The development of an organism and its adult homeostasis rely on regulatory mechanisms that control the underlying gene expression programs. In certain biological contexts, such as germ cell development, gene expression regulation is largely executed at the post-­‐transcriptional level. This relies on RNA-­‐binding proteins (RBPs), whose activity and expression are also heavily controlled. While the RNA-­‐binding potential of RBPs is currently of intense scrutiny, surprisingly little is known to date about the molecular mechanisms that control RNA-­‐binding proteins abundance in the context of germ cell development. This work identifies the molecular mechanisms that shape expression patterns of two evolutionarily conserved RNA-­‐binding proteins, CPB-­‐3 and GLD-­‐ 1, which belong to CPEB and STAR protein family, respectively. By focusing on their regulation in the C. elegans germ line, this work reveals an involvement of the proteasome in reducing levels of CPB-­‐3/CPEB and GLD-­‐1/STAR at the pachytene-­‐to-­‐diplotene transition during meiotic prophase I. Furthermore, it documents that CPB-­‐3 and GLD-­‐1 are targeted to proteasomal degradation by a conserved SCF ubiquitin ligase complex that utilises SEL-­‐10/Fbxw7 as a substrate recognition subunit. Importantly, destabilisation of both RBPs is likely triggered by their phosphorylation, which is regulated by the mitogen-­‐activated protein kinase, MPK-­‐1, and restricted to the meiotic timepoint of pachytene exit. Lastly, this work investigates the potential consequences of target mRNA regulation upon delayed RBP degradation. Altogether, the collected data characterise a molecular pathway of CPEB and STAR protein turnover, and suggest that MPK-­‐1 signaling may couple RBP-­‐mediated regulation of gene expression to progression through meiosis during oogenesis.

Plant reproductive development begins when vegetative shoot apical meristems change their fate to inflorescence meristems which develop floral meristems on the flanks. This process of meristem fate change and organ development involves regulated activation and/or repression of many cell fate determining factors that execute down-stream gene expression cascades. Flowers are formed when floral organs are specified on the floral meristem in four concentric whorls. In the model dicot plant Arabidopsis, the identity and pattern of floral organs is determined by combined actions of MADS-domain containing transcription factors of the classes A, B, C, D and E. Rice florets are produced on a compact higher order branch of the inflorescence and have morphologically distinct non-reproductive organs that are positioned peripheral to the male and female reproductive organs. These unique outer organs are the lemma and palea that create a closed floret internal to which are a pair of lodicules that are asymmetrically positioned fleshy and reduced petal-like organs. The unique morphology of these rice floret organs pose intriguing questions on how evolutionary conserved floral meristem specifying and organ fate determining factors bring about their distinct developmental functions in rice. We have studied the functions for two rice MADS-box proteins, OsMADS2 and OsMADS1, to understand their role as master regulators of gene expression during rice floret meristem specification and organ development. OsMADS2; a transcriptional regulator of genes expression required for lodicule development Arabidopsis B-function genes AP3 and PI are stably expressed in the whorl 2 and 3 organ primordia and they together with other MADS-factors (Class A+E or C+E) regulate the differentiation of petals and stamens (Jack et al, 1992; Goto and Meyerowitz, 1994). Rice has a single AP3 ortholog, SPW1 (OsMADS16) but has duplicated PI-like genes, OsMADS2 and OsMADS4. Prior studies in our lab on one of these rice PI-like genes OsMADS2 showed that it is needed for lodicule development but is dispensable for stamen specification (Kang et al., 1998; Prasad and Vijayraghavan, 2003). Functional divergence between OsMADS2 and OsMADS4 may arise from protein divergence or from differences in their expression patterns within lodicule and stamen whorls. In this study, we have examined the dynamic expression pattern of both rice PI-like genes and have examined the likelihood of their functional redundancy for lodicule development. We show OsMADS2 transcripts occur at high levels in developing lodicules and transcripts are at reduced levels in stamens. In fully differentiated lodicules, OsMADS2 transcripts are more abundant in the distal and peripheral regions of lodicules, which are the tissues that are severely affected in OsMADS2 knock-down florets (Prasad and Vijayraghavan, 2003). The onset of OsMADS4 expression is in very young floret meristems before organ primordia emergence and this is expressed before OsMADS2. In florets undergoing organogenesis, high level OsMADS4 expression occurs in stamens and