Tesis sobre el tema "Pou5f1"

Embryonic stem cells (ESCs) are characterized by their unlimited capacity for self-renewal and the ability to contribute to every lineage of the developing embryo. The promoters of developmentally regulated loci within these cells are marked by coincident epigenetic modifications of gene activation and repression, termed bivalent domains. Trithorax group (TrxG) and Polycomb Group (PcG) proteins respectively place these epigenetic marks on chromatin and extensively colocalize with Oct4 in ESCs. Although it appears that these cells are poised and ready for differentiation, the switch that permits this transition is critically held in check. The derepression of bivalent domains upon knockdown of Oct4 or PcG underscores their respective roles in maintaining the pluripotent state through epigenetic regulation of chromatin structure. The mechanisms that facilitate the recruitment and retention of Oct4, TrxG, and PcG proteins at developmentally regulated loci to maintain the pluripotent state, however, remain unknown. Oct4 may function as either a transcriptional activator or repressor. Prevailing thought holds that both of these activities are required to maintain the pluripotent state through activation of genes implicated in pluripotency and cell-cycle control with concomitant repression of genes required for differentiation and lineage-specific differentiation. More recent evidence however, suggests that the activator function of Oct4 may play a more critical role in maintaining the pluripotent state (Hammachi et al., 2012). The purpose of the studies described in this dissertation was to clarify the underlying mechanisms by which Oct4 functions in transcriptional activation and repression. By so doing, we wished to contextualize its role in pluripotent cells, and to provide insight into how changes in Oct4 function might account for its ability to facilitate cell fate transitions. As a result of our studies we find that Oct4 function is dependent upon post-translational modifications (PTMs). We find through a combination of experimental approaches, including genome-wide microarray analysis, bioinformatics, chromatin immunoprecipitation, functional molecular, and biochemical analyses, that in the pluripotent state Oct4, Akt, and Hmgb2 participate in a regulatory feedback loop. Akt-mediated phosphorylation of Oct4 facilitates interaction with PcG recruiter Hmgb2. Consequently, Hmgb2 functions as a context dependent modulator of Akt and Oct4 function, promoting transcriptional poise at Oct4 bound loci. Sumoylation of Oct4 is then required to maintain Hmgb2 enrichment at repressed loci and to transmit the H3K27me3 mark in daughter progeny. The expression of Oct4 phosphorylation mutants however, leads to Akt inactivation and initiates the DNA Damage Checkpoint response. Our results suggest that this may subsequently facilitate chromatin reorganization and cell fate transitions. In summary, our results suggest that controlled modulation of Oct4, Akt, and Hmgb2 function is required to maintain pluripotency and for the faithful induction of transcriptional programs required for lineage specific differentiation.

La préservation de la fertilité suscite une inquiétude croissante. Depuis les dernières décennies, la fertilité a diminué dans les pays industrialisés. En conséquence, un nombre croissant de couples a recours à la procréation médicalement assistée, sans grand succès par manque de connaissances sur les cellules reproductrices (cellules germinales). L’étude de ces cellules constitue un socle essentiel pour des applications médicales, mais jusqu’à présent, les mécanismes moléculaires qui régissent leur développement restent peu compris en raison de l’absence de modèles d'étude physiologiques.Les cellules germinales sont produites au cours du développement embryonnaire, se multiplient pour constituer un stock suffisant, se différencient dans la gonade en fonction du sexe de l'embryon, puis entrent en méiose pour devenir des gamètes fertiles, les ovocytes (femelles) et les spermatozoïdes (mâles). Mon laboratoire d’accueil a démontré que la voie de signalisation canonique WNT/β-catenin est nécessaire au développement de l'ovaire in vivo et qu'en absence d'activation de cette voie dans l'ovaire embryonnaire de souris, la prolifération et la différenciation des cellules germinales primordiales sont perturbées.Tirant partie de notre expertise dans l'analyse de modèles de différenciation gonadique, nous nous sommes intéressés au mécanisme d'action de la voie WNT/β-catenin dans la différenciation des cellules germinales, en analysant au moyen de modèles murins de perte et gain de fonction de β-catenin les conséquences de la modification de son expression dans les cellules de la gonade.Dans un premier temps, nous avons démontré que le maintien, après la naissance, de l'activité de la voie WNT/β-catenin dans les cellules souches spermatogoniales du testicule de souris stimule leur prolifération de manière massive, provoquant un défaut de différenciation en spermatozoïdes. Dans un deuxième temps, nous avons montré que l'ablation génétique du gène Ctnnb1 (codant pour β-catenin) dans les cellules germinales primordiales in vivo entraîne une perte précoce de leur pluripotence et une différenciation prématurée en ovogonies. Nous avons démontré, pour la première fois in vivo, que tandis que le développement ovarien progresse, β-catenin forme des complexes protéiques avec POU5F1 et CDH1 qui transitent du noyau des cellules germinales à leur membrane, permettant ainsi leur sortie de pluripotence et leur différenciation. Nos résultats indiquent que l'E3-ubiquitine ligase ZNRF3 régule la pluripotence des cellules germinales par une boucle de rétroaction négative.Ces données indiquent que dans l'ovaire comme dans le testicule, une régulation fine de l'activité de la voie de signalisation WNT/β-catenin est requise pour déterminer la fenêtre de différenciation des cellules germinales primordiales in vivo. De plus, la voie WNT/β-catenin contrôle la sortie de pluripotence des cellules germinales primordiales via une activité non-transcriptionnelle de β-catenin, permettant à terme de coordonner le développement des cellules somatiques et germinales de la gonade. Dans les années à venir, nos travaux pourraient aider à la génération des gamètes in vitro
Since the last decennials, fertility is decreasing in industrial countries, and nowadays, infertility reaches a prevalence of 9 to 18% of the general population, with a significant proportion of cases being due to defective gametogenesis. Accordingly, fertility preservation raises a growing concern. Nowadays, a growing number of couples undergo Assisted Reproductive Technology with little success due to the lack of knowledge on mechanisms orchestrating germ cell differentiation. Little is known about how primordial germ cells become gametogenesis-competent cells (gonocytes), because of the lack of suitable physiological models. Primordial germ cells give rise to the next generation by differentiating from pluripotent progenitors into highly specialized cells, the gametes, which in turn generate a totipotent zygote after fertilization. After specification in the early embryo, primordial germ cells colonize the gonad, lose pluripotency and gain their capacity for irreversible sexual differentiation. Gonocytes then become either female (oogonia) or male (spermatogonia), and eventually progress into meiotic divisions. So far, the mechanisms of germ cell changes in potency remain largely elusive.My host laboratory has demonstrated that activation of the canonical WNT/β-catenin signalling is required for ovarian development. Thus, absence of WNT/β-catenin activation eventually triggers defects in germ cell proliferation and sexual differentiation. However, genetic models of cell-specific ablation of Ctnnb1 (encoding β-catenin) were still missing to assess the contribution of WNT/β-catenin in the germ cells. We decided to analyse the role of this signalling pathway by generating specific mouse models for β-catenin loss or gain of function and by investigating the consequences of WNT/β-catenin deregulation in either the somatic or the germ cells of the developing gonad. We first demonstrated that WNT/β-catenin regulates spermatogonial stem cell proliferation and differentiation in the post-natal testis, demonstrating that this signalling activity must be finely tuned over time to ensure spermatogenesis.Secondly, we showed that genetic elimination of Ctnnb1 in mouse primordial germ cells in vivo leads to a precocious loss of pluripotency and to premature germ cell differentiation into gonocytes. We demonstrated, for the first time in vivo, that while ovarian development is getting forward, β-catenin forms proteic complexes containing POU5F1 and CDH1 that transit from the nucleus of the germ cells to their membrane, thus allowing primordial germ cells to exit pluripotency and eventually differentiate. Our results also reveal that the ZNRF3 E3-ubiquitine ligase negatively regulates germ cell exit from pluripotency through a negative feedback loop.Collectively, our results show that the WNT/β-catenin signalling is necessary for determining the proper window of differentiation in both somatic and germ cells. Moreover, WNT/β-catenin controls the germ cell exit from pluripotency through β-catenin non-transcriptional activity, eventually coordinating the development of the different cell types of the fetal ovary. In the future, our results might help to recapitulate gametogenesis in vitro

