During tissues development multipotent progenitor cells establish tissue-specific gene expression programmes, leading to differentiation into specialized cell types. It has been previously shown that the transcription factor p63, a master regulator of skin development, controls the expression of adhesion molecules and essential cytoskeleton components. It has also been shown that p63 plays an important role in establishing distinct three-dimensional conformations in the Epidermal Differentiation Complex (EDC) locus (Fessing et al., 2011). Here we show that in p63-null mice about 32% of keratinocytes showed altered nuclear morphology. Alterations in the nuclear shape were accompanied by decreased expression of nuclear lamins (Lamin A/C and Lamin B1), proteins of the LINC complex (Sun-1, nesprin-2/3) and Plectin. Plectin links components of the nuclear envelope (nesprin-3) with cytoskeleton and ChIP-qPCR assay with adult epidermal keratinocytes showed p63 binding to the consensus binding sequences on Plectin 1c, Sun-1 and Nesprin-3 promoters. As a possible consequence of the altered expression of nuclear lamins and nuclear envelope-associated proteins, changes in heterochromatin distribution as well as decrease of the expression of several polycomb proteins (Ezh2, Ring1B, Cbx4) has been observed in p63-null keratinocytes. Moreover, recent data in our lab have showed that p63 directly regulates Cbx4, a component of the polycomb PRC1 complex. Here we show that mice lacking Cbx4 displayed a skin phenotype, which partially resembles the one observed in p63-null mice with reduced epidermal thickness and keratinocyte proliferation. All together these data demonstrate that p63-regulated gene expression program in epidermal keratinocytes includes not only genes encoding adhesion molecules, cytoskeleton proteins (cytokeratins) and chromatin remodelling factors (Satb1, Brg1), but also polycomb proteins and components of the nuclear envelope, suggesting the existence of a functional link between cytoskeleton, nuclear architecture and three dimensional nuclear organization. Other proteins important for proper epidermal development and stratification, are cytokeratins. Here, we show that keratin genes play an essential role in spatial organization of other lineage-specific genes in keratinocytes during epidermal development. In fact, ablation of keratin type II locus from chromosome 15 in epidermal keratinocytes led to changes in the genomic organization with increased distance between the Loricrin gene located on chromosome 3 as well as between Satb1 gene located on chromosome 17 and keratin type II locus, resulting in a more peripheral localization of these genes in the nucleus. As a possible consequence of their peripheral localization, reduced expression of Loricrin and Satb1 has also been observed in keratins type II-deficient mice. These findings together with recent circularized chromosome conformation capture (4C) data, strongly suggest that keratin 5, Loricrin and Satb1 are part of the same interactome, which is required for the proper expression of these genes and proper epidermal development and epidermal barrier formation. Taken together these data suggest that higher order chromatin remodelling and spatial organization of genes in the nucleus are important for the establishment of lineage-specific differentiation programs in epidermal progenitor cells. These data provide an important background for further analyses of nuclear architecture in the alterations of epidermal differentiation, seen in pathological conditions, such as psoriasis and epithelial skin cancers.

A central problem in the bioinformatics is to find the binding sites for regulatory motifs. This is a challenging problem that leads us to a platform to apply a variety of data mining methods. In the efforts described here, a combined motif discovery method that uses mutual information and Gibbs sampling was developed. A new scoring schema was introduced with mutual information and joint information content involved. Simulated tempering was embedded into classic Gibbs sampling to avoid local optima. This method was applied to the 18 pieces DNA sequences containing CRP binding sites validated by Stormo and the results were compared with Bioprospector. Based on the results, the new scoring schema can get over the defect that the basic model PWM only contains single positioin information. Simulated tempering proved to be an adaptive adjustment of the search strategy and showed a much increased resistance to local optima.

3 ABSTRACT LEDGF/p75 protein is a human transcriptional co-activator and epigenetic reader associated with transcriptionally active chromatin. It is crucial for HIV integration and MLL1 fusion-driven leukemia development. Interactions of LEDGF/p75 with HIV integrase (HIV IN) and MLL1-menin complex are considered an attractive therapeutic target for drug development. LEDGF/p75 interacts with both HIV IN and MLL1-menin complex through its integrase binding domain (IBD). While the pathophysiological interactions of LEDGF/p75 IBD were intensively studied, little was known about the physiological ones. In addition to HIV IN and MLL1, the LEDGF/p75 IBD also interacts with JPO2, PogZ, ASK and MLL2. In search for specific inhibitors of LEDGF/p75 IBD interaction with HIV IN and MLL1, it is essential to obtain detailed information about its interactions with all binding partners. The IBD-MLL1-menin complex has been structurally characterized, but only partially. Using NMR spectroscopy, we identified and mapped a novel part of the IBD-MLL1 interface. This additional interface is able to maintain the interaction between LEDGF/p75 and MLL1 even without the presence of menin, which was considered necessary. Moreover, colony forming assays of primary leukemic blasts revealed that this additional interface is essential for...

