Dissertations / Theses on the topic 'ANKRD11'

Platelets (thrombocytes) mediate clot formation following vessel injury. Inherited thrombocytopenias, characterised by platelet counts below 150 x 109 L-1, have variable bleeding severity. Whole exome sequencing of two cousins with severe thrombocytopenia identified a shared single-codon deletion in ANKRD18A. ANKRD18A’s function is unknown. We hypothesise that this mutation in ANKRD18A is the aetiology of these children’s thrombocytopenia. Despite limited patients’ blood, quantitative real-time polymerase chain reaction demonstrated reduced ANKRD18A mRNA levels in patient leukocytes compared to controls. Patients’ platelets demonstrated decreased activity when stimulated with adenosine diphosphate, collagen related peptide and the synthetic peptide SFLLRN, which mimics the N-terminal sequence of protease activated receptors. Electron microscopy demonstrated macrothrombocytopenia. To assist characterisation studies, a recombinant fragment of ANKRD18A, the ankyrin repeat domain, was purified to generate a polyclonal antibody, α-25277. Experiments demonstrated possible specificity of α-25277. Cellular functions of ANKRD18A remain unclear, yet the availability of α-25277, once better characterised, will be instrumental in demonstrating the potential link between ANKRD18A and the observed thrombocytopenia. Another in-house antibody, α-21234, may also be effective. Antibody specificity can be proven by immunoblotting lysates of cells, where ANKRD18A is silenced by RNAi. Should both antibodies not be specific, generating a monoclonal antibody is requisite.

Les thrombopénies héréditaires sont une classe de maladies hématologiques congénitaux affectant principalement la lignée mégacaryocytaire. Elle s’accompagnant d'une diminution du nombre de plaquettes. Près de 50 gènes différents ont été associés à des thrombopénies héréditaires, et d'énormes différences existent entre chaque maladie, en ce qui concerne la manifestation clinique et la pathobiologie. Mes recherches ont porté sur deux thrombopénies héréditaires différentes: Filaminopathie A et THC2. La première maladie est un syndrome X-linked, associé à des mutations dans le gène FLNA (Filamine A). Ces patients, sur le point de vue hématologique, présentent une macrothrombopénie associée à une tendance à saigner toute la vie. La seconde maladie est transmise selon une mécanisme autosomale dominante, et elle est provoquée par des mutations dans le 5’ UTR du gène ANKRD26. Les patients montre une dysmégacaryopoïèse, une thrombopénie et un risque accru de développer des tumeurs malignes myéloïdes. Pour étudier la physiopathologie de ces deux maladies rares, j'ai exploité la technologie des cellules souches pluripotentes induites, pour développer plusieurs lignées cellulaires spécifiques au patient. Ces outils expérimentaux ont permis la compréhension de la physiopathologie pour les deux maladies, et m'ont permis de décrire en grand détail les mécanismes moléculaires sous-tendant le defaut de la formation de proplaquettes pour la Filaminopatie A, et la prédisposition à la leucémie pour la THC2. Pour effectuer de telles études, nous avons conçu un protocole de différenciation robuste, récapitulant efficacement la différenciation hématopoïetique et facilement adapté à la différenciation in vitro des plusieurs lignées cellulaires. En plus, nous avons exploité une technique de modification du génome pour introduire efficacement différents mutants, afin de disséquer le rôle moléculaire de la FLNa dans la mégacaryopoïèse.En ce qui concerne la filaminopathie A, nous avons pu décrire une relation originale entre une intégrine membranaire (IIb3), la Filamine A et une voie de signalization curciale (RhoA) pour la mégacaryopoïèse. Nos données supportent un modèle où l’absence de la FLNa induit une activité anormale de la voie RhoA, en réponse au lien entre l'intégrine IIb3 et le fibrinogène. Concernant la THC2, nous avons décrit un nouveau mécanisme associant l'augmentation de l'expression de ANKRD26 à une activité dérégulée de la voie de signalisation dépendante du G-CSF. Cette anomalie affecte la granulopoïèse et conduit à une amplification anormale de cette lignée cellulaire, augmentant éventuellement le risque d'acquisition d'autres mutations et de progression vers une leucémie myéloïde.En conclusion, avec ce travail, nous offrons une preuve de concept de la potentialité de cellules souches pluripotentes induites pour la modélisation de maladies. Nos résultats ouvrent la voie à d'autres études susceptibles de faire progresser notre compréhension de la physiopathologie des troubles plaquettaires héréditaires