carpels and transcripts are at low level in lodicules (Yadav, Prasad and Vijayraghvan, 2007). Thus, we show that these paralogous genes differ in the onset of their activation and their stable transcript distribution within lodicules and stamens that are the conserved expression domains for PI-like genes. Since the expression of OsMADS4 in OsMADS2 knock-down florets is normal, our results show OsMADS2 has unique functions in lodicule development. Thus our data show subfunctionalization of these paralogous rice PI-like genes. To identify target genes regulated by OsMADS2 that could contribute to lodicule differentiation, we have adopted whole genome transcript analysis of wild-type and dsRNAiOsMADS2 panicles with developing florets. This analysis has identified potential down-stream targets of OsMADS2 many of which encode transcription factors, components of cell division cycle and signalling factors whose activities likely control lodicule differentiation. The expression levels of few candidate targets of OsMADS2 were examined in various floret organs. Further, the spatial expression pattern for four of these down-stream targets of OsMADS2 was analysed and we find overlap with OsMADS2 expression domains (Yadav, Prasad and Vijayraghvan, 2007). The predicted functions of these OsMADS2 target genes can explain the regulation of growth and unique vascular differentiation of this short fleshy modified petal analog. OsMADS1, a rice E-class gene, is a master regulator of other transcription factors and auxin and cytokinin signalling pathways In Arabidopsis four redundant SEPALLATA factors (E-class) are co-activators of other floral organ fate determining MADS-domain factors (classes ABCD) and thus contribute to floral meristem and floral organ development (Krizek and Fletcher, 2005). Among the grass-specific sub-clade of SEP-like genes, rice OsMADS1 is the best characterized. Prior studies in our lab showed that OsMADS1 is expressed early throughout the floret meristem before organ primordia emergence and later is restricted to the developing lemma and palea primordia with weak expression in carpel (Prasad et al, 2001). Stable expression continues in these floret organs. OsMADS1 plays critical non-redundant functions to specify a determinate floret meristem and also regulates floret organ identities (Jeon et al., 2000; Prasad et al, 2001; 2005; Agarwal et al., 2005; Chen et al., 2006). In the present study, we have adopted two different functional genomic approaches to identify genes down-stream of OsMADS1 in order to understand its mechanism of action during floret development. We have studied global transcript profiles in WT and dsRNAiOsMADS1 panicles and find OsMADS1 is a master regulator of a significant fraction of the genome’s transcription factors and also a number of genes involved in hormone-dependent cell signalling. We have validated few representative genes for transcription factors as targets regulated by OsMADS1. In a complementary approach, we have determined the consequences of induced-ectopic over-expression of a OsMADS1:ΔGR fusion protein in shoot apical meristems of transgenic plants. Transcript levels for candidate target genes were assessed in induced tissues and compared to mock-treated meristems and also with meristems induced for OsMADS1:ΔGR but blocked for new protein synthesis. These analyses show that OsMADS55 expression is directly regulated by OsMADS1. Importantly, OsMADS55 is related to SVP that plays an important role in floral transition and floral meristem identity in Arabidopsis. OsHB3 and OsHB4, homeodomain transcription factors, with a probable role in meristem function, are also directly regulated by OsMADS1. The regulation of such genes by OsMADS1 can explain its role in floret meristem specification. In addition to regulating other transcription factors, OsMADS1 knock-down affects expression of genes encoding proteins in various steps of auxin and cytokinin signalling pathways. Our differential expression profiling showed OsMADS1 positively regulates the auxin signalling pathway and negatively regulates cytokinin mediated signalling events. Through our induced ectopic expression studies of OsMADS1:ΔGR, we show OsMADS1 directly regulates the expression of OsETTIN2, an auxin response transcription factor, during floret development. Overall, we demonstrate that OsMADS1 modulates hormonal pathways to execute its functions during floret development on the spikelet meristems. Functional studies of OsMGH3; an auxin-responsive indirect target of OsMADS1 To better understand the contribution of auxin signalling during floret development, we have functionally characterized OsMGH3, a down-stream indirect target of OsMADS1, which is a member of the auxin-responsive GH3 family. The members of this family are direct targets of auxin response factors (ARF) class of transcription factors. GH3-proteins inactivate cellular auxin by conjugating them