Accounting for 14% of all new cancer diagnosis in the United States, prostate cancer is the most commonly diagnosed cancer and the second leading cause of cancer related death in the United States. Prognosis for patients diagnosed with metastatic disease is especially poor, since no effective treatments have been developed (1). In this study, we examined the expression and function of POU5F1B, a protein-encoding pseudogene of the homeodomain transcription factor Oct4, in prostate cancer. POU5F1B is located at 8q24, a "gene desert" containing numerous alleles associated with prostate cancer risk. A recent study has indicated that a number of these risk alleles are correlated with POU5F1B expression and prostate cancer susceptibility. The role of POU5F1B in prostate cancer carcinogenesis and progression, however, is not known. In our study, we found that POU5F1B expression is upregulated in prostate cancers and highly overexpressed by high grade (Gleason ≥8) and metastatic prostate cancers. We cloned POU5F1B from prostate cancer cell lines, which contains prostate cancer risk associated SNPs, including a missense mutation inside the homeobox DNA binding domain, to study the functional effects of POU5F1B overexpression in prostate cancers. Here, we report that POU5F1B from prostate tumor encodes functional proteins that exhibit gene transactivation activity comparable to its parent gene, Oct4. Further, we report that POU5F1B overexpression in prostate cancer cell lines increases prostate cancer cell proliferation, migration, anchorage independent growth, and drug resistance in vitro and tumor xenograft growth in vivo. Conversely, shRNA mediated knockdown of endogenous POU5F1B expression in prostate cancer cells inhibit cell proliferation in vitro and tumor growth in vivo, as well as prolong tumor free survival in animal models. The data provide compelling evidence that POU5F1B is an important mediator of prostate cancer progression. We further examined the molecular mechanism behind POU5F1B driven prostate cancer progression. Our studies found that POU5F1B overexpression suppresses E-Cadherin expression at both mRNA and protein levels. Our studies further found POU5F1B overexpression in prostate cancer cells increases Wnt1, TCF1, and TCF4 expression, as well as increased Wnt/β-Catenin signaling - indicating the induction of epithelial-to-mesenchymal transition (EMT) in POU5F1B overexpressing cells(2). Consistently, qPCR analysis found that POU5F1B overexpression significantly increased the expressions of numerous EMT related genes and prostate cancer stem cell markers. Functional studies further confirmed that the transactivation activity of Nanog, another stem cell related transcription factor, is dramatically increased in POU5F1B overexpressing cells. Taken together, our data strongly suggests that POU5F1B overexpression drives prostate cancer progression through the induction of EMT and conferment of stem-cell properties to tumor cells. In summary, our data demonstrated that POU5F1B is overexpressed in prostate tumors, especially high-grade and metastatic tumors, and is a functional driver of prostate cancer progression by inducing EMT in prostate cancer cells. Our study also showed that POU5F1B can potentially be targeted to treat prostate cancer. Based on our findings, depletion of POU5F1B may reduce the risk of metastatic disease or tumor recurrence when used with concurrent therapies in early state tumors and may attenuate treatment resistance in diseases at advanced stages.