Inherited thrombocytopenias are a class of congenital haematological disorders affecting primarily the megakaryocytic lineage and accomunated by a decrease in platelet numbers. Almost 50 different genes have been associated to inherited platelet disorders, and huge differences exist between each disorder, in regard to clinical manifestation and pathobiology. My research interest have been focused on two different congenital thrombocytopenias: Filaminopathy A and Thrombocytopenia 2. The first disease is a X-linked syndrome associated to mutations in the gene FLNA (Filamin A), and patients display a mild to severe macrothrombocytopenia, associated with a lifelong bleeding tendency. The second disorder is an automal dominant condition caused by mutations in the 5’ UTR of the ANKRD26 gene. It is associated with dysmegakaryopoiesis, mild to severe thrombocytopenia and an increased risk to develop myeloid malignancies. To study the physiopathology of those two rare diseases, we have exploited the induced pluripotent stem cell technology to develop several patient specific cell lines. Those experimental tools revealed invaluable for the understanding of the disease physiopathology, and allowed us to describe in great details the molecular mechanisms underlying the reduction in proplatelet formation for Filaminopathy A and the predisposition to leukemia for Thrombocytopenia 2. To perform such studies, we devised a robust differentiation protocol, recapitulating efficiently the haematopoietic differentiation and easily adapted to the in vitro differentiation of multiple cell lineages. Furthermore, we exploited a genome editing technique to introduce efficiently different protein mutants, in order to dissect the molecular role of Filamin A in megakaryopoiesis. In regard of Filaminopathy A, we have been able to describe an original and novel relationship between a membrane integrin (IIb3), Filamin A and a crucial signalling pathway (RhoA) for megakaryopoiesis. Our data support a model where the absence of FLNa induces an abnormal activity of the RhoA pathway, in response to the integrin IIb3 binding to fibrinogen. Concerning the thrombocytopenia 2, we described a novel mechanism that associated the increased expression of ANKRD26 to a deregulated activity of the G-CSF-dependent signalling pathway. This anomaly impacts the normal granulopoiesis and lead to an abnormal amplification of this cell lineage, possibly increasing the risk of acquiring other mutational hits and progress towards a myeloid malignancy.In conclusion, with this work we offer a proof of concept of the potentiality of disease modeling via induced pluripotent stem cells. Our results pave the way for further studies that could advance our understanding of the physiopathology of inherited platelet disorders

Les récepteurs couplés aux protéines G (RCPG) constituent la plus grande famille de protéines transmembranaires et régulent une panoplie de processus physiologiques. La densité de récepteurs exprimés à la surface d'une cellule dicte l’intensité de la réponse cellulaire suite à l’activation par un ligand. Un débalancement dans les niveaux d’expressions protéiques est la cause de plusieurs pathologies. C’est pourquoi il est d’une importance capitale de comprendre les mécanismes qui régissent les niveaux d’expressions totaux et membranaires des RCPG. Dans un premier temps, nos travaux ont permis de caractériser la dynamique d’internalisation et de recyclage du récepteur CRTH2. Nous avons identifié les domaines qui sont impliqués dans la régulation du trafic du récepteur. Des essais d’expression de surface ont révélé les cibles majeures des GRK2, GRK5, de la PKC et de l’arrestine-3 pour l’internalisation de CRTH2 suite à une stimulation du récepteur. De plus, nous rapportons la présence d’un motif de recyclage interne de type PDZ dans la queue cytoplasmique de CRTH2. Avant de se rendre à la membrane plasmique, les RCPG doivent être synthétisés au réticulum endoplasmique (RE), subir des étapes de maturation au Golgi et être transportés à la membrane plasmique. Les évènements coordonnant ces processus sont encore très peu étudiés. La dernière partie de nos travaux a permis d’élucider différents mécanismes régulant le contrôle qualité des RCPG à un stade précoce de synthèse. Tout d’abord, une nouvelle stratégie de purification protéomique a été développée et validée dans le but d’identifier des nouveaux partenaires d’interaction des RCPG dans un contexte cellulaire. Les résultats des criblages ont montré une interaction entre le récepteur [beta indice inférieur 2]AR et l’ubiqutine ligase RNF5. Ceci a conduit à la découverte d’un nouveau mécanisme de contrôle qualité des RCPG faisant intervenir les protéines RNF5 et JAMP comme étant des composantes importantes d’un système de régulation qui module la stabilité des RCPG. En dernier lieu, une nouvelle protéine, ANKRD13A, régulerait l’expression des RCPG par un mécanisme encore inconnu. Des évidences nous permettent de croire que les protéines RNF5, JAMP et ANKRD13A feraient partie d’un complexe protéique régulant le contrôle qualité des RCPG au RE.

