOV expression and block apoptosis in ACC.Altogether, these findings identifiy SF1 and E2F1 as two independentpartners of EZH2, inducing repression of proapoptotic factors, and activation ofcell cycle genes respectively, thus leading to aggressiveness of ACC.