A transição endotélio-mesenquimal (EndMT) é uma forma especializada da transição epitéliomesenquimal (EMT) e é caracterizada pela alteração da morfologia celular para um formato fibroblastoide, perda da expressão dos marcadores endoteliais e ganho da expressão dos marcadores mesenquimais, bem como a aquisição de propriedades invasivas e migratórias. Entretanto, o mecanismo molecular envolvido nesse processo ainda não está totalmente elucidado. O objetivo desse trabalho foi avaliar os fatores indutores da EMT em células endoteliais (CEs) de fontes distintas por meio da superexpressão do fator de transcrição SNAIL e do tratamento com TGF-?2, bem como identificar os mecanismos moleculares envolvidos nesse processo. Para tal, as linhagens de CE da artéria pulmonar (HPAEC), pool de CE primária de veia de cordão umbilical (PHUVEC), CE da aorta (PAEC) e CE da artéria coronária (CAEC) foram induzidas em três condições distintas: I) TGF-?2; II) superexpressão do fator de transcrição SNAIL; III) superexpressão do fator de transcrição SNAIL associado ao tratamento com TGF-?2 (SNAIL+TGF-?2). Após a indução, a expressão dos genes relacionados com a EndMT foi analisada por PCR em tempo real (qPCR) e as CAECs foram as células que apresentaram maior mudança no perfil de expressão gênica, no qual o grupo SNAIL+TGF-?2 apresentou um aumento dos marcadores mesenquimal FN1, SM22, CNN1 e CD90. O grupo SNAIL+TGF-?2 também mostrou uma diminuição dos marcadores endoteliais CD31 e CDH5 por Western blot. Em seguida, a técnica de microarray foi realizada nas CAECs induzidas à EndMT e as análises revelaram um dendrograma cujo perfil mostrou que SNAIL e SNAIL+TGF-?2 se agrupam separadamente das outras condições. Os dados de microarray resultaram em uma rede na qual os genes mesenquimais COL1A1, COL1A2, FN1 e CNN1 estavam aumentados no grupo SNAIL+TGF-?2 comparado com o grupo controle. Os genes diferencialmente expressos entre a análise CT vs. SNAIL+TGF-?2 foram analisados quanto a participação em vias canônicas e a via de regulação da EMT foi uma das mais representadas, a qual inclui a via de sinalização Notch e Wnt. Nos dados de microarray, NOTCH3 e WNT5B estavam superexpressos no grupo SNAIL+TGF-?2 comparado com o controle. Sabendo que Wnt5b pode inibir a via ?-catenina, a expressão de NOTCH3, WNT5B e ?-CATENINA foi avaliada por qPCR e a expressão de NOTCH3 e WNT5B confirmou os dados do microarray e nenhuma diferença estatística foi observada na expressão de ?- CATENINA. Ainda, as CAECs induzidas foram submetidas ao ensaio de migração e de capacidade de formação de estruturas semelhantes a capilares. Foi observado que as CAECSNAIL+ TGF-?2 migraram significativamente comparadas com as outras condições e nenhuma das células induzidas (TGF-?2, SNAIL e SNAIL+TGF-?2) foram capazes de formar estruturas semelhantes a capilares. Alguns microRNAs foram selecionados e avaliados por qPCR. O miR-let7a foi significativamente expresso no grupo SNAIL e SNAIL+TGF-?2. O ensaio de perda e ganho de função do miR-let7a foi realizado, entretanto, a repressão ou a indução do miR-let7a não alterou a EndMT. Esses resultados sugerem que as CEs de fontes anatômicas distintas apresentam respostas diferentes quando estimuladas a sofrerem EndMT. Ademais, a associação entre SNAIL+TGF-?2 é um potente indutor para EndMT e essa indução pode ser mediada pelas vias de sinalização Notch e Wnt não canônica. / Endothelial-mesenchymal transition (EndMT) is a specialized form of epithelialmesenchymal transition (EMT) which is characterized by changes in cell morphology as a fibroblastoid conversion, expression of endothelial markers decreased, expression of mesenchymal markers increased and acquirement of invasive and migratory properties. However, the molecular mechanism associated with this process is not completely elucidated. The aim of this study was to evaluate the EMT-inducing factors in the endothelial cells (ECs) from different sources through the overexpression of the transcription factor SNAIL and through the treatment with TGF-?2, as well as to identify the molecular mechanisms involved in EndMT. For this purpose, primary pulmonary artery EC (HPAEC), primary pooled umbilical vein EC (PHUVEC), primary aortic EC (PAEC), primary coronary artery EC (CAEC) lineages were induced under three distinct conditions: I) TGF-?2; II) ectopic expression of SNAIL; III) ectopic expression of SNAIL associated with TGF-?2 (SNAIL+TGF- ?2). After the EndMT induction, the expression of the genes associated with EndMT was analyzed by Real time PCR (qPCR) and CAECs showed the most prominent alterations on their gene expression profile which showed that SNAIL+TGF-?2 group presented an increase of mesenchymal markers FN1, SM22, CNN1, and CD90 expression. CAEC-SNAIL+TGF-?2 group also showed a decrease of endothelial markers CD31 and CDH5 by western blot. Then, microarray was performed in CAECs after EndMT induction and hierarchical clustering analysis showed that the ectopic expression of SNAIL and SNAIL+TGF-?2 clustered separately from the other conditions. Microarray data resulted in a network which presented an upregulation of the mesenchymal genes such as COL1A1, COL1A2, FN1, and CNN1 in the CAEC-SNAIL+TGF-?2 compared to control cells. We analyzed the canonical pathways related to the differentially regulated genes between CAEC- SNAIL+TGF-?2 and control cells and the regulation of EMT pathways was the most represented, which includes Notch and Wnt signaling pathway. In the microarray data, NOTCH3 and WNT5B were overexpressed in CAEC-SNAIL+TGF-?2 compared to control. It is known that Wnt5b might inhibit the ?- catenin pathway. Therefore, NOTCH3, WNT5B and ?-CATENIN gene expression were analyzed by qPCR. NOTCH3 and WNT5B gene expression confirmed the microarray data and no statistical difference were observed in ?-CATENIN expression. Moreover, all the CAECs conditions were subjected to scratch migration assay and the formation of capillary-like structures assay. CAEC-SNAIL+TGF-?2 had a significant migration compared to other conditions and the three EndMT inductions (TGF-?2, SNAIL, and SNAIL+TGF-?2) were not able to form capillary-like structures. Some microRNAs were selected and evaluated by qPCR. The miR-let7a was significantly expressed in the SNAIL and SNAIL+TGF-?2 groups. The assay of gain or loss of function of miR-let7a was realized; however, the repression or induction of miR-let7a did not change the EndMT. These results suggest that endothelial cells from distinct anatomical sources have different responses when stimulated to undergo the EndMT. Moreover, the association between SNAIL+TGF-?2 is a potent inductor for EndMT and this induction can be mediated by Notch and non-canonical Wnt signaling pathway activation.