The outer segment is a specialized region of rod and cone photoreceptor cells located in vertebrate retina. It features stacks of membranous discs containing visual pigment molecules, and the membranous structures undergo continuous renewal process. In order to better understand the cellular mechanisms in the outer segment, Kwok et al. (2008) used tandem mass spectrometry on bovine rod outer segment preparations, and identified many proteins of unknown function, one of which was ankyrin repeat domain 33 (ankrd33). Ankrd33 belongs to the ankyrin repeat protein class, which has been described to be involved in a variety of functions, such as cell-cell signalling and cytoskeleton structure. However, the function and localization of Ankrd33 have not been previously investigated. In this project, ankrd33 was cloned from bovine retinal cDNA and RT-PCR experiments showed a retina specific gene expression of this protein. In addition, monoclonal antibodies were raised against N and C-termini of ankrd33. These antibodies were used to localize the protein in retina. In addition, they were used to identify the interacting partners of ankrd33 in photoreceptors. Ankrd33 was found to be exclusively expressed in the outer segments of photoreceptor cells and co-immunoprecipitation studies identified hippocalcin like 1 protein (HPCAL-1) as one of the interacting partners. HPCAL-1 belongs to the family of proteins called neuronal calcium sensors (NCS) which are EF-hand containing Ca2+-binding proteins and expressed in different neuronal cells. These proteins are involved in calcium modulation of numerous cellular activities based on the cell type and their interacting partners. As a first step in identifying the possible cellular pathways that ankrd33 and HPCAL-1 might be involved in, monoclonal antibodies were produced against the full length of HPCAL-1 and were used for immunofluorescence studies and in vitro confirmation of the interaction between the two proteins. Immunofluorescence studies showed labelling of ankr33 and HPCAL-1 in rod and cone outer segment with cone outer segment having a stronger signal. These results showed that ankrd33 and HPCAL-1 are both highly expressed in the cone outer segment and these proteins may be involved in calcium dependent-cone specific pathways.

RA is a chronic and disabling disease with no known cure. The disease has a strong genetic component and modern genetic studies have successfully identified over 100 loci associated with the onset of RA. Despite the number of associations identified, the full genetic component of RA is not known, and for the majority of the loci the causal variant remains unknown. The overall aim of this study was to utilise well-powered genetic data, in order to identify novel loci, refine genetic associations, and generate robust evidence for the causal SNP and causal gene at a selected RA locus. An initial analysis was undertaken utilising 3870 RA cases and 8430 controls from the UK-ImmunoChip, a study designed for comprehensive fine-mapping of confirmed RA susceptibility loci. Analysis of the UK-ImmunoChip data identified a novel finding with the TYK2 locus, and proved informative to refining association signals, illustrating the utility of fine-mapping and implicated SNPs with putative regulatory function. The UK-ImmunoChip was subsequently expanded to incorporate samples from five additional cohorts in a study led by Dr. Stephen Eyre. In additional to novel loci discovery, this study provided evidence for SNPs putatively associated with RA (P smaller or equal to5E-05 < 5E-08). In a combined meta-analysis of 17,581 cases and 20,160 controls, convincing evidence was obtained for two novel RA loci, BACH2 and RAD51B.The newly identified genes implicate two novel pathways in RA (B-cell differentiation and DNA repair) and add to the growing number of loci associated with multiple AIDs. These findings are important to aid comprehensive pathway analysis and add to the knowledge of RA risk genes. The third most associated RA locus in both serological subtypes of disease, with an uncharacterised protein, ANKRD55, was subsequently selected for in-depth characterisation. Utilising genetic and haplotypic analysis the association at this locus was refined to a single signal, with four SNPs in strong LD (r2 > 0.8). Through bioinformatic analysis, two SNPs rs6859219 and rs10065637 showed evidence for functional activity, with evidence of being located in an enhancer element, supported by histone marks, DNAse hypersensitivity, evidence of transcription factor binding and eQTL. The use of RNA and ChIP experiments have established a testable hypothesis that the presence of the putative causal variants rs6859219 and rs10065637, act to weaken the strength of the enhancer element in which they are located, (evidenced by diminished H3k4me1 modification), which in turn down-regulates the transcriptional output of the target gene ANKRD55 (evidenced by eQTL in both whole blood and CD4+ T cells).In summary this study has led to the identification of three novel loci, highlighted the importance of fine-mapping and developed a successful systemic strategy for the characterisation of the 5q11 risk locus associated with RA.