with amino acids and thus regulate auxin homeostasis in Arabidopsis (Staswick et al., 2005). OsMGH3 expression in rice florets overlaps with that of OsMADS1 (Prasad et al, 2005). In this study, we have demonstrated the consequences of OsMGH3 over-expression and knock-down. The over-expression of OsMGH3 during vegetative development causes auxin-deficient phenotypes such as dwarfism and loss of apical dominance. Its over-expression in developing panicles that was obtained by driving its expression from tissue-specific promoters created short panicles with reduced branching. The latter is a phenotype similar to that observed upon over-expression of OsMADS1. In contrast, the down-regulation of endogenous OsMGH3 through RNA-interference produced auxin over-production phenotypes such as ectopic rooting from aerial nodes. Knock-down of OsMGH3 expression in florets affected carpel development and pollen viability both of which affect floret fertility. Taken together, this study provides evidence for the importance of auxin homeostasis and its transcriptional regulation during rice panicle branching and floret organ development. Our analysis of various conserved transcription factors during rice floret development suggest that factors like OsMADS2, OsMADS4 and OsMADS1 are master regulators of gene expression during floret meristem specification and organ development. The target genes regulated by these factors contribute to development of morphologically distinct rice florets.

Le système ubiquitine-protéasome joue un rôle central dans le contrôle de la progression du cycle cellulaire par la dégradation régulée de nombreuses protéines. Dans ce système, Fbw7 (aussi appelée Fbxw7, hCdc4, hAgo, Sel-10), est l'une des protéines F-box qui sert d'adaptateur de substrats pour l'une des plus importantes familles d'ubiquitine ligases : les complexes SCF (Skp1/Cullin/ F-box). Fbw7 assure la dégradation de plusieurs régulateurs positifs du cycle cellulaire : la cycline E, cMyc, c-Jun, Notch, Aurora A, mTOR, MCL1. En conséquence, l'altération des fonctions de Fbw7 conduit à des défauts de prolifération cellulaire, de différenciation et à de l'instabilité génomique. La mutation de Fbw7 dans les cancers entraîne une dérégulation de l'expression périodique cycline E qui n'est alors plus restreinte à la transition G1/S du cycle cellulaire. Nos résultats montrent qu'une isoforme, Fbw7, est exprimée dans les œufs de xénope matures arrêtés en métaphase II mais n'est pas fonctionnelle, expliquant la présence de grande quantité de cycline E dans les œufs à cette phase mitotique. Nous montrons que Fbw7 est maintenue inactive sous forme poly-ubiquitylée suite à sa phosphorylation par une PKC jusqu'à la fin des cycles embryonnaires rapides, au moment où la cycline E est brutalement dégradée. Nous montrons que la régulation négative de Fbw7 par PKC est conservée au cours des cycles cellulaires somatiques des cellules humaines, et contribue à l'expression périodique de la cycline E. Ces résultats mettent en évidence un nouveau mécanisme critique pour la régulation de Fbw7 au cours du cycle cellulaire et suggèrent que les fonctions de Fbw7 peuvent être altérées par une dérégulation de PKC, un phénomène observé dans de nombreux types de tumeurs humaines. / The ubiquitin-proteasome system plays a central role in the control of cell cycle progression through the regulated degradation of numerous critical proteins. In this process, one key family of ubiquitin ligases are the SCF (Skp1/Cul-1/F-box) complexes, in which F-box-bearing proteins act as substrate-recruiting factors. Fbw7 (also known as Fbxw7, hCdc4, hAgo, Sel-10) is one such F-box protein. It controls the stability and thus the levels of several positive regulators of the cell cycle, including cyclin E, cMyc, c-Jun, Notch, Aurora A, mTOR, Mcl1. As a consequence of its biological roles, alterations of the functions of Fbw7 lead to defects in cellular proliferation, differentiation and genetic instability. As seen in cancers, mutation of Fbw7 leads to deregulation of cyclin E expression, which is no more restricted to the G1-S phase boundary of the cell cycle. Here we report that Fbw7, although expressed in mature Xenopus eggs arrested in metaphase II, is not functional, explaining why cyclin E can be stockpiled in this mitotic-like phase. We found that, in these eggs as well as in early Xenopus embryos, Fbw7 is maintained under a PKC-dependent poly-ubiquitylated state until the end of the early rapid cleavage cycles where cyclin E is abruptly degraded. Importantly, we show that this PKC-dependent negative regulation of Fbw7 is conserved during human somatic cell cycles, resulting into the periodic expression of cyclin E. These findings reveal a novel mechanism critical for the temporal regulation of Fbw7 and suggest that the key functions of Fbw7 can be altered by PKC dysregulation, a mechanism known to occur in many types of human tumours.