Orientador: Rafael Henrique Nóbrega
Resumo: As células tronco são classificadas em dois grandes grupos de acordo com sua origem e capacidade de diferenciação. Células tronco embrionárias (CTE) são originadas do zigoto, e podem ser classificadas como totipotentes, isto é, capazes de originar um indivíduo inteiro, ou pluripotentes, quando originam os três folhetos embrionários (ecto, meso e endoderme). As células tronco adultas (CTA) são as células tronco encontradas nos tecidos fetais e adultos; classificadas como uni, oligo ou multipotentes dependendo da variedade de tecidos originados a partir delas. Marcadores de células tronco, como antígenos de superfície específicos, fatores de trancrição como OCT4 e NANOG são expressos em CTE e algumas CTA, mas são rapidamente reprimidos à medida que as células se diferenciam. O presente trabalho tem como objetivo identificar marcadores de células tronco e com isso, os efeitos do hormônio endócrino Fsh e do fator parácrino GDNF na atividade proliferativa e gênica dessas populações de células e também de células de Sertoli. Foi observado que os marcadores Pou5f3 e Gfra1a são principalmente expressos em espermatogônias tronco indiferenciadas e que sua expressão reduz significativamente sob efeito do recombinante zebrafish Fsh. Por outro lado, genes como o igf3, nanos3 e nanog tiveram sua expressão aumentada significativamente. O recombinante humano GDNF não altera significativamente a expressão desses genes, porém estimula a proliferação de espermatogônias tipo Aund e Adiff e célul... (Resumo completo, clicar acesso eletrônico abaixo)
Mestre

Chez la souris, Six6 et Lhx2 sont exprimés dans l’œil et la glande pituitaire en développement. Par une approche "gènes candidats", les ADN de patients avec un phénotype proche de celui de souris invalidées pour ces gènes ont été séquencés. Aucune mutation a été mise en évidence pour SIX6. Deux variations hétérozygotes faux-sens de LHX2 ont été identifiées mais n’ont pas d'effet (tests in vitro). LHX2 a un rôle régulateur transcriptionnel in vitro sur deux gènes pituitaires (PRL, POU1F1), et en action synergique avec POU1F1. Deux mutations hétérozygotes composites de LHX3 chez un patient non consanguin ont permis d’assigner à ce gène un syndrome décrit uniquement chez des patients consanguins. Une de ces mutations a un effet dominant négatif. POU1F1, impliqué dans la différenciation pituitaire terminale, est associé en pathologie humaine à un déficit en hormone de croissance (GH), PRL et TSHβ. L’expression de GH est régulée par la fixation de POU1F1 sur son promoteur et sur un « Locus Control Region » mais ses cofacteurs ne sont pas connus. Deux mutations faux-sens identifiées dans le domaine de transactivation (TAD) de POU1F1 sont associées à un déficit isolé en GH. La résonnance plasmonique de surface a permis de définir les interactions de POU1F1 (normal et mutés) sur ses séquences cibles ; des extraits nucléaires sont passés avec POU1F1 (normal et mutés) afin d’identifier (par spectrométrie de masse) ses partenaires au locus GH. Une cristallographie du TAD a débuté pour analyser sa structure tridimensionnelle qui est probablement altéré par les mutations identifiées
In mice, Six6 and Lhx2 were shown to be expressed in developing eye and pituitary gland. Using a candidate gene approach, DNA from patients with phenotypic features reminiscent of those reported in invalidated mice were sequenced. Any mutation was identified in SIX6 gene. Two LHX2 heterozygous missense variations were identified but are not deleterious (in vitro tests). A transcriptional regulator role of LHX2 was shown in vitro on two pituitary genes (PRL, POU1F1), and a synergic action with POU1F1 on these promoters. Two compound heterozygous LHX3 mutations in a non consanguineous patient permitted us to implicate this gene in syndrome described in other consanguineous patients. A dominant negative effect of one of these mutations was shown. POU1F1, implicated in terminal pituitary differentiation is associated, in human pathology, with growth hormone (GH), PRL and TSHβ deficiencies. GH expression is regulated by POU1F1 binding on its promoter and on a locus control region but its co-factors are not known. Two new missense mutations located in the POU1F1 transactivation domain (TAD) were identified and associated with isolated growth hormone deficiency. Surface plasmon resonance permitted us to define interaction properties between POU1F1 (wild-type and mutated) on its target sequences. Nuclear extracts from pituitary cell lines are used with POU1F1 (wild-type and mutated) to identified (by mass spectrometry) co-factors at GH locus. A crystallographic study was started to determine the TAD tridimensional structure which is probably altered by the identified mutations

A ce jour, un certain nombre d'adénomes hypophysaires somatotropes échappent encore au contrôle de la maladie. Il reste nécessaire d'étudier plus finement les différentes voies de contrôle de la sécrétion hormonale et de la prolifération tumorale afin d'identifier des traitements alternatifs susceptibles de contrôler ces symptômes tumoraux. De même, un certain nombre de patients porteurs de prolactinomes (environ 10%), à l'origine de dysfonctionnement gonadique et sexuels, échappent au contrôle de l'hypersécrétion de prolactine par les traitements actuels. Pour ces deux types de tumeurs, s'ajoutent des complications neurologiques consécutives à l'accroissement des volumes tumoraux. Dans une perspective de contrôle de l'hypersécrétion tumorale nous nous sommes intéressés à deux stratégies faisant intervenir un transfert de gènes dans les cellules. POU1F1 est un facteur de transcription crucial pour le développement et le maintien des lignages somatotrope, lactotrope et thyréotrope et son expression est retrouvée dans les adénomes hypophysaires. De plus, des mutations de ce gène, notamment le mutant dominant négatif R271W, ont été identifiés chez des patients présentant des déficits hypophysaires combinés en GH, en PRL et en TSH. Une première partie de ma thèse a donc porté sur l'étude du transfert du mutant dominant négatif de POU1F1 par un vecteur lentiviral dans les tumeurs hypophysaires somatotropes et lactotropes afin d'évaluer son impact sur la sécrétion hormonale et sur la viabilité cellulaire. Nous avons montré que l'inactivation de ce facteur de transcription entrainait une diminution de la viabilité cellulaire et de la sécrétion de toutes les tumeurs testées
To this date, some somatotroph adenomas do not respond to the current treatment of pituitary tumors. It still necessary to study the different ways to control the hormonal secretion and the tumor progression to identify alternate therapy capable to manage these symptoms. Likewise, some patient with prolactinomas (about 10%) responsible of gonad and sexual dysfunction do not respond to the control of prolactin hypersecretion. In addition, some neurological complications are finding due to the neighboring structures compression for both of these tumors. To better control the hormonal secretion we investigate two different strategies involving a gene transfer in the cells. POU1F1 is a major transcription factor involved in the pituitary development and the management of somatotroph, thyreotroph and lactotroph cell lines. Its expression is found in pituitary adenomas belonging of these 3 cell lines. Moreover some mutations like the R271W mutant are identified in patients with combined pituitary hormonal deficiency (CPHD). In the first part of my thesis we transfer this dominant negative mutant via a lentiviral transfer in the somatotroph and lactotroph adenomas to evaluate its impact on hormonal secretion and cell viability. We show an inhibitory effect of the POU1F1 inactivation on the secretion and the cell viability of all our tumors. Somatostatin analogs are the major medical therapy for somatotroph adenomas and control GH secretion in 60% of these patients. Octreotide resistance is highly correlated to a low expression of the sst2 receptor