This thesis investigates epigenetics in cancer with particular emphasis on breast cancer. There are two major themes, see Figure above. The first theme relates to the potential for assessing and developing more efficient epigenetic drugs while the second theme investigates mechanism of downregulation of ANKRD11, a putative tumour suppressor gene, in human breast cancer. This thesis is in the publication format with Chapters 1 and 3 as published articles, Chapter 2 submitted for publication and Chapter 4 as a manuscript in preparation. Theme 1: To improve the epigenetic-based therapeutic approach (Chapter 1 and 2) One of the roles that epigenetics plays in cancer development is the inhibition of transcription of tumour suppressor genes. Chapter 1, published as a review in Biodrugs, examines the knowledge of currently available therapeutic approaches related to epigenetic mechanisms such as DNA methylation for cancer treatment. Drug-related issues that could influence the application of therapeutics for clinical use are reviewed and possible developments to improve the clinical use of the drugs explored. Epigenetic-based drugs are emerging as anti-cancer therapies in the clinic. Existing demethylating agents have poor pharmacological properties that limit their clinical use, and the application of nano-based encapsulation to resolve these issues is discussed. Chapter 2, submitted as an original research article to Biodrugs, presents the development and assessment of an assay to allow comparison of epigenetic-related drugs in a high throughput format. Decitabine is encapsulated in a liposomal formulation and the potency of this newly formulated decitabine and existing drugs are effectively compared using the developed assay system. Further development and validation of the assay system and the liposomal formulated decitabine, not included in the submitted manuscript are included as supplementary data. Theme 2: Investigation of gene silencing mechanism of tumour suppressor ANKRD11 (Chapter 3 and 4) ANKRD11 is novel gene that was previously characterised in our laboratory, and found to be a putative tumour suppressor gene and a p53-coactivator (Neilsen et al. 2008). Chapter 3, published in European Journal of Cancer, investigates the mechanism of downregulation of ANKRD11 in human breast cancer. This chapter identifies the promoter sequence of ANKRD11, demonstrates the critical region of the ANKRD11 promoter subjected to DNA methylation, and associates the DNA methylation levels of ANKRD11 with its gene expression and clinical data. Further analysis of the DNA methylation pattern of this gene revealed a putative GLI1 transcription-factor binding site within the localised region of the promoter that is methylated. Chapter 4, presented as a manuscript in preparation, further explores the relationship between ANKRD11 and GLI1 in breast cancer. GLI1 is a Hedgehog signalling transcription factor, which has been shown to be involved in breast cancer development. This study analyses the transcriptional activity of ANKRD11 in the cells overexpressed with GLI1 and quantifies differential expression of these two genes in different stages of breast cancer. Future experiments to confirm and extend these exciting preliminary findings are discussed. The final chapter of this thesis summarises the findings of these studies and possible future research directions. The impact of these findings for the development of anti-cancer drugs, and the possible role of expression of ANKRD11 and GLI1 in breast cancer are highlighted.
Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2012

Loss of heterozygosity (LOH) on the long arm of chromosome 16 is frequently observed during the onset of breast cancer. Our laboratory has recently identified both ANKRD11 and FBXO31 as candidate tumour suppressor genes in the chromosome band 16q24.3, which is the smallest region of overlap for breast cancer LOH. This thesis focuses on the functional analysis of these two novel genes and implicates a role for them as breast cancer tumour suppressors. ANKRD11: a novel p53 coactivator involved in the rescue of mutant p53. The ability of p53 to act as a transcription factor is critical for its function as a tumour suppressor. Ankyrin repeat domain 11 (ANKRD11) was found to be a novel p53-interacting protein which enhanced the transcriptional activity of p53. ANKRD11 expression in breast cancer cell lines was shown to be down-regulated when compared to ANKRD11 expression in finite life-span HMECs and non-malignant immortalized breast epithelial cells. Restoration of ANKRD11 expression in MCF-7 (p53 wild-type) and MDA-MB-468 (p53[superscript R273H] mutant) cells suppressed the oncogenic properties of these breast cancer cell lines through enhancement of p21[superscript waf1] expression. ShRNA-mediated silencing of ANKRD11 reduced the ability of p53 to activate p21[superscript waf1] expression in response to DNA damage. ANKRD11 was shown to associate with the p53 acetyltransferase, P/CAF, and exogenous ANKRD11 expression increased the levels of acetylated p53. Exogenous ANKRD11 expression enhanced the DNA-binding properties of the p53[superscript R273H] mutant to the CDKN1A promoter, implicating a role for ANKRD11 in the restoration of mutant p53[superscript R273H] function. These findings demonstrate a role for ANKRD11 as a p53 coactivator and illustrate the potential of ANKRD11 in the restoration of mutant p53[superscript R273H] function. ANKRD11 has roles beyond that of p53 coactivation. This thesis also presents preliminary findings to suggest that ANKRD11 may be involved in the regulation of eukaryotic cell division. Furthermore, ANKRD11 was shown to function as an estrogen receptor coactivator. Taken together, these finding suggest that ANKRD11 is a multi-functional cancer-related protein. FBXO31: the 16q24.3 senescence gene. A BAC located in the 16q24.3 breast cancer loss of heterozygosity region was previously shown to restore cellular senescence when transferred into breast tumour cell lines. We have shown that FBXO31, although located just distal to this BAC, can induce cellular senescence in the breast cancer cell line MCF-7 and is the likely candidate senescence gene. Exogenous FBXO31 expression inhibited the oncogenic properties of the MCF-7 breast cancer cell line. In addition, compared to the relative expression in normal breast, levels of FBXO31 were down-regulated in breast tumour cell lines and primary tumours. FBXO31 protein levels were cell cycle regulated, with maximal expression from late G2 to early G1 phase. Ectopic expression of FBXO31 in the breast cancer cell line MDA-MB-468 resulted in the accumulation of cells at the G1 phase of the cell cycle. FBXO31 was also shown to be a component of a SCF ubiquitination complex. We propose that FBXO31 functions as a tumour suppressor by generating SCF[superscript FBXO31] complexes that target particular substrates, critical for the normal execution of the cell cycle, for ubiquitination and subsequent degradation.
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Thesis (Ph.D.) -- University of Adelaide, School of Medicine, Discipline of Medicine, 2008

Loss of heterozygosity (LOH) on the long arm of chromosome 16 is frequently observed during the onset of breast cancer. Our laboratory has recently identified both ANKRD11 and FBXO31 as candidate tumour suppressor genes in the chromosome band 16q24.3, which is the smallest region of overlap for breast cancer LOH. This thesis focuses on the functional analysis of these two novel genes and implicates a role for them as breast cancer tumour suppressors. ANKRD11: a novel p53 coactivator involved in the rescue of mutant p53. The ability of p53 to act as a transcription factor is critical for its function as a tumour suppressor. Ankyrin repeat domain 11 (ANKRD11) was found to be a novel p53-interacting protein which enhanced the transcriptional activity of p53. ANKRD11 expression in breast cancer cell lines was shown to be down-regulated when compared to ANKRD11 expression in finite life-span HMECs and non-malignant immortalized breast epithelial cells. Restoration of ANKRD11 expression in MCF-7 (p53 wild-type) and MDA-MB-468 (p53[superscript R273H] mutant) cells suppressed the oncogenic properties of these breast cancer cell lines through enhancement of p21[superscript waf1] expression. ShRNA-mediated silencing of ANKRD11 reduced the ability of p53 to activate p21[superscript waf1] expression in response to DNA damage. ANKRD11 was shown to associate with the p53 acetyltransferase, P/CAF, and exogenous ANKRD11 expression increased the levels of acetylated p53. Exogenous ANKRD11 