The development of an organism and its adult homeostasis rely on regulatory mechanisms that control the underlying gene expression programs. In certain biological contexts, such as germ cell development, gene expression regulation is largely executed at the post-­‐transcriptional level. This relies on RNA-­‐binding proteins (RBPs), whose activity and expression are also heavily controlled. While the RNA-­‐binding potential of RBPs is currently of intense scrutiny, surprisingly little is known to date about the molecular mechanisms that control RNA-­‐binding proteins abundance in the context of germ cell development. This work identifies the molecular mechanisms that shape expression patterns of two evolutionarily conserved RNA-­‐binding proteins, CPB-­‐3 and GLD-­‐ 1, which belong to CPEB and STAR protein family, respectively. By focusing on their regulation in the C. elegans germ line, this work reveals an involvement of the proteasome in reducing levels of CPB-­‐3/CPEB and GLD-­‐1/STAR at the pachytene-­‐to-­‐diplotene transition during meiotic prophase I. Furthermore, it documents that CPB-­‐3 and GLD-­‐1 are targeted to proteasomal degradation by a conserved SCF ubiquitin ligase complex that utilises SEL-­‐10/Fbxw7 as a substrate recognition subunit. Importantly, destabilisation of both RBPs is likely triggered by their phosphorylation, which is regulated by the mitogen-­‐activated protein kinase, MPK-­‐1, and restricted to the meiotic timepoint of pachytene exit. Lastly, this work investigates the potential consequences of target mRNA regulation upon delayed RBP degradation. Altogether, the collected data characterise a molecular pathway of CPEB and STAR protein turnover, and suggest that MPK-­‐1 signaling may couple RBP-­‐mediated regulation of gene expression to progression through meiosis during oogenesis.

Einer der Gründe für das Scheitern derzeitiger Behandlungsmethoden beim Brustkrebs ist die Resistenz gegenüber der angewandten Chemotherapie. Eine große Rolle bei der Entstehung der Multiplen Medikamentenresistenz spielt das MDR1-Gen und sein Genprodukt, das P-Glykoprotein. Das Y-Box-Protein YB-1 reguliert die Expression des MDR1-Gens; eine Überexpression und nukleäre Lokalisation von YB-1 geht im Brustkrebs mit einer gesteigerten P-Glykoprotein Expression einher. In dieser Arbeit wurden Gewebeproben von 83 Brustkrebspatientinnen auf eine YB-1 Überexpression im Tumor und im peritumoralen Epithel untersucht. YB-1 wurde mittels der immunhistochemischen APAAP-Methode an Formalin-fixierten, in Paraffin eingebetteten Brustkrebsgewebeproben nachgewiesen. Die klinische Relevanz der YB-1 Expression wurde untersucht, indem sie mit dem klinischen Verlauf in einem mittleren Beobachtungszeitraum von 61 Monaten und etablierten biologischen Tumorfaktoren wie Lymphknotenstatus, histologisches Grading, Tumorgröße, Hormonrezeptorstatus, uPA und PAI-1 verglichen wurde. In der Kohorte der Patientinnen mit einer postoperativen adjuvanten Chemotherapie zeigte sich eine 5-Jahres-Rezidivrate von 68 % bei einer hohen YB-1 Expression im Tumor und eine Rückfallrate von 39 % bei einer niedrigen YB-1 Expression. Unter Beachtung auch der YB-1 Expression im peritumoralen Epithel konnte ein noch größerer Unterschied hinsichtlich der 5-Jahres-Rezidivrate festgestellt werden. Diese betrug bei Patientinnen mit einer hohen YB-1 Expression 66 %, während bei Patientinnen mit einer niedrigen YB-1 Expression im Nachbeobachtungszeitraum kein Rezidiv festgestellt wurde. Bei