La synthèse d’ARN messagers résulte d’une cascade d’évènements temporellement et spatialement orchestrée. Au moment de l’initiation de la transcription, divers facteurs tels que les facteurs généraux de transcription, le complexe Médiateur, des co-activateurs, des facteurs de remodelage de la chromatine ainsi que l’ARN polymérase II sont recrutés au niveau de la région promotrice du gène. Certains facteurs de la voie NER de réparation de l’ADN sont également recrutés. En utilisant des cellules de patients porteurs de mutations dans les gènes MED12 (sous-unité du Médiateur) ou XPC (facteur initiant la voie NER), nous avons pu étudier le rôle de ces protéines dans la transcription. Les patients MED12 sont notamment caractérisés par une lourde déficience intellectuelle et des malformations congénitales. Nous avons montré que MED12 est impliqué dans le contrôle de certains gènes de réponse immédiate comme JUN, qui contribue notamment au développent et à la plasticité cérébrale. L’expression de ce dernier est affectée par les mutations de MED12, mais différemment en fonction de la position de la mutation, apportant une possible indication sur l’origine des variations phénotypiques observées chez les patients. En parallèle, les patients XPC se caractérisent par une forte photosensibilité. Nous avons montré que la protéine XPC, en collaboration avec le facteur E2F1, est impliquée dans le recrutement de l’histone acetyl-transférase GCN5 au niveau du promoteur d’un certain nombre de gènes. Cette dernière permet notamment l’a modification de l’environnement chromatinien, en coopération avec le facteur général de transcription TFIIH et participe ainsi à l’initiation de la transcription. En plus d’approfondir la compréhension des mécanismes régissant la transcription, ces résultats ont permis de mieux comprendre l’étiologie des maladies associées aux mutations. / The synthesis of messenger RNA is a highly regulated process. During transcription initiation, a large number of proteins are recruited to gene promoter, including the RNA polymerase II, general transcription factors, co-activators, chromatin remodellers and the Mediator complex. Some DNA repair factors from the NER pathway are also recruited. Using cells derived from patients bearing mutations in either MED12 gene or XPC gene, we studied the roles of such proteins in transcription. MED12 patients are mostly characterised by intellectual disability and developmental delay. We showed that MED12 is implicated in the transcription regulation of immediate early genes like JUN, known for its role in neurological development and neuronal plasticity. JUN expression is markedly altered by MED12 mutations. We also showed that the position of the mutation influences this alteration, bringing possible explanation for inter-patients symptom variability. Meanwhile, XPC patients are mostly characterized by photosensitivity. We showed that XPC protein, which engages one of the NER pathways, is implicated in chromatin post-translational modification. Together with E2F1, it helps the recruitment of GCN5 acetyl-transferase to promoter of a certain set of genes. On the promoter, GCN5 notably cooperates with TFIIH to modify the chromatin environment during transcription initiation. In addition to help the comprehension of the transcription mechanisms, these results bring knew insight into the aetiology of mutations associated diseases.