expression enhanced the DNA-binding properties of the p53[superscript R273H] mutant to the CDKN1A promoter, implicating a role for ANKRD11 in the restoration of mutant p53[superscript R273H] function. These findings demonstrate a role for ANKRD11 as a p53 coactivator and illustrate the potential of ANKRD11 in the restoration of mutant p53[superscript R273H] function. ANKRD11 has roles beyond that of p53 coactivation. This thesis also presents preliminary findings to suggest that ANKRD11 may be involved in the regulation of eukaryotic cell division. Furthermore, ANKRD11 was shown to function as an estrogen receptor coactivator. Taken together, these finding suggest that ANKRD11 is a multi-functional cancer-related protein. FBXO31: the 16q24.3 senescence gene. A BAC located in the 16q24.3 breast cancer loss of heterozygosity region was previously shown to restore cellular senescence when transferred into breast tumour cell lines. We have shown that FBXO31, although located just distal to this BAC, can induce cellular senescence in the breast cancer cell line MCF-7 and is the likely candidate senescence gene. Exogenous FBXO31 expression inhibited the oncogenic properties of the MCF-7 breast cancer cell line. In addition, compared to the relative expression in normal breast, levels of FBXO31 were down-regulated in breast tumour cell lines and primary tumours. FBXO31 protein levels were cell cycle regulated, with maximal expression from late G2 to early G1 phase. Ectopic expression of FBXO31 in the breast cancer cell line MDA-MB-468 resulted in the accumulation of cells at the G1 phase of the cell cycle. FBXO31 was also shown to be a component of a SCF ubiquitination complex. We propose that FBXO31 functions as a tumour suppressor by generating SCF[superscript FBXO31] complexes that target particular substrates, critical for the normal execution of the cell cycle, for ubiquitination and subsequent degradation.
Thesis (Ph.D.) -- University of Adelaide, School of Medicine, Discipline of Medicine, 2008

碩士
國立陽明大學
生化暨分子生物研究所
101
Ankyrin repeat domain containing protein 17 (Ankrd17) gene encodes a ubiquitously expressed protein with two clusters of ankyrin repeats. The Ankrd17-deficient (Ankrd17-/-) mice die around embryonic day (E) 11.5 due to vascular development defects with severe hemorrhage. The blood vessels of Ankrd17-/- embryo has a normal pattern of endothelial cells (ECs) but less vascular smooth muscle cells (vSMCs) surround ECs compare with that in wild-type. These results suggest that Ankrd17 might play a functional role in regulating cell differentiation or migration of vSMCs. Using a gene knockdown approach in C3H/10T1/2 cells, I found that Ankrd17 could repress smooth muscle α-actin (α-SMA) expression but it was not caused by regulating the transcriptional activity of α-SMA promoter. Furthermore, downregulation of Ankrd17 could suppress the proliferation but not the migration ability in C3H/10T1/2 cells. Finally, I generated Ankrd17 SMC and EC-specific conditional knockout (CKO) mice to examine the role of Ankrd17 in embryonic vascular development. Interestingly, Ankrd17 CKO generated by SM22-, Tie2- or Mesp1-Cre did not cause vascular defect phenotypes as that in conventional knockout mice. Put together, my study provided some clues for the relationship between Ankrd17 and vSMC, but the biological and biochemical function of Ankrd17 in regulating vascular development still needs more works to investigate.

博士
國防醫學院
生命科學研究所
97
Ankyrin repeat domain 17 (Ankrd17) encodes an ubiquitously expressed protein with two clusters of ankyrin repeats. In order to elucidate its function(s), gene targeting strategy was used to ablate the Ankrd17 gene in mouse. The Ankrd17-deficient mice died between embryonic day (E) 10.5 and E11.5 due to cardiovascular defects. Serious hemorrhages were detected and the vascular smooth muscle cells (vSMCs) surrounding the vessels were drastically reduced in the Ankrd17-deficient embryos, suggesting that the vascular maturation was not completed. Interestingly, vSMC differentiation marker genes were up-regulated in the Ankrd17 mutant embryos. These genes were also up-regulated in a mouse vSMCs precursor cell line (C3H/10T1/2) when RNAi was applied to knockdown the Ankrd17 expression in these cells. Significantly, this up-regulation phenomenon was accompanied by a switch of the subcellular location of the Krüppel-like factor 4 (KLF4), a known repressor of the vSMC differentiation marker genes and an important player for the phenotype switching of vSMCs. Furthermore, 10T1/2 cells with knockdown of Ankrd17 expression exhibited reduced migration in a wound healing assay. These data together suggest that Ankrd17 participates in the maintenance of the dedifferentiation state of the vSMCs by restricting the expression of vSMC differentiation marker genes through control of the subcellular location of KLF4. Loss-of-function of Ankrd17 would then lead to the promiscuously high level of vSMC differentiation marker gene expression and consequently the defective migration of vSMCs from their originating sites to the vessel tubes.

