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Background: Patellar luxation is an orthopedic disorder in which the patella moves out of its normal location within the femoral trochlea of the knee and it can lead to osteoarthritis, lameness, and pain. In dogs it is a heritable trait, with both environmental and genetic factors contributing to the phenotype. The prevalence of patellar luxation in the Dutch Flat-Coated Retriever population is 24%. In this study, we investigated the molecular genetics of the disorder in this population. Results: Genome-wide association analysis of 15,823 single nucleotide polymorphisms (SNPs) in 45 cases and 40 controls revealed that patellar luxation was significantly associated with a region on chromosome CFA07, and possibly with regions on CFA03, CFA31, and CFA36. The exons of the genes in these regions, 0.5 Mb combined, were analyzed further. These exons from 15 cases and a pooled sample from 15 controls were enriched using custom genomic hybridization arrays and analyzed by massive parallel DNA sequencing. In total 7257 variations were detected. Subsequently, a selection of 144 of these SNPs were genotyped in 95 Flat-Coated Retrievers. Nine SNPs, in eight genes on CFA07 and CFA31, were associated with patellar luxation (P

Quantitative and qualitative protein characteristics are regulated at genomic, transcriptomic, and post-transcriptional levels. Here, we integrated in-depth transcriptome and proteome analyses of liver tissues from two rat strains to unravel the interactions within and between these layers. We obtained peptide evidence for 26,463 rat liver proteins. We validated 1,195 gene predictions, 83 splice events, 126 proteins with nonsynonymous variants, and 20 isoforms with nonsynonymous RNA editing. Quantitative RNA sequencing and proteomics data correlate highly between strains but poorly among each other, indicating extensive nongenetic regulation. Our multilevel analysis identified a genomic variant in the promoter of the most differentially expressed gene Cyp17a1, a previously reported top hit in genome-wide association studies for human hypertension, as a potential contributor to the hypertension phenotype in SHR rats. These results demonstrate the power of and need for integrative analysis for understanding genetic control of molecular dynamics and phenotypic diversity in a system-wide manner.

Background Recognition of histone modifications by specialized protein domains is a key step in the regulation of DNA-mediated processes like gene transcription. The structural basis of these interactions is usually studied using histone peptide models, neglecting the nucleosomal context. Here, we provide the structural and thermodynamic basis for the recognition of H3K36-methylated (H3K36me) nucleosomes by the PSIP1-PWWP domain, based on extensive mutational analysis, advanced nuclear magnetic resonance (NMR), and computational approaches. Results The PSIP1-PWWP domain binds H3K36me3 peptide and DNA with low affinity, through distinct, adjacent binding surfaces. PWWP binding to H3K36me nucleosomes is enhanced approximately 10,000-fold compared to a methylated peptide. Based on mutational analyses and NMR data, we derive a structure of the complex showing that the PWWP domain is bound to H3K36me nucleosomes through simultaneous interactions with both methylated histone tail and nucleosomal DNA. Conclusion Concerted binding to the methylated histone tail and nucleosomal DNA underlies the high- affinity, specific recognition of H3K36me nucleosomes by the PSIP1-PWWP domain. We propose that this bipartite binding mechanism is a distinctive and general property in the recognition of histone modifications close to the nucleosome core.

E2F transcription factors are known to be important for timely activation of G 1 /S and G 2 /M genes required for cell cycle progression, but transcriptional mechanisms for deactivation of cell cycle-regulated genes are unknown. Here, we show that E2F7 is highly expressed during mid to late S-phase, occupies promoters of G 1 /S-regulated genes and represses their transcription. ChIP-seq analysis revealed that E2F7 binds preferentially to genomic sites containing the TTCCCGCC motif, which closely resembles the E2F consensus site. We identified 89 target genes that carry E2F7 binding sites close to the transcriptional start site and that are directly repressed by short-term induction of E2F7. Most of these target genes are known to be activated by E2Fs and are involved in DNA replication, metabolism and DNA repair. Importantly, induction of E2F7 during G 0 -G 1 /S resulted in S-phase arrest and DNA damage, whereas expression of E2F7 during G 2 /M failed to disturb cell cycle progression. These findings provide strong evidence that E2F7 directly controls the downswing of oscillating G 1 /S genes during S-phase progression.

Item does not contain fulltext Cell Biology, 26 mei 1999 Promotor : Wieringa, B. Co-promotor : Hendriks, W.J.A.J. 177 p.