Chez la souris, Six6 et Lhx2 sont exprimés dans l'œil et la glande pituitaire en développement. Par une approche "gènes candidats", les ADN de patients avec un phénotype proche de celui de souris invalidées pour ces gènes ont été séquencés. Aucune mutation a été mise en évidence pour SIX6. Deux variations hétérozygotes faux-sens de LHX2 ont été identifiées mais n'ont pas d'effet (tests in vitro). LHX2 a un rôle régulateur transcriptionnel in vitro sur deux gènes pituitaires (PRL, POU1F1), et en action synergique avec POU1F1. Deux mutations hétérozygotes composites de LHX3 chez un patient non consanguin ont permis d'assigner à ce gène un syndrome décrit uniquement chez des patients consanguins. Une de ces mutations a un effet dominant négatif. POU1F1, impliqué dans la différenciation pituitaire terminale, est associé en pathologie humaine à un déficit en hormone de croissance (GH), PRL et TSHβ. L'expression de GH est régulée par la fixation de POU1F1 sur son promoteur et sur un " Locus Control Region " mais ses cofacteurs ne sont pas connus. Deux mutations faux-sens identifiées dans le domaine de transactivation (TAD) de POU1F1 sont associées à un déficit isolé en GH. La résonnance plasmonique de surface a permis de définir les interactions de POU1F1 (normal et mutés) sur ses séquences cibles ; des extraits nucléaires sont passés avec POU1F1 (normal et mutés) afin d'identifier (par spectrométrie de masse) ses partenaires au locus GH. Une cristallographie du TAD a débuté pour analyser sa structure tridimensionnelle qui est probablement altéré par les mutations identifiées

The fate of cells is determined by a distinct group of transcription factors (TFs) which control the target gene expression in response to cellular stimuli during development and after injury. Moreover, TFs can physically interact with each other, thereby modifying target gene transcription for the fine­‐-tuning of cell fate determination depending on the specific cellular signals. The Brn­‐-3 family of TFs, B rn‐-­3aand Brn-­‐3b, was o riginallyisolated and characterised in the neurons of the central and peripheral nervous system, where they play a critical role in differentiation and cell survival during the nervous development. Studies have since shown that Brn­‐- 3TFs are expressed in the murine heart during cardiac development, however, the roles of these TFs in the heart following injury are yet to be elucidated. Further to the direct control of gene transcription, Brn-­‐3a also physically interacts with p53 and alters cellular response by modifying p53 target gene expression. Thus, up onco-­‐expression with p53, Brn -­‐3 aprotects neuronal cells from apoptosis and enhances survival by inhibiting p53­‐-mediated transcription of pro-¬‐ apoptotic prot einsBax and Noxa, while increasing expression of cell cycle arrest gene p21cip1/waf1 In this study, we investigated the potential role for Brn­‐-3a in protecting cardiomyocytes from p53-­‐mediated apoptosis following injurious stimuli such as ischemia. W efound that p53 a ndit spr o-­‐apoptotic target g enesBax, Noxa and Puma were markedly induced in the infarcted m yocardiumof the injured mouse heart by 1 week after permanent ischemia. In contrast, pro­‐-survival Brn-­‐3 awas upregulated in the non-­‐infarct, remote area of the heart, but interestingly, Brn-­‐3a was found to be downregulated in the infarcted tissues where upregulation of the pro-­‐apoptotic proteins was most evident. Importantly, immunostaining analysis revealed that Brn­‐-3a was co-¬‐ localised with p53 in a distinct region of myocardium bordering th einfarct and non­‐- infarct areas. Moreover, transfection studies indicated that Brn­‐-3a enhanced activity of p53 to activate thep21cip1/waf1 promoter in H9c2 cells. In conclusion, our results suggest that Brn­‐-3a is co­‐-expressed with p53 in a subpopulation o fcardiomyocytes following ischemic injury and that this pro-­‐survival TF may play a role in protecting cardiomyocytes from ischemia­‐-induced apoptosis through modulation of p53­‐-dependent transcription of apoptosis­‐-associated genes.