Meiosis is a specialized cell division that produces haploid cells (gametes) from diploid progenitors. During meiosis parental chromosomes (homologs) need to pair, synapse and eventually segregate. Faithful chromosome segregation depends on chromosome recombination. In the beginning of prophase I programmed double strand breaks (DSBs) are introduced in meiotic cells by SPO11 enzyme. DSBs are positioned at hotspot sites that are specified by that action of DNA-binding histone methyltransferase PRDM9. Specific enzymes act at the site of breaks to create 5’ single stranded DNA ends. With the assistance of the strand exchange proteins DMC1 and RAD51 these ends invade homologous DNA sequence and DSB repair is initiated. DSB repair can be completed either as a crossover (reciprocal exchange of DNA) or as a non-crossover. Crossover events lead to the formation of chiasmata between homologs and ensure proper segregation during the first meiotic division. An interesting feature in male meiosis is the XY chromosomes. The shared region between sex chromosomes is short and is called pseudoautosomal region (PAR). Due to their large non synapsed region, XY chromosomes need to be transcriptionally silenced. Thus they are covered with the phosphorylated histone variant H2AX (γH2AX) forming the so called sex body. PAR region has higher density of DSBs than autosomes and it had been shown that sex chromosomes undergo delayed homologous pairing. Nevertheless little is known how meiotic recombination is regulated in PAR region of sex chromosomes. In close proximity with sex body it has been found a structure named dense body (DB). There are few reports suggesting that DB contains RNAs/proteins but no DNA. Its role in meiosis was unclear because no structural component had been described. In the present thesis the role of two meiotic expressed genes is described. In our group after performing RNA screens we identified several genes that are highly expressed during meiotic prophase I. Based on the expression profile we selected polycomb-related sex comb on midleg like 1 (Scml1) gene and the ankyrin repeat domain 31 (Ankrd31) to study their role in mammalian meiosis.:List of figures i List of abbreviations ii 1. Introduction 1 1.1 Gametogenesis 1 1.2 Meiotic prophase I 2 1.2.1 Meiotic recombination 4 1.2.2 Regulation of meiotic recombination 7 1.2.2.1 Meiotic recombination hotspots and PRDM9 activity 7 1.2.2.2 Meiotic surveillance mechanisms 8 1.3 Unique properties of XY recombination 9 1.4 Sex chromatin associated structure: The dense body 10 1.5 Aim of the thesis 11 2. Publications 12 3. Discussion 92 4. Summary 98 5. References 102 Acknowledgements 108 Declarations 109

Les anomalies du tube neural (ATN) sont des malformations congénitales parmi les plus fréquentes chez l’humain en touchant 1-2 nouveau-nés par 1000 naissances. Elles résultent d’un défaut de fermeture du tube neural pendant l’embryogenèse. Les formes les plus courantes d'ATN chez l'homme sont l'anencéphalie et le spina-bifida. Leur étiologie est complexe impliquant à la fois des facteurs environnementaux et des facteurs génétiques. Un dérèglement dans la signalisation Wnt, incluant la signalisation canonique Wnt/β-caténine et non-canonique de la polarité planaire cellulaire (PCP), peut causer respectivement le cancer ou les anomalies du tube neural (ATN). Les deux voies semblent s’antagoniser mutuellement. Dans cette étude, nous investiguons les rôles de Lrp6 et deANKRD6, entant qu’interrupteurs moléculaires entre les deux voies de signalisation Wnt, et CELSR1, en tant que membre de la PCP, chez la souris mutante Skax26m1Jus, générée par l’agent mutagène N-Ethyl-N-Nitrosuera, et dans une cohorte de patients humains ATN. Pour Lrp6, nous avons démontré que Skax26m1Jus représente un allèle hypermorphe de Lrp6 avec une augmentation de l’activité de la signalisation Wnt/canonique et une diminution de l’activité JNK induite par la voie PCP. Nous avons également montré que Lrp6Skax26m1Jus interagit génétiquement avec un mutant PCP (Vangl2Lp) où les doubles hétérozygotes ont montré une fréquence élevée d’ATN et des défauts dans la polarité des cellules ciliées de la cochlée. Particulièrement, notre étude démontre l'association des nouvelles et rares mutations faux-sens dans LRP6 avec les ATN humaines. Nous