A carcinogênese oral é um processo multifásico, onde componentes genéticos levam a desregulação de vias de sinalização celular que controlam funções celulares básicas, como divisão, diferenciação e morte celular. Uma das maneiras de compreender a natureza biológica dos cânceres, além do curso clínico, é através do entendimento do processo de progressão e metástase destas neoplasias. Este estudo teve como objetivo avaliar a participação da proteína Twist no desenvolvimento e progressão dos carcinomas epidermóides orais. Com tal proposta, também foi avaliada a participação das proteínas caderina-E e p-Akt, e sua possível interação com Twist no processo de carcinogênese oral. O trabalho em questão analisou a expressão imuno-histoquímica destas proteínas em 30 espécimes de displasia oral, 20 de carcinoma epidermóide oral e 10 de mucosa oral normal, e avaliou também a possível inter-relação dessas proteínas em linhagens derivadas de carcinoma epidermóide de cabeça e pescoço por meio dos ensaios de Western Blotting e imunofluorescência. Os resultados deste estudo demonstraram uma relação inversamente proporcional entre Twist e caderina-E desde os estágios mais precoces da carcinogênese oral. Tal afirmação baseou-se na presença de diferenças significantes entre a expressão imuno-histoquímica de Twist e Caderina-E na amostras de epitélio oral, epitélio displásico e nos espécimes de carcinoma epidermóide oral. Adicionalmente, foi observada a relação inversa entre Twist e a Caderina-E nas linhagens de carcinoma epidermóide de cabeça e pescoço, sendo este evento constatado pelo decréscimo nos níveis protéicos da Caderina-E frente a uma elevação de Twist. Estes resultados sugerem um importante papel de Twist na progressão do carcinoma epidermóide oral, e juntamente com a Caderina-E, pode representar um relevante marcador biológico do câncer oral. / The oral carcinogenesis is a multi-stage process, where genetic components leads to deregulation of cell signaling pathways that control basic cellular functions such as division, differentiation and cell death. One way to understand the biological nature of cancers, besides the clinical course, is through understanding the process of progression and metastasis of these neoplasms. This study aimed to evaluate the role of Twist protein in the development and progression of oral squamous cell carcinomas. With this proposal, was also evaluated the involvement of E-cadherin and p-Akt proteins, and its possible interaction with Twist in the process of oral carcinogenesis. The work in question examined the immunohistochemical expression of these proteins in 30 specimens of oral dysplasia, 20 oral squamous cell carcinoma and 10 normal oral mucosa, and also evaluated the possible interrelationship of these proteins in lines derived from squamous cell carcinoma of head and neck by means of Western blotting assays and immunofluorescence. The results of this study showed an inverse relationship between Twist and E-cadherin since the earliest stages of oral carcinogenesis. These results were based on the presence of significant differences between the immunohistochemical expression of Twist and ECadherin in samples of oral epithelium, dysplastic epithelium and in specimens of oral squamous cell carcinoma. In addition, we observed the inverse relationship between Twist and E-Cadherin in the lines of squamous cell carcinoma of head and neck; this event was evidenced by the decrease in protein levels of E-Cadherin forward to a high of Twist. These results suggest an important role of Twist in the progression of oral squamous cell carcinoma, and along with E-cadherin may represent a relevant biomarker of oral cancer.

Les lymphocytes T CD4+ auxiliaires ou T helper en anglais sont capables de soutenir une grande diversité de fonctions grâce à leur capacité à se différencier en différents sous-types effecteurs en fonction de l’antigène rencontré et de l’environnement cytokinique dans lequel ils se trouvent. Les connaissances actuelles sur la différenciation des cellules T helper mettent en avant l’existence de réseaux transcriptionnels particulièrement complexes et spécifiques à chaque sous-ensemble T helper. En 2008, les cellules T CD4 sécrétrices d’IL-9 (Th9) sont identifiées comme un nouveau sous-type de cellules T helper. Différenciées en présence d’IL-4 et TGF-β, les cellules Th9 sécrètent de l’IL-9 et de l’IL-21, et contribuent au développement de maladies auto-immunes et allergiques. Les lymphocytes Th9 présentent également des propriétés anti-tumorales particulièrement intéressantes.Le réseau transcriptionnel des cellules Th9 résulte d’un équilibre entre les voies de signalisation induites par les différentes cytokines nécessaires à sa polarisation. L’IL-4 permet l’activation de STAT6 et l’expression de GATA3 et IRF4, tandis que le TGF-β conduit à l’activation de la voie des Smad et l’expression du facteur PU.1. Le module transcriptionnel IRF4/BATF ainsi que le facteur PU.1 sont des messagers indispensables au développement des cellules Th9 et à la sécrétion d’IL-9.IRF8 est un facteur de transcription critique pour le développement des cellules myéloïdes et des lymphocytes B. Récemment, il est apparu qu’IRF8 était impliqué dans la polarisation de sous-ensembles T helper. En effet, IRF8 limite la sécrétion d’IL-17 par les cellules