Chez le xénope, le pronéphros constitue le rein fonctionnel du têtard. Le sang est filtré par le glomus et l'urine est modifiée lors de son transit dans le tubule. Au cours de ma thèse, nous avons analysé l'expression des quatre membres de la famille pou3f au cours du développement embryonnaire chez le xénope. Nous avons montré que leur expression neurale, otique ou encore épidermique est conservée entre le xénope et la souris, de même que l'expression rénale de pou3f3. Nous avons également mis en évidence une expression pronéphrique de pou3f4. Nous avons ensuite analysé le rôle de Pou3f4 au cours du développement du pronéphros. La perte de fonction de Pou3f4 entraine des défauts de différenciation terminale du tubule intermédiaire et distal. Sa surexpression aboutit au contraire à une augmentation de l'expression des marqueurs de différenciation slc12a1 et clcnkb1. Les morphants Pou3f4 présentent aussi des défauts de morphogenèse du tubule intermédiaire caractérisés par des circonvolutions réduites. Nos résultats montrent que Pou3f4 contrôle l'expression de l'ephrine efnA3 dans le tubule pronéphrique. La perte de fonction d'EfnA3 conduit à des défauts de morphogenèse du tubule, suggérant que Pou3f4, par la régulation de l'expression d'efnA3, est impliqué dans les mouvements cellulaires nécessaires à l'élongation antérieure du tubule. Enfin, nous avons observé que pou3f3 et pou3f4 ne se régulent pas l'un de l'autre. Nous avons en revanche mis en évidence une redondance fonctionnelle de ces gènes dans la morphogenèse et la différenciation du tubule puisque la perte de fonction combinée de Pou3f3 et Pou3f4 entraine un phénotype plus sévère que chaque simple perte de fonction
In Xenopus, pronephros is the functional kidney at tadpole stage. The blood is filtrated by the glomus and the urine is modifed all along the tubule. Pou3f3 is required for intermediate and distal tubule formation in mouse. During my PhD, we have analyzed the expression of the four pou3f genes during Xenopus embryonic development. We found that neural, otic, or epidermic expression of the various pou3f genes is conserved between Xenopus and mouse. Pou3f3 expression in the pronephros is similar to that observed in mouse. We futher showed for the first time pou3f4 expression in the developping kidney. Then, we analyzed the role of Pou3f4 during pronephros development. Pou3f4 depletion inhibits the expression of terminal differentiation marker genes in the intermediate and distal tubule. Pou3f4 upregulates the expression of slc12a1 and clcnkb1 in the tubule. Moreoer, we found that Pou3f4 loss of function leads to intermediate tubule morphogenesis defects. While ephrin signaling pathway is largely described as playing crucial role in cellular movement, Pou3f4 controls efnA3 expression in the intermediate and distal tubule. EfnA3 depletion phenocopies Pou3f4 loss of function, suggesting that Pou3f4, by regulationg efnA3 expression, is implicated in cell movements necessary for anterior elongation of the tubule. Finally, we find that pou3f3 and pou3f4 do not regulate each other expression. However, they act redundantly in pronephros development since the combined Pou3f3 and Pou3f4 loss of function results in a more severe phenotype than each loss of function alone

Hair cells are the mechanosensory cells of the inner ear whose loss causes irreversible hearing loss in mammals. POU4F3, a POU-domain transcription factor, is only expressed in hair cells in the inner ear and is essential for hair cell terminal maturation in mice. In humans, it is essential for hair cell survival as all three identified families with a POU4F3 mutation show autosomal dominant adult-onset progressive hearing loss. The pathways by which POU4F3 has this effect are unknown as little is known about its target genes (only four such genes are known: Bdnf, Ntf3, Gfi1 and Lhx3). Therefore, identification of unknown POU4F3 target genes would aid the understanding of hair cell maturation and survival. Prior to starting my doctoral work, a subtractive hybridization was carried out in an inner ear cell line (UB/OC-2) which was manipulated to either over- or under-express POU4F3 to identify unknown POU4F3 target genes. The clones produced by this screen were first matched to their corresponding gene; the presence of these genes in POU4F3-expressing UB/OC-2 cells was then confirmed by western blot and/or reverse transcriptase PCR. Where possible, expression was also confirmed in cochlear hair cells by immunofluorescence microscopy. Subsequently, the interaction of POU4F3 with these genes was characterised. Bioinformatics software was used to identify putative POU4F3 recognition elements in target gene promoters, and these sequences were then synthesised and used in a DNA binding assay (EMSA) to assess POU4F3 binding. The ability of POU4F3 to regulate putative gene promoters was also tested using luciferase assays. By these methods, one gene (Nr2f2) was shown to be a direct target of POU4F3 in vitro and two other genes were shown to be likely POU4F3 targets. The identification of these genes improves our understanding of the mechanism of POU4F3 function and further investigation of these networks may yield therapies for deafness caused by hair cell loss.

Understanding signalling pathways that control pancreatic endocrine tumour (PET) development and proliferation may reveal novel targets for therapeutic intervention. The pathogenesis for sporadic and hereditary PETs, apart from mutations of the MEN1 and VHL tumour suppressor genes, is still elusive. The protein product of the MEN1 gene, menin, regulates many genes. The aim of this thesis was to identify genes involved in pancreatic endocrine tumourigenesis, with special reference to Notch signalling.

Messenger RNA and protein expression of NOTCH1, HES1, HEY1, ASCL1, NEUROG3, NEUROD1, DLK1, POU3F4, PDX1, RPL10, DKK1 and TPH1 were studied in human PETs, sporadic and MEN 1, as well as in tumours from heterozygous Men1 mice. For comparison, normal and MEN1 non-tumourous human and mouse pancreatic specimens were used. Nuclear expression of HES1 was consistently absent in PETs. In mouse tumours this coincided with loss of menin expression, and there was a correlation between Men1 expression and several Notch signalling factors. A new phenotype consisting of numerous menin-expressing endocrine cell clusters, smaller than islets, was found in Men1 mice. Expression of NEUROG3 and NEUROD1 was predominantly localised to the cytoplasm in PETs and islets from MEN 1 patients and Men1 mice, whereas expression was solely nuclear in wt mice. Differences in expression levels of Pou3f4, Rpl10 and Dlk1 between islets of Men1 and wt mice were observed.