In this thesis, we focus on using driver and/or passenger mutations detected from WGS to develop machine learning classifiers for cancer diagnostics (Chapter 2 and Chapter 3), as well as to study cancer development (Chapter 4 and Chapter 5). Chapter 2 describes a Classifier of HOmologous Recombination Deficiency (CHORD) that detects HRD using various mutation types including microhomology deletions, small structural deletions and large structural duplications. Using CHORD, we found that HRD was most common in ovarian, breast, prostate and pancreatic cancer. We also found that HRD was often associated with loss-of-heterozygosity in all cancer types, with increased contribution of deep deletions in prostate cancer. Chapter 3 describes Cancer of Unknown Primary Location Resolver (CUPLR), a machine learning model that predicts tumor tissue of origin using a wide range of genomic features, including RMD, mutational signatures, driver gene mutations, aneuploidy, viral insertions, and various SV types. We found that SVs were important for and improved the performance of tumor tissue of origin classification for cancer types where SV related features were important, such as in pilocytic astrocytomas (characterized by KIAA1549-BRAF fusions) or cervical cancer (characterized by human papillomavirus DNA insertions). In Chapter 4, we use liver organoids to study mutation accumulation and its contribution to cancer development in three liver diseases, alcoholic cirrhosis, non-alcoholic steatohepatitis (NASH), and primary sclerosing cholangitis (PSC). We find surprisingly that these liver diseases do not contribute to detectable alterations in the mutation landscape. In Chapter 5, we perform a pan-cancer comparison of primary versus metastatic cancer and find that metastatic tumors differ from primary tumors mainly in their increased burden of small mutations and SVs as a result of treatment exposure and cancer type specific endogenous mutational processes. Lastly, in Chapter 6 we discuss the limitations and challenges faced in using WGS data to understand and diagnose cancer, and provide directions for future research.

Our genomes are 99.5% identical, and yet we are different. Part of this variation can be explained by genetic variation. The genomic locations where a certain nucleotide (a different 'letter' in the DNA sequence) is associated with a phenotype can be found with a genome-wide association study (GWAS): we compare - at genomic locations with a lot of variation (single nucleotide polymorphisms; SNPs) - people with and without the phenotype (cases respectively controls). In this thesis I show the added value of defining the phenotype differently. In chapter 1 we look for genetic associations with the subtypes of ischemic stroke. Three existing methods to diagnose the subtype do not always agree with each other. We compared these methods, and the intersect and union. The intersect (where cases were diagnosed with the subtype by all methods) is a stricter definition that only counts people as a case if they quite certainly have a subtype. In the case of ischemic stroke it seems more important to know the subtype with high certainty than it is to have a large sample. In chapter 2 we describe a GWAS of the age at onset of ischemic stroke. We split the cases in women and men, and found a SNP that is associated with a 1.6 years lower age at onset of ischemic stroke in women. The last chapters are on pleiotropic SNPs; they have an association with multiple phenotypes. Chapter 3 describes methods that can find pleiotropic SNPs. Chapter 4 describes a method we have developed; PolarMorphism. We use a different approach than previously described methods. PolarMorphism is faster then the method that previously could find the most pleiotropic SNPs, without finding less SNPs.

Technological advances in DNA sequencing techniques provide new resources and opportunities to study genetic contributions to disease. Still, it remains a challenge to determine which of the many detected genetic changes have an effect and through which mechanism they contribute to phenotypes and disease. This is especially challenging for structural variants, which are larger genomic changes that can affect both coding and non-coding regions on the genome. Cancer is a genetic disease with generally unstable genomes thereby continuing to accumulate somatic DNA changes including structural variants. In this thesis, we studied recurrent structural alterations in the aryl hydrocarbon receptor (AHR) gene that we identified in the whole-genome sequencing data of urinary tract cancer tumors. We propose activating alterations in the AHR gene as a driver event in urinary tract cancer. Furthermore, we studied the causative relationship between certain cancer driver genes and the accumulation of patterns of structural variants in various tumor types to gain insights into the molecular mechanisms of the development of SVs and their contribution to tumorigenesis. Structural variants are also present in the germline and although de novo SVs in the germline are a rare event, such structural variations are causative for congenital disorders such as intellectual disability and autism spectrum disorders. Integration of different layers of genomic and phenotypic information was found to provide additional benefit in determining the molecular consequences and interpreting pathogenicity of structural variations in patients with congenital disorders. Finally, we describe the discovery of inherited splice site mutations in the POLR3GL gene that cause teeth and bone abnormalities that fit in the spectrum of POLR3 pathologies and suggest that POLR3GL mutations should be considered in patients with POLR3 pathology.