Th17, de même qu’il réprime l’expression de l’Il4 et l’Il17 dans les cellules Treg. Structurellement proche d’IRF4, IRF8 interagit avec des cofacteurs tels que PU.1 ou BATF afin de réguler l’activité transcriptionnelle.Ce travail présenté ici révèle que le facteur IRF8 participe à la polarisation des cellules Th9 in vitro et in vivo. Le TGF-β nécessaire à la différenciation des cellules Th9 régule directement l’expression d’Irf8 grâce à l’activation de Smad3. Comme dans d’autres types cellulaires, la fonction transcriptionnelle d’IRF8 est dépendante de ces partenaires d’interaction. Nous montrons qu’en présence des facteurs PU.1, IRF4 et BATF, IRF8 participe à un complexe multiprotéique nécessaire à l’induction des cytokines caractéristiques des cellules Th9, notamment l’Il9 et l’Il21. Nous démontrons également qu’en présence de la protéine ETV6, IRF8 est capable de former un complexe initiant la répression de l’activité transcriptionnelle de l’Il4. Nous soulignons ainsi le rôle bivalent joué par IRF8 dans le développement des cellules Th9 dépendamment de ses partenaires. Pour finir, l’expression d’Irf8 est nécessaire aux cellules Th9 pour exercer leurs fonctions anti-tumorales. / CD4 helper T cells support a wide range of functions due to their ability to differentiate into different effector subsets depending on the antigen encountered and the cytokine environment in which they are. Current knowledge on the differentiation of helper T cells highlights the existence of complex transcriptional networks specific to each T helper subset. In 2008, IL-9 secreting CD4 T cells (Th9) are identified as a new helper T cell subtype. Differentiated in the presence of IL-4 and TGF-β, Th9 cells secrete IL-9 and IL-21, and contribute to the development of autoimmune and allergic diseases. Th9 lymphocytes also exhibit strong anti-tumor properties.The transcriptional network of the Th9 cells results from a balance between the signaling pathways induced by the different cytokines required for its polarization. IL-4 allows activation of STAT6 and expression of GATA3 and IRF4, whereas TGF-β leads to activation of the Smad pathway and expression of the transcription factor PU.1. The IRF4 / BATF transcriptional module and the PU.1 factor are essential messengers for the development of Th9 cells and IL-9 secretion.IRF8 is a crucial transcription factor for the development of myeloid cells and B lymphocytes. Recently it appeared that IRF8 was involved in helper T subset polarization. Indeed, IRF8 limits the secretion of IL-17 by Th17 cells, as well as repressing the expression of Il4 and Il17 in Treg cells. Structurally close to IRF4, IRF8 interacts with cofactors such as PU.1 or BATF in order to regulate transcriptional activity.This work reveals that the IRF8 transcription factor contributes to the polarization of Th9 cells in vitro and in vivo. The TGF-β needed for Th9 cell differentiation activate Smad3 pathway which directly modulates the Irf8 expression. As in many cellular subtypes, the transcriptional function of IRF8 is dependent on these interaction partners. We show that in the presence of the transcription factors PU.1, IRF4 and BATF, IRF8 participates in a multiprotein complex essential for the induction of the Th9 cytokines, Il9 and Il21. We also demonstrate that in the presence of the ETV6 protein, IRF8 is able to form a complex responsible for the repression of Il4 expression. We underline the bivalent role played by IRF8 in the development of Th9 cells depending on its partners. Finally, expression of Irf8 is crucial for Th9 cells to exercise their antitumor functions.

骨骼肌分化是由一个包括转录因子、表观遗传调控子和非编码RNA在内的复杂网络共同调控的。YY1能够通过募集PRC2抑制一系列肌肉结构基因的表达，进而抑制肌肉分化。miRNA是一组转录后调控基因表达的小片段非编码RNA，miRNA与转录因子的相互作用已经被广泛证实。在本次研究中，我们证实了一个YY1和肌肉特异性miRNA（miR-1,miR-133和miR-206）的调控回路。实验证实，YY1通过肌肉特异性miRNA增强子区域的YY1结合位点募集PRC2来抑制肌肉特异性miRNA的表达。YY1调控miR-1在体外和体内肌肉分化均被证实有重要意义。另外，我们还证实miR-1能够负反馈作用于YY1，抑制YY1的表达。 / 为了阐述YY1在基因组转录中的作用，我们做了肌肉中YY1的ChIP-seq。测序结果表明在C2C12肌肉母细胞中有1820个YY1结合位点，其中很大部分位于基因间的区域。进一步研究发现，基因间YY1的结合可能调控一些lincRNA，而这些lincRNA在肌肉发育的作用目前尚不清楚。进一步研究这些可能受YY1调节的lincRNA，我们证实了YY1能够正调控两个新的lincRNA，YAM-1和YAM-2。YAM-1在肌肉分化过程中逐渐下调，并且通过正调控他的临近基因miR-715，抑制肌肉分化，而YAM-2能够促进早期的肌肉分化。 / 总之，我们第一次在肌肉细胞中进行了YY1的ChIP-seq，并且证实在肌肉分化过程中转录因子和非编码RNA相互作用的重要性和普遍性。 / Skeletal muscle cell differentiation is a process orchestrated by a complex network of transcription factors, epigenetic regulators and non-coding RNAs. As a repressor of myogenesis, Yin Yang 1 (YY1) silences a number of muscle structural genes through recruiting Polycomb repressive complex2 (PRC2) in proliferating myoblasts. microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally, and mounting evidences support the prevalence and functional significance of their interplay with transcription factors (TFs). Here we describe the identification of a regulatory circuit between muscle miRNAs (miR-1, miR-133 and miR-206) and Yin Yang 1 (YY1). The subsequent experimental results demonstrate that YY1 indeed represses muscle miRs expression in myoblasts and the repression is mediated through multiple enhancers and recruitment of Polycomb complex to several YY1 binding sites. YY1 regulating miR-1 is functionally important for both in vitro and in vivo myogenesis. Furthermore, we demonstrate that miR-1 in turn targets YY1, thus forming a negative feedback loop. / To