der Gegenüberstellung der 5-Jahres-Rezidivraten in der Kohorte der Patientinnen ohne Zytostatikatherapie zeigte sich eine Rückfallrate von 30 % bei einer hohen YB-1 Expression und eine Rückfallrate von 0 % bei einer niedrigen YB-1 Expression. Eine hohe YB-1 Expression war demnach in beiden Kohorten mit einer schlechteren klinischen Prognose assoziiert. Das Ergebnis in der Gruppe der Patientinnen ohne postoperative Chemotherapie zeigt, dass YB-1 mit der Tumoraggressivität beim Brustkrebs korreliert. Eine Korrelation zwischen der YB-1 Expression und den etablierten prognostischen Faktoren Lymphknotenstatus, Tumorgröße und histologisches Grading konnte nicht festgestellt werden. Es wurde jedoch eine signifikante negative Korrelation zwischen der YB-1 Expression und dem Hormonrezeptorstatus und eine positive Korrelation zwischen YB-1 und den Faktoren uPA und PAI-1 gefunden. In dieser Arbeit wurde gezeigt, dass YB-1 eine klinische Relevanz besitzt mit Hinblick sowohl auf eine prognostische als auch eine prädiktive Bedeutung bei der Identifikation von Hoch-Risiko-Patientinnen im Brustkrebs in Ab- und Anwesenheit einer postoperativen Chemotherapie. / Intrinsic or acquired resistance to chemotherapy is one of the reasons for failure of current treatment regimens in breast cancer patients. P-glycoprotein and its gene mdr1 plays a major role in the development of a multi-drug resistant tumor phenotype. The Y-box protein YB-1 regulates the expression of mdr1. In human breast cancer, overexpression and nuclear localization is associated with upregulation of P-glycoprotein. In this study, tissues of 83 breast cancer patients have been analyzed with regard to YB-1 overexpression in tumor tissue and in surrounding benign breast epithelial cells. YB-1 has been detached by the immunohistochemical APAAP-method using formalin-fixed, paraffin-embedded breast cancer tissues. Clinical relevance of YB-1 expression was analyzed by comparing it with clinical outcome after a median follow-up of 61 months and with tumor biological factors lymph-node status, tumor size, histological grading, hormone-receptor status and the factors uPA and PAI-1. In patients who received postoperative chemotherapy, the 5-year-relapse rate was 68% in patients with high YB-1 expression in tumor cells and 39% in patients with low expression. With regard to YB-1 expression in surrounding benign breast epithelial cells, the 5-year-relapse rate was 66% in patients with high YB-1 expression whereas in patients with low expression no relapse has been observed so far. YB-1 thus indicates clinical drug resistance in breast cancer. In patients who received no chemotherapy, the 5-year-relapse rate was 30% in patients with high YB-1 expression whereas in patients with low YB-1 expression no relapse occurred. YB-1 thus correlates with breast cancer aggressiveness. In both groups high YB-1 expression was associated with poor clinical outcome. A correlation between YB-1 and tumor biological factors lymph-node status, tumor size and histological grading has not been found. But a significant negative correlation has been observed between YB-1 and hormone-receptor status and a positive correlation between YB-1 and uPA and PAI-1. This dissertation could show the clinical relevance of YB-1 with regard to a prognostic and predictive significance by identifying a high-risk group of breast cancer patients both in presence and absence of postoperative chemotherapy.

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