In addition, combined RNA interference and microarray expression analysis in the pancreatic endocrine cell line BON1 identified 158 target genes of ASCL1. For two of these, DKK1 (a negative regulator of the WNT/β-catenin signalling pathway) and TPH1, immunohistochemistry was performed on PETs. In concordance with the microarray finding, DKK1 expression showed an inverse relation to ASCL1 expression.

Altered subcellular localisation of HES1, NEUROD1 and NEUROG3 and down-regulation of DKK1 may contribute to tumourigenesis.

碩士
長庚大學
生物醫學研究所
98
Abstract Previously, we reported that EBV oncoprotein latent membrane protein 1 (LMP1) activates DNA methyltransferase 1 (DNMT1), which induces DNA hypermethylation and causes gene silencing in nasopharyngeal carcinoma (NPC). Aberrant DNA methylation is considered one of the major causes that lead to gene inactivation; hence, hypermethylation of tumor suppressor genes (TSGs) may promote cancer formation. Here, we identified a differential methylated gene, POU3F1, a transcription factor regulating keratinocyte differentiation and neurogenesis, in NPC tumor. Bisulfite sequencing results indicated that POU3F1 methylation percentage was relatively higher in NPC tumor when compared with adjacent normal tissues. The results were consistent with quantitative RT-PCR results where POU3F1 mRNA expression level was relatively low in NPC. Addition of 5’Aza-dC, a DNA methylation inhibitor, restored mRNA expression in NPC cells suggesting POU3F1 promoter activity could be regulated by DNA methylation. We further analyzed the regulation of POU3F1 promoter by Luc-reporter assay. There are four potential p300/CBP binding sites located at proximal POU3F1 promoter; over-expression of p300/CBP induced POU3F1 promoter activity in vitro. Preliminary functional study indicated that POU3F1 may reduce NPC cell proliferation rate. Its biological function in NPC will be discussed.

碩士
長庚大學
生物醫學研究所
100
Epstein-Barr virus (EBV) infection is associated with the nasopharyngeal carcinoma (NPC). An important oncoprotein of EBV, latent membrane protein 1 (LMP1) activates DNA methyltransferase (DNMT) and in turn leads to gene silencing due to aberrant DNA hypermethylation. Here, we identified a hypermethylated gene, POU3F1, a transcription factor that belongs to POU family, in NPC. Its mRNA expression is downregulated in tumor when compared with adjacent normal tissues. We performed bisulfite sequencing using NPC and adjacent normal tissues to locate POU3F1 differential methylated region. Our data showed that DMR is within POU3F1 open reading frame. To test whether DMR had enhancer activity, we constructed a series of POU3F1 promoter Luc reporter constructs: DMR sequence was inserted before or after POU3F1 promoter construct; and one promoter construct had reverse DMR sequence. The results showed that DMR could activate POU3F1 promoter activity independently of position or orientation, indicating DMR may act as an enhancer. To investigate the biological function of POU3F1, we generated lentivirus-based NPC stable cells expressing POU3F1 or vector control. Our results revealed that POU3F1 overexpression in NPC cells inhibited cell proliferation, reduced colony formation and delayed cell cycle progression. Moreover, in animal study, the POU3F1 -expressing NPC cells exhibited smaller tumor formation when compared with vector control. Together, these results demonstrated that POU3F1 may act as tumor suppressor in normal cell. Thus, silencing of POU3F1 via hypermethylation can inactivate this gene and in turn promote NPC progression.

碩士
國立中興大學
生物醫學研究所
104
Because cancer is the top ten caused death, the treatment is concern of every one of us. The treatment of cancer can be divided into three classes, surgical treatment, radiation therapy, and target therapy. Target therapy is different from other therapies. It can inhibit cancer cells proliferation but not normal cells by targeting a specific protein in cancer cells. Generally, these protein targets in cancer cells mutation proteins that may either gain of function or lose of function to resilt in uncontrolled cell growth. Those mutation proteins enhance cell proliferation and/or reduce cell death. Therefore, therapeutic agents that induces cell death by targeting those mutated proteins may help in cancer management. However most of cancer target therapies still have their limitations, therefore, it is important for us to find new target proteins. In our laboratory, we have identified that tNOX is only expressed in cancer cells and enhances cell proliferation , migration and metastasis. Therefore, it is important to understand the transcriptional regulation of this tumor associated tNOX protein. POU binding sites have been found in tNOX promoter region. In previous studies, POU2F1 is found to be a ubiquitous protein but other POU protein POU3F2 can recognize this POU2F1 binding site to modulate protein expressions. POU3F2 is classified to oncoprotein. POU3F2 and POU2F1 can cause different outcomes in the same gene. We decided to investigate whether POU3F2 regulates expression of tNOX. In this study, when we overexpressed POU3F2 in TMK cells, the tNOX expression did not change. However, tNOX protein increased when POU3F2 was expressed in AGS cells. On the other hand, we knocked down POU3F2 in A375 cells and found a reduction in tNOX expression. Subsequently, ERK and P-AKT were reduced which are important for cell proliferation regulation when tNOX were down regulated. Similarly, SIRT1 a deacetylase for p53 that is important for inhibiting apoptosis, was reduced when tNOX was downregulated which can deacetylated P53 and prevent apoptosis also showed the amount of decrease as the reduction of tNOX. Together, We propose that POU3F2 regulates expression of tNOX and affects cell survival and proliferation.