montrons que trois mutations de LRP6 causent une activité canonique réduite et non-canonique élevée. Pour ANKRD6, nous avons identifié quatre nouvelles et rares mutations faux-sens chez 0,8% des patients ATN et deux chez 1,3% des contrôles. Notamment, seulement deux, des six mutations validées (p.Pro548Leu et p.Arg632His) ont démontré un effet significatif sur l’activité de ANKRD6 selon un mode hypomorphique. Pour CELSR1, nous avons identifié une mutation non-sens dans l'exon 1 qui supprime la majeure partie de la protéine et une délétionde 12 pb. Cette perte de nucléotides ne change pas le cadre de lecture et élimine un motif putatif de phosphorylation par la PKC " SSR ". Nous avons également détecté un total de 13 nouveaux et rares variants faux-sens qui avaient été prédits comme étant pathogènes in silico. Nos données confirment le rôle inhibiteur de Lrp6 dans la signalisation PCP pendant la neurulation et indiquent aussi que les mutations faux-sens identifiées chez LRP6 et ANKRD6 pourraient affecter un équilibre réciproque et un antagonisme très sensible à un dosage précis entre les deux voies Wnt. Ces variants peuvent aussi agir comme facteurs prédisposants aux ATN. En outre, nos résultats impliquent aussi CELSR1 comme un facteur de risque pour les anomalies du tube neural ou l’agénésie caudale. Nos résultats fournissent des preuves supplémentaires que la voie de signalisation PCP a un rôle pathogène dans ces malformations congénitales et un outil important pour mieux comprendre leurs mécanismes moléculaires.
Neural tube defects (NTDs) are among the most common congenital malformations in humans affecting 1–2 infants per 1000 births. NTDs are caused by failure of the neural tube to close during embryogenesis. The most common forms of NTDs in humans are anencephaly and spina bifida. Their etiology is complex implicating environmental and genetic factors. Wnt signaling has been classified as canonical Wnt/ β-catenin dependent or non-canonical planar cell polarity (PCP) pathway. Misregulation of either pathway is linked mainly to cancer or neural tube defects (NTDs) respectively. Both pathwaysseem to antagonize each other. In this study, we investigate the role of Lrp6andANKRD6 as molecular switches between both Wnt pathways as well as CELSR1 as PCP member, in a novel ENU mouse mutant of Lrp6 (Skax26m1Jus) and in human NTDs. For Lrp6, we demonstrate that Skax26m1Jus represents a hypermorphic allele of Lrp6 with increased Wnt canonical and abolished PCP-induced JNK activities. We also show that Lrp6Skax26m1Jusgenetically interacts with a PCP mutant (Vangl2Lp) where double heterozygotes showed an increased frequency of NTDs and defects in cochlear hair cells’ polarity. Importantly, our study also demonstrates the association of rare and novel missense mutations in LRP6 that is an inhibitor rather than an activator of the PCP pathway with human NTDs. We show that three LRP6 mutations in NTDs led to a reduced Wnt canonical activity and enhanced PCP signaling. For ANKRD6: We identified four rare missense mutations in 0.8% of the NTD patients and 2 rare missense mutations in 1.3% of the controls. Notably, when all 6 mutations were validated, only two mutations identified in NTD patients, p.Pro548Leu, p.Arg632His, significantly altered DIVERSIN activity in Wnt signaling assays in a hypomorphic fashion. For CELSR1: We identified one nonsense mutation in exon 1 of CELSR1 that truncates the majority of the protein in one NTD patient and one in-frame 12 bp deletion that removes a putative PKC phosphorylation“SSR” motif in one caudal agenesis patient. We also detected a total of 13 novel missense variants in 12 patients (11 NTDs and 1 caudal agenesis) that were predicted to be pathogenic in silico. Our data confirm an inhibitory role of Lrp6 in PCP signaling in neurulation and indicate that rare missense mutations in LRP6 and ANKRD6 could affect a balanced reciprocal and a highly dosage sensitive antagonism between both Wnt pathways in neurulation and act as predisposing factors to NTDs in a subset of patients. Also, our findings implicate CELSR1 as a risk factor for NTDs or caudal agenesis. Our findings provide additional evidence for a pathogenic role of PCP signaling in thesemalformations and an important tool for better understanding their molecular mechanisms.