elucidate its role on genome-wide regulation of transcription, here in the second part of this study we performed ChIP-Seq for YY1 in muscle cells. Our results revealed 1820 YY1 binding peaks genome-wide in myoblasts, with a large portion residing in the intergenic region. A close analysis of the intergenic region bound by YY1 uncovered that YY1 may regulate a large number of lincRNAs (Long Intergenic non-coding RNAs), whose roles in skeletal myogenesis have not been explored yet. As further elucidation of the functional roles of YY1-lincRNA regulation, we identified two novel lincRNAs, YAM-1 and YAM-2 as positively regulated by YY1. YAM-1 was found to be down-regulated upon myogenic differentiation and acts as an inhibitor of myoblast differentiation. We further demonstrated that YAM-1 functions by its in cis regulation on a downstream gene, miR-715 which promotes differentiation. YAM-2, on the other hand, appears to promote myogenesis. / Together, our studies not only provide the first genome-wide picture of YY1 association in muscle cells but also uncovered novel regulatory circuits required for skeletal myogenesis and reinforce the idea that regulatory circuitry involving non-coding RNAs and TFs is essential components of myogenic regulatory network. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lu, Leina. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 144-167). / Abstract also in Chinese. / Abstract / 摘要 / Acknowledgement / Publications / List of figures / List of tables / Abbreviations / Table of content / Chapter Chapter 1: --- INTRODUCTION / Chapter 1.1 --- Skeletal Myogenesis --- p.1 / Chapter 1.2 --- Transcriptional Regulation of myogenic differentiation --- p.3 / Chapter 1.2.1 --- Transcriptional regulatory network in myogenic differentiation --- p.3 / Chapter 1.2.2 --- YY1 as a transcription factor in myogenic differentiation --- p.5 / Chapter 1.3 --- Epigenetic Regulation during skeletal muscle differentiation --- p.6 / Chapter 1.4 --- microRNA: Post-transcriptional regulation on myogenic differentiation --- p.11 / Chapter 1.4.1 --- Muscle specific miRNAs in skeletal myogenic differentiation --- p.15 / Chapter 1.4.2 --- Non-muscle specific miRNAs in skeletal myogenic differentiation --- p.20 / Chapter 1.4.3 --- miRNAs and skeletal muscle diseases --- p.23 / Chapter 1.5 --- Long Non-coding RNAs --- p.26 / Chapter 1.5.1 --- Long Non-coding RNAs and lincRNAs --- p.26 / Chapter 1.5.2 --- LincRNAs in muscles --- p.30 / Chapter Chapter 2: --- MATERIALS AND METHODS / Chapter 2.1 --- C2C12 cell line --- p.32 / Chapter 2.2 --- Primary Myoblast isolation and in vitro culture --- p.32 / Chapter 2.3 --- Animal studies --- p.33 / Chapter 2.4 --- RNA extraction --- p.34 / Chapter 2.5 --- RT-PCR and Real-Time RT-PCR --- p.35 / Chapter 2.6 --- Transfection and infection --- p.37 / Chapter 2.7 --- Oligonucleotides --- p.38 / Chapter 2.8 --- Dual-luciferase reporter assay --- p.43 / Chapter 2.9 --- Immunofluorencence staining --- p.44 / Chapter 2.10 --- Antibodies --- p.45 / Chapter 2.11 --- Protein extraction and Western blotting --- p.46 / Chapter 2.12 --- DNA constructs --- p.48 / Chapter 2.13 --- Mutagenesis --- p.49 / Chapter 2.14 --- RNA-Fluorescence In Situ Hybridization (RNA-FISH) --- p.51 / Chapter 2.15 --- C2C12 cells with YY1-stably knocked down --- p.52 / Chapter 2.16 --- Rapid Amplification of cDNA Ends (RACE) --- p.53 / Chapter 2.17 --- Chromatin Immunoprecipitation (ChIP) --- p.55 / Chapter 2.18 --- ChIP-PCR --- p.58 / Chapter 2.19 --- ChIP-sequencing --- p.58 / Chapter 2.20 --- Northern blotting --- p.59 / Chapter 2.21 --- Prediction of miRNA targets --- p.60 / Chapter 2.22 --- Statistical analysis --- p.60 / Chapter Chapter 3: --- Results / Chapter 3.1 --- YY1-miR-1/133 regulatory circuitry in skeletal myogenesis --- p.61 / Chapter 3.1.1 --- YY1 decreases miR-1/133 during skeletal muscle differentiation --- p.61 / Chapter 3.1.1.1 --- Negative correlation between YY1 and miR-1/133 during C2C12 differentiation --- p.61 / Chapter 3.1.1.2 --- Negative correlation between YY1 and miR-1/133 in primary cell differentiation --- p.63 / Chapter 3.1.1.3 --- Negative correlation between YY1 and miR-1/133 in postnatal muscle development and mdx mouse model --- p.65 / Chapter 3.1.1.4 --- Deletion of YY1 upregulates miR-1/133 both in C1C12 and primary myoblast --- p.68 / Chapter 3.1.1.5 --- Deletion of YY1 upregulates miR-1/133 at the transcriptional level --- p.70 / Chapter 3.1.2 --- YY1 represses miR-1/133 by binding to 4 enhancers --- p.72 / Chapter 3.1.2.1 --- Four enhancers of miR-1/133 with potential YY1 targeting sites --- p.72 / Chapter 3.1.2.2 --- YY1 represses the four enhancers’ activities --- p.75 / Chapter 3.1.2.3 --- Depletion of YY1 up-regulates the four enhancers’ activities --- p.77 / Chapter 3.1.2.4 --- YY1 directly binds to the putative binding sites and mediates the repression on miR-1/133 --- p.79 / Chapter 3.1.2.5 --- YY1 recruits Ezh2 to the enhancers which subsequently causes histone modification --- p.82 / Chapter 3.1.3 --- YY1 repressing miR-1/133 is functionally significant in myogenesis --- p.84 / Chapter 3.1.3.1 --- Negative correlation between YY1 and miR-1/133 in CTX induced muscle regeneration model --- p.84 / Chapter 3.1.3.2 --- Depletion of YY1 in CTX induced muscle regeneration model promotes miR-1/133 