In this thesis, we focused on developing new bioinformatics algorithms with the ultimate aim of creating a better understanding of cancer. Our research targeted 3 unsolved problems. First, a main difficulty in treating cancer is that it is often unclear which mutations are present. As a result, cells with undetected mutations may escape therapy and re-grow. Our solution involves sampling from multiple regions of the tumor to capture a larger number of mutations, and constructing a tree that represents the order in which these mutations originated. Second, many cancers present with structural variants in non-coding regions, yet their function is unknown. We found that these structural variants often disrupt the folding of the genome, thereby enabling novel interactions between genes and regulatory elements, such as enhancers. We introduce an algorithm that uses machine learning to detect from gene expression data which structural variants may be causal for cancer. Third, genes often lose function through non-genetic mechanisms, such as methylation. However, obtaining such data types is costly, and therefore often only genetic disruptions, i.e. mutations, can be detected. We found that genes with loss of function often leave behind specific signatures of mutations and structural variants in the genome. Using this information, we developed machine learning algorithms that can detect aberrant gene function even if loss cannot be measured directly.

Cancer genomics is a thriving field with constant methodological and technological advances. These developments enable personalized and targeted treatments for cancer patients based on the unique genomic profiles of tumors. In this thesis I leveraged novel technologies for the advancement of cancer research and care. In chapter 1 I introduced the different types of genomic variation, the genomic characteristics of cancer, the importance of genomic technology for personalized medicine and the potential of liquid biopsies for low-invasive cancer monitoring. I also introduced model systems used in cancer research, including patient-derived organoids (PDOs), detailing the potential for their use particularly in ovarian cancer (OC). Lastly, I introduced the role of somatic SV in cancer and the different sequencing technologies that are used to detect them, along with the challenges that this presents. In the first part of this thesis, we used organoid technology to advance OC research. In chapter 2 we established and characterized a PDO biobank that faithfully represented the disease, and presented its applications. Next, we expanded the OC-PDO biobank and used those PDOs for extensive drug screening in chapter 3. We performed screening assays on 36 PDO lines derived from 23 patients and retrospectively compared their drug responses to clinical responses of the patients. In the second part of the thesis I focused on somatic structural variation in cancer. Accurate detection of structural variants (SVs) is still challenging, and truth sets and standardized workflows are lacking. We tackled the challenge of accurate somatic SV detection in cancer genomes and generated a truth set of somatic SVs that can be used for method development and benchmarking, which I presented in chapter 4. Furthermore, we developed methods to utilize long-read sequencing and somatic structural variants (SVs) for cancer dynamics after treatment and minimal residual disease tracing. In chapter 5, we developed an assay that leveraged nanopore sequencing technology for rapid detection of somatic SVs from a tumor.We also developed an assay that leverages CRISPR-Cas9 based enrichment of genomic targets in pediatric leukemias from the lymphoid lineage. In chapter 6, we targeted loci recurrently involved in genomic rearrangements in these leukemias, such as the immunoglobulin (Ig) and T-cell receptor (TCR) loci, and the KMT2A and SIL-TAL1 fusion-gene loci. Finally, in chapter 7 I discussed and reflected on the technological advances presented in the previous chapters. I explained the advantages of PDO technology in OC research, but also the challenges for its further clinical implementation in clinical care. Similarly, I identify current challenges and propose several solutions for enhancing the knowledge of the role of somatic SVs in cancer and implementation of long-read sequencing. In conclusion, this thesis proposes several cancer genomics technological opportunities to advance cancer research and develop personalized diagnostic assays to improve patients’ outcomes.

E3 ubiquitin ligases are enzymes that confer specificity to the ubiquitin-proteasome system by directly binding the proteins that are targeted for degradation. E3s are encoded by over 650 human genes, exceeding the number of genes encoding for protein kinases. Accordingly, E3 ubiquitin ligases have been linked to the control of virtually all cellular processes. In spite of their fundamental importance, our knowledge of the biological function and mechanism of action for most E3 ubiquitin ligases is still poor. Increasing our knowledge of the biological role of ubiquitin ligases is fundamental because experimental and clinical data have shown that their abnormal functions is involved in the pathogenesis of many human cancers. During my PhD, I have investigated the function of SCF ubiquitin ligases and elucidated the molecular mechanisms by which deregulated functions of SCF ubiquitin ligases contribute to the unchecked proliferation typical of cancer cells. I have demonstrated that SCFβTrCP targets for proteasomal degradation the basic helix-loop-helix transcription factor DEC1. In particular, I have found that the regulated proteolysis of DEC1 controls the DNA damage checkpoint. I have shown that in response to DNA damage, DEC1 is stabilized by the USP17 deubiquitylating enzyme and that during recovery from the G2 DNA damage checkpoint DEC1 is targeted for degradation by SCFβTrCP in collaboration with Casein Kinase 1a. I have also found that the same SCF ubiquitin ligase, SCFβTrCP, is responsible for the destruction of another basic helix-loop-helix transcription factor, namely TFAP4, and demonstrated that the SCFβTrCP-mediated degradation of TFAP4 is required for G2 progression and mitotic entry. In addition, I have performed a systematic analysis of the gene expression profile of 67 F-box proteins, substrate receptor subunits of SCF ubiquitin ligases, in human primary tumors and studied the biological role of a number of F-box proteins that are found overexpressed in cancer cells.