碩士
國立中興大學
生物醫學研究所
101
Neurotrophin-3 (NTF3) is a member of neurotrophin factors, a family of growth factors involved in nervous system and regulating diverse functions of central nervous system (CNS). NTF3 affects development of neurons in the embryo when it is expressed in human placenta and produced to maintain the adult nervous system functions, survival, differentiation, growth and synaptic plasticity. NTF3 also contributes in facilitating memory and implicated engaging in the pathophysiology of a wide variety of neurodegenerative and psychiatric disorders. The transcriptional regulation of NTF3 is poorly known. Here we hypothesize that POU domain, class 3, transcription factor 2 (POU3F2) may involves in the transcriptional regulation of NTF3 through bioinformatic prediction. POU3F2 is an important transcription factor that specifically expressed in central nervous system (CNS) and neuronal cell lineages and engaged in early stage of differentiation. POU3F2 regulates terminal hypothalamic neuronal differentiation and neuronal phenotypes, including activation of neuron specific gene expression, axonal excitation, and maintenance of neuronal viability. In current study, we try to validate the transcriptional regulation of NTF3 is modulated by POU3F2 and further to investigate its effect in neuron. At first, we establish an inducible neuron differentiation model by NT2/D1 cell line and confirm the characteristics of the differentiated neuron. Promoter-luciferase reporter assay is applied to examine transcriptional regulation role of POU3F2 on the promoter of NTF3 gene. The DNA-protein binding assay of NTF3 promoter and POU3F2 are also performed by EMSA assay. Furthermore, alteration of NTF3 and POU3F2 expression level during neuron differentiation process is also been measured. Results from this study will contribute to the understanding of POU3F2-mediated NTF3 regulation in CNS and provide important insights in related neuropsychiatric diseases.

碩士
國立中興大學
生物醫學研究所
100
POU3F2 belongs to a large family of brain-specific homeobox transcription factors that bind to an octameric DNA sequence. POU3F2 gene is expressed in the central nervous system (CNS) during neuronal development and in adult brain. POU3F2 has been proposed to participate in several important modulation of CNS-related gene expression, including melination of schwann cells, cell fate decision of neural precursor cells, melanoma metastasis and so on. Above evidences suggest its important role in regulating neuronal gene expression and may involved in pathogenesis of various neuron related diseases. Variations profiling of POU3F2 in our own population is crucial for further genetic association studies of a variety of neuron-related diseases. Here we applied the direct sequencing method to systematically screen the sequence variations of POU3F2 gene in Chinese Han population. We designed primers to amplify and sequence the single exon and up to 1 kb of 5’ regulatory regions of the human POU3F2 gene. We have systematically sequenced most of the functional regions of the human POU3F2 gene in a panel of 54 samples, We also resort our study to faster gene variation screening technology which are meant to be more effective in this type of research. High resolution melting (HRM) technology is utilized to screen POU3F2 variation region. It is interesting to know whether HRM could be more effetive than the traditional sanger sequencing technology in screening the POU3F2 gene variation. We have identified 12 single nucleotide variations (SNV) of POU3F2 gene in the chinese Han population. And two of 12 SNVs have not been reported in any public databases or literature according to our knowledge, thus they are new and probably population-specific polymorphisms in Han Chinese. Among the twelve SNVs of POU3F2 gene, three are located on 5’ flanking region, one is located on coding region, and eight are located on the 3’ untranslated region. In addition, bioinformatic tools were applied to predict the putative function of POU3F2 variations, and will be validated further functional assays. Our study will provide information that could contribute to further genetic illustration of POU3F2 gene with different CNS-related phenotype/diseases. The data of POU3F2 gene variation and genetic analysis in our research, may be a good reference for Taiwan genetic research.

碩士
國立中興大學
生物醫學研究所
107
Taiwan is one of the regions with high prevalence of gastric cancer. However, the pathogenesis of gastric cancer is extremely complicated and has not been thoroughly studied. POU3F2 is a POU-homeodomain transcription factor, and is thought to play important roles in neuronal differentiation, melanocyte growth, differentiation and tumorigenesis. However, the function of POU3F2 in gastric cancer has not been well studied. We apply the bioinformatics tools to predict the transcriptional target genes of POU3F2, and found that POU3F2 may regulate several apoptosis genes including GADD45A. Here, we report that overexpression of POU3F2 increase apoptosis in gastric cancer AGS cells by Flow Cytometry. The RNA and protein level of the known POU3F2 transcriptional target gene GADD45A are increased by the overexpression of POU3F2, and lead to the activation of the downstream protein p38. In addition, in POU3F2-overexpressed AGS cells, increase of the phosphorylated p53, APAF-1 expression, PARP cleavage, and downregulation of the anti-apoptotic protein Bcl-2 appeared to be associated with the increase in apoptosis response, and vice versa. Furthermore, both p53 knockdown and p38 inhibiton can prevent the apoptosis induced by overexpression of POU3F2. And the overexpression of POU3F2 in gastric cancer KATO III cells (p53-null) can increase the expression of GADD45A and the phosphorylation of p38, but cannot promote apoptosis. We also found UVB radiation and alkylating agent will increase POU3F2 expression and promote the following apoptosis pathway through GADD45A─p38─p53 axis. Taken together, our results provide the first evidence for a molecular mechanism of POU3F2 acts through GADD45A─p38─p53 axis to induce apoptosis in gastric cancer cells.

博士
國防醫學院
生命科學研究所
103
The association between breast cancer risk and genetic variants of fibroblast growth factor receptor 2 (FGFR2) has been identified and repeatedly confirmed; however, the mechanism underlying the involvement of FGFR2 in breast tumorigenesis remains obscure. Given that breast tumorigenesis is particularly related to DNA double-strand break repair (DSBR), we examined the hypothesis that FGFR2 is involved in DSBR. Our results showed that expression of Mre11, a vital exonuclease in DSBR, was downregulated by FGFR2, which was further linked to decreased DSBR. Analysis of the Mre11 promoter revealed that POU1F1 mediated FGFR2-induced Mre11 downregulation. Furthermore, ERK, which functions downstream of FGFR2, directly interacted with and phosphorylated POU1F1, increasing POU1F1 binding capacity to the Mre11 promoter and repressing Mre11 expression, which consequently affected DSBR and sensitized breast cancer cells to chemotherapeutic treatments. The importance of the FGFR2-Mre11-DSBR link in cancer progression is suggested by the finding that genotypes of FGFR2 and Mre11 were associated with the survival of breast cancer patients and that FGFR2 expression correlated with cancer prognosis specifically in patients receiving chemotherapy. This study yields important insight into the role of FGFR2 in breast tumorigenesis and may facilitate development of a useful therapeutic approach for breast cancer.