expression --- p.87 / Chapter 3.1.3.3 --- Depletion of YY1 in CTX induced muscle regeneration model promotes muscle differentiation --- p.89 / Chapter 3.1.4 --- miR-1 can target YY1 forming a feedback loop --- p.92 / Chapter 3.1.5 --- miR-1 can repress Pax7 by targeting two binding sites on 3’UTR --- p.95 / Chapter 3.1.5.1 --- miR-1 targets Pax7 by binding to two target sites --- p.95 / Chapter 3.1.5.2 --- miR-1 represses Pax7 forming an YY1-miR-1-Pax7 regulating circuitry in skeletal myogenesis --- p.98 / Chapter 3.1.6 --- Conclusion: YY1-miR-1-Pax7 regulatory circuitry in skeletal myogenesis --- p.100 / Chapter 3.2 --- ChIP-seq reveals YY1-lincRNA regulation in skeletal myogenesis --- p.102 / Chapter 3.2.1 --- ChIP-seq uncovered a large number of genes under YY1 regulation --- p.102 / Chapter 3.2.2 --- ChIP-seq reveals that YY1 associates with lincRNA loci --- p.105 / Chapter 3.2.2.1 --- YY1 associates with lincRNA-YAM loci --- p.105 / Chapter 3.2.2.2 --- YY1 positively regulates YAM-1 and YAM-2 both in vitro and in vivo --- p.107 / Chapter 3.2.3 --- YY1-YAM-1-miR-715 regulatory pathway in muscle differentiation --- p.109 / Chapter 3.2.3.1 --- Genomic organization and cellular localization of YAM-1 --- p.109 / Chapter 3.2.3.2 --- Expression of YAM-1 decreases during myogenic differentiation --- p.112 / Chapter 3.2.3.3 --- YAM-1 represses myogenic differentiation both in vitro and in vivo --- p.115 / Chapter 3.2.3.3.1 --- YAM-1 inhibits C2C12 differentiation --- p.115 / Chapter 3.2.3.3.2 --- YAM-1 inhibits muscle differentiation in vivo --- p.117 / Chapter 3.2.3.4 --- A functional YY1-YAM-1-miR-715 regulatory axis in skeletal myogenic differentiation --- p.119 / Chapter 3.2.3.4.1 --- miR-715 is down-regulated during muscle differentiation --- p.119 / Chapter 3.2.3.4.2 --- miR-715 is under the regulation of YY1-YAM-1 --- p.122 / Chapter 3.2.3.4.3 --- miR-715 represses muscle differentiation forming a YAM-1-miR-715 regulatory axis during muscle differentiation --- p.124 / Chapter 3.2.4 --- YAM-2 promotes early myogenic differentiation --- p.126 / Chapter 3.2.4.1 --- Genomic organization and cellular localization of YAM-2 --- p.126 / Chapter 3.2.4.2 --- YAM-2 is regulated during myogenic differentiation --- p.129 / Chapter 3.2.4.3 --- YAM-2 promotes early myogenic differentiation --- p.131 / Chapter Chapter 4: --- DISCUSSION / Chapter 4.1. --- YY1-miRNA regulatory circuit in skeletal myogenesis --- p.133 / Chapter 4.2 --- YY1 mediates epigenetic modification in skeletal myogenesis --- p.135 / Chapter 4.3 --- miRNAs in skeletal myogenesis --- p.136 / Chapter 4.4 --- YY1 regulates long intergenic non-coding RNAs in skeletal myogenesis --- p.138 / Chapter Chapter --- 5: SUMMARY AND FUTURE WORK --- p.142 / REFERENCE --- p.144

Afin de mieux comprendre les mécanismes impliqués dans l'initiation des racines coronaires chez le riz nous avons recherché des gènes régulés par le facteur de transcription CROWN ROOTLESS 1 (CRL1) qui contrôle leur initiation en réponse à l'auxine. Différentes approches de transcriptome ont été mises en œuvre. La première a consistée à rechercher les gènes différenciellement exprimés dans les bases de tige du mutant crl1 par rapport au sauvage. La seconde a consisté à rechercher des gènes régulés par l'auxine de manière CRL1-dépendant. La troisième a consisté à rechercher des gènes dont l'expression est induite dans les bases de tige du mutant crl1 après expression conditionnelle ectopique du gène CRL1. Parmi les gènes identifiés des expériences de qRT-PCR nous ont permis de valider 11 gènes comme étant induit par l'auxine de manière CRL1 dépendant et des expérience d'hybridation in situ, 10 gènes qui s'expriment de manière spécifique dans les primordium de racine coronaires. La majorité de ces gènes codent pour des facteurs de transcription et des éléments de transduction de signaux. Certains sont des modulateurs de la stucture de la chromatine d'autre des transporteurs d'auxine. Ces résultats éclairent sur les cibles régulées par le facteur de transcription CRL1 et apportent des éléments nouveaux sur les mécanismes moléculaires impliqués dans la régulation de l'initiation des racines coronaires chez le riz. / In order to understand better the mechanisms involved in crown root initiation in rice we researched the genes regulated by the CROW ROOT LESS 1 (CRL1) transcription factor that controls their initiation in response to auxin. Several transcript profiling approaches have been used. The first was to look for the genes differentially expressed in crl1 stem bases relatively to the wild type. The second one was to research genes that are CRL1-dependant auxin responsive. The last one consisted to research genes that are up-regulated in crl1 stem bases just after the inducible ectopic expression of CRL1. Among identified genes RT-qPCR experiments allowed to validate 11 CRL1-dependant auxin responsive genes and in situ hybridization experiments ten genes that are specifically expressed in crown root primordia. Most of these genes encodes transcription factors or components of transduction signal patways. Some of them encode chromating modulling factors or auxin transporters. These results give new knowledge about the gene regulatory network acting down-stream CRL1 and about the molecular mechanisms involved in crown root initiation in rice.

E4F1 est un facteur de transcription liant l'ADN, exprimé de façon ubiquitaire par tous les tissus, et qui possède une activité E3 ubiquitine ligase atypique dirigée contre le suppresseur de tumeur p53. La protéine E4F1 interagit directement avec plusieurs suppresseurs de tumeurs cellulaires et des oncogènes viraux (p53, pRb, DRAL, RASSF1A, p19ARF, BMI1, HBX et GAM1...), suggérant qu’elle est elle-même impliquée dans la tumorigenèse. La perte d’E4F1 dans des fibroblastes embryonnaires (Mefs) transformés ou dans des cellules de sarcomes histiocytaires déficientes pour la voie p53, entraine la mort de ces cellules. La même inactivation d'E4F1 dans les cellules normales n'affecte pas leur survie mais entraine un arrêt de prolifération. Des analyses transcriptomiques et de liaison à l'ADN à l'échelle du génome entier (ChIP-seq et analyses différentielles des transcriptomes de cellules E4F1 WT et KO) nous ont permis d’identifier une centaine de gènes liés et régulés directement par E4F1. Ces gènes codent notamment pour des protéines mitochondriales impliquées dans le métabolisme et l'homéostasie de cette organelle, dont plusieurs composants et régulateurs de l'enzyme multimérique, pyruvate déshydrogénase. Un second groupe de gênes cibles d’E4F1 est impliqué dans la réponse aux dommages à l'ADN, dont le gène codant pour la kinase CHK1 qui joue un rôle essentiel dans le contrôle de la stabilité du génome. En accord avec la fonction de ces gènes cibles, la perte d’E4F1 entraine des perturbations du métabolisme cellulaire et des checkpoints de réponse aux stress génotoxiques. Dans les cellules déficientes pour la voie p53, ces perturbations conduisent à des stress oxydatifs (surproduction de ROS mitochondriaux) et énergétiques, suivis de dommages aux protéines et à l'ADN, et in fine, à une mort cellulaire massive. Dans les cellules compétentes pour la voie p53 ces altérations sont fortement atténuées et conduisent à un arrêt de la prolifération. Une partie des effets protecteurs de p53 est due à sa capacité à stimuler l'expression du gène ALDH4a1 qui code pour une aldehyde dehydrogenase impliquée dans le catabolisme de la proline et dont l'activité possède des propriétés anti-oxydantes.Mes travaux mettent également en évidence que le niveau d'expression de la protéine E4F1 et son activité augmentent lors de la transformation cellulaire ainsi qu'en réponse à des stress énergétiques, génotoxiques ou oxydatifs. Dans ces trois dernières conditions, la phosphorylation d'E4F1 est également augmentée au niveau de plusieurs sérines qui ont été identifiées par spectrométrie de masse. En résumé, tous ces éléments indiquent qu'E4F1 est un acteur important du contrôle de l'homéostasie métabolique et de la réponse aux stress, particulièrement essentiel pour la survie des cellules cancéreuses déficientes pour la voie p53. Mes observations suggèrent également qu'E4F1 est activé en réponse à différents stress et qu'il pourrait jouer un rôle essentiel dans la capacité des cellules cancéreuses à s'adapter aux multiples stress environnementaux auxquels elles sont exposées au cours de la tumorigenèse. / The ubiquitously expressed E4F1 protein acts as a transcription factor that binds a consensus DNA sequence at promoters, and as an atypical E3-ligase for the tumor suppressor p53. E4F1 physically interacts with several bona fide cellular tumor suppressors and viral oncoproteins (including p53, pRb, DRAL, RASSF1A, p19ARF, BMI1, HBX and GAM1...), suggesting that it might itself be involved in tumorigenesis. E4F1 genetic inactivation in transformed mouse embryo fibroblasts (Mefs) and in hematopoietic tumors deficient for the p53 pathway, results in massive cell death. Importantly, inactivation of E4F1 in normal cells does not affect cell survival. Genome wide approaches (ChIP-seq profiling and comparative transcriptomics performed on E4F1 WT and KO cells) identified a limited list (100) of genes that are bound and directly regulated by E4F1. Several E4F1 target genes code for mitochondrial proteins involved in mitochondria metabolism and homeostasis, including several components of the pyruvate dehydrogenase complex. Another set of E4F1 target genes codes for factors involved in DNA repair and damage checkpoints, including the checkpoint kinase CHK1. Accordingly, both mitochondrial and checkpoint functions are altered in E4F1 KO cells. In proliferating cells deficient for the p53 pathway, these defects lead to energetic and oxidative stresses, protein and DNA damages, and in fine, massive cell death. In p53-proficient cells, these alterations are attenuated and lead to growth arrest. Part of this protective effect of p53 is mediated by ALDH4a1, a p53 target gene encoding an aldehyde dehydrogenase involved in proline catabolism and that exhibits antioxidant properties.In this thesis, I also demonstrate that E4F1 protein level and activity is increased during cell transformation and upon exposure to genotoxic, energetic or oxidative stresses. These stresses also lead to E4F1 phosphorylation at specific serine residues that were identified by mass spectrometry. All together this work shows that E4F1 controls cellular functions that are important for mammalian cells metabolic homeostasis and stress responses, and that are essential for the survival of p53-deficient cancer cells. This work also suggests that E4F1 is activated in response to various stresses and therefore, that it could play an essential role in allowing cancer cells to adapt to environmental stresses.