碩士
國立中興大學
生物醫學研究所
102
Abstract Bipolar affective disorder (BPD) is one of the most common and complex mood disorder, characterized by mood swinging back and forth between mania and depression episodes. Little is known about the pathophysiology of BPD. However, the previous twins, family, and adoption studies of BPD indicated that BPD has strong genetic components. Increased of amygdala volume, larger ventricles volume and deficit of GABAergic interneurons in cerebral cortex were observed in BPD patients in neuroimaging and postmortem studies. These findings imply some defect on neuronal development may contribute to BPD pathogenesis. POU3F2 is a transcriptional factor that most likely expressed in CNS, and plays an important role in regulating neuronal differentiation related gene expression, including cell fate decision of neuronal precursor cells, Schwann cell development, and so on. Although POU3F2 is involved in neuronal lineage determination, limited studies regarding POU3F2 gene and its effects on neuropsychiatric disorder have been reported. Here we aim to analyze the role of POU3F2 gene in etiology of BPD. Our team have systematically screened the POU3F2 locus, and identified twelve variants of POU3F2 gene. Here, we select six SNPs to perform BPD association study. Single locus association, linkage disequilibrium analysis, and haplotypic association study were applied to test the genetic association between POU3F2 and BPD. We found the different LD patterns of SNP g.-291G>A between BPD and controls. For studying the biological function of g.-291G>A, promoter reporter functional assay is done and find g.-291G>A of POU3F2 promoter will affect the gene expression in neuroblastoma cells. Results from our current study imply POU3F2 gene has a role in BPD.

碩士
國立中興大學
生物醫學研究所
104
Cancer has been ranked as the first leading cause of death, and gastric cancer is the seventh most common cause of cancer-related death in Taiwan. Although complete surgical resection remains the only curative modality for early stage gastric cancer, surgery alone only provides long-term survival in 20% of patients with advanced-stage disease. The mechanism in gastric cancer is still unclear. Our study helps us to understand more about the mechanisms of gastric cancer. POU3F2 is a POU-Homeodomain transcription factor, and has been proved to play a critical role in melanoma growth via promote cell proliferation. The POU3F2 gene has been found in several melanoma cell lines, but not in melanocytes, and evidences implicate its role in melanoma growth via promote cell proliferation, migration and invasion, while inhibits POU3F2 expression shown opposite results. However, the role of POU3F2 in gastric cancer has not been investigated. In this study, we attempted to clarify the role of POU3F2 in gastric cancer. We found the POU3F2 also promote gastric adenocarcinoma AGS cell proliferation by direct cell counting ,cell doubling time (fluorometric) and xCELLigence System (electrical impedance) cell viability assay, and we also observed that POU3F2 promote level of phosphorylated Akt and phosphorylated ERK. Moreover, overexpression POU3F2 also promote invasion and migration by transwell assay in AGS cell. We will further elucidating the multiple role of POU3F2 in gastric cancer progression, and to observe the effects on gastric cancer by using shPOU3F2 to knowdown POU3F2.

碩士
國立中興大學
生物醫學研究所
107
Gastric cancer (GC) is one of the most severe cancers in the world with high incidence and low survival rate. Elucidation of the molecular mechanisms underlying GC pathogenesis is important and could contribute to improve clinical practice of GC. POU3F2 is a POU-homeodomain transcription factor mainly expressed in the central nervous system (CNS) during neuronal development and in adult brain.. Recently, accumulating evidence suggests that POU3F2 is also involved in different types of cancer. Our laboratory has previously found that POU3F2 can promote proliferation, invasion and metastasis of gastric cancer cells. Although POU3F2 seems to be important for gastric cancer progression, little is known about the downstream genes and underlying mechanisms involved in these tumorigenic effects. In this study, we applied the bioinformatic tools to predict the POU3F2 putative target genes and found several genes involved in autophagy pathway may under the POU3F2 transcriptional regulation. Using flow cytometry, we show that overexpression of POU3F2 in gastric cancer AGS cells repress autophagic process and RNA/protein expression level of several autophagy related genes, ATG5 and ATG7 included. In contrast, knockdown of POU3F2 upregulate ATG7 expression level and autophagic process. Using the luciferase reporter assay, we further demonstrate that POU3F2 can affect the transcriptional regulation of ATG7. Therefore, we propose that POU3F2 regulates the ATG7 gene through transcriptional regulation and participates in the inhibition of autophagy in gastric cancer cells.

Genetic and Hormonal Regulation of Children's Growth MUDr. Jan Vosáhlo Abstract Growth in childhood is a complex process of changing the body, which can be disrupted by various illnesses including endocrine disorders, particularly growth hormone deficiency. Tumors or other processes affecting hypothalamic-pituitary area can be a postnatal cause of GHD; prenatal causes include 1) developmental disorders of the pituitary as part of complex syndromes, 2) developmental disorders of the pituitary due to defects in regulatory genes and 3) defects in genes involved in the synthesis and secretion of GH. The first topic of the thesis was septo-optic dysplasia - a complex syndrome involving optic nerve hypoplasia, structural brain abnormalities and pituitary dysfunctions. We extensively described phenotype in 11 Czech patients; we observed both complete SOD and incomplete forms variously combining two of the three main components of the syndrome. The cohort then became a part of an international study of 68 patients, in which we studied the phenotype in dependence on the brain morphology. We found correlation between the severity of clinical symptoms and the degree of septum pellucidum abnormities and also a correlation between hippocampus and falx abnormities and neurological symptoms. As the second topic we studied...