Recent reports show that formation of COPII coated vesicles and ER exit are regulated by kinases and that more generally, many components of the secretory pathway have been found phosphorylated. However the conditions under which these events occur are so far poorly understood. As the main aim of this thesis, we asked how the function and organization of the early secretory pathway respond to nutrient stress. In particular, we examine the behavior the components of the early secretory pathway under nutrient restriction, their contribution to other processes that are also regulated by the same stress (such as protein translation arrest and stress granule formation), and the nature of the signaling events regulating these responses. We follow up on the results of a kinase screen designed to identify regulators of the early secretory pathway and find that the extracellularly regulated kinase 7 (Erk7, also known as MAPK15) mediates the response of the ERES to serum starvation. These findings establish that the early secretory pathway is sensitive to nutrient signals, that pathways sensing nutrient abundance regulate its functional organization and that the key ERES component, Sec16 is the platform that integrates these signals. These results fuelled the further experiments to examine the behavior of the ERES components upon amino-acid starvation. We find that this response is even more dramatic than the one observed with serum deprivation. Indeed the ERES are remodeled into the Sec bodies, a novel, non-membrane bound, reversible structure that does not support protein transport and acts as a reservoir for ERES components. We find that the formation of Sec bodies is critical to cell survival and re-adaptation to normal growth conditions after the stress is relieved. Our quest to understand the nature of this novel structure reveals that it behaves like a liquid droplet, linking it to the cytoplasmic reorganization that occurs during stress, a well-documented manifestation of which is the assembly of stress granules. The similarities we observed between the Sec bodies and the stress granules prompted us to investigate the hypothesis that the formation of two structures is somehow linked. Indeed, we find that even though Sec bodies and stress granules are distinct both morphologically and functionally the specific ERES components that are required for Sec body assembly are also necessary for stress granule formation. These observations reveal a so far unexplored link between ER exit and mRNA sorting and turnover. Furthermore we study the stress granule assembly under a different type of stress, heat exposure, which does not affect the early secretory pathway. We find that TORC2 signaling is required for their formation. Having characterized the heat sensitivity phenotype of Rictor mutant flies we set out to understand the molecular mechanisms of this phenotype. Using S2 cells initially and then confirming our observations in Drosophila tissues we show that during heat stress TORC2 mediates the assembly of stress granules, possibly via its effector, Akt.

Cancer is caused by damage to a cells’ DNA. This damage can be as small as a change or deletion of one single DNA base, as big as duplications or deletions of a full chromosome, or more complex such as erroneous reassembly of broken chromosomes. All these events can lead to unbridled cell proliferation and defective cell repair mechanisms: a tumor cell. Because of DNA sequencing, we now know several genes that are frequently affected by DNA damage in cancer, and also know the effect of such events on the cell. This has led to the development of so called ‘targeted treatments’, treatments that are specifically aimed at the defective gene. By applying these types of treatment, we can fight the tumor more effectively because they block the exact processes that make a specific cell cancerous. Unfortunately, targeted treatment has not been as successful as expected, especially not for patients with metastatic disease. In many cases, patients initially respond really well to treatment, but their tumors become resistant after some time, or a subset of their tumors does respond to therapy while other lesions continue to grow. This is likely due to genetic differences between tumors within a single patient, or even between cells within the same tumor: tumor heterogeneity. As a tumor continues to grow, the DNA damage builds up. At each cell division, the damaged DNA is copied to the new cell, and additional defects can occur on top of that which finally leads to large differences between cells. Obviously, these genetic differences will cause the cells to behave differently as well. In this thesis, we investigate the extent of tumor heterogeneity in patients with advanced metastatic disease, by comprehensively characterizing samples from multiple tumor sites or multiple time points. We combine several techniques to capture the various types of DNA damage, and aim to unravel the effect of the genetic changes on the behavior of the tumor by adding gene expression analysis or therapy response measurements. We also present a technique to extract more information from a single sequencing experiment, which increases the efficiency of routine screening for targetable genetic defects. Our results demonstrate the progress that has been made in the past few years, both on the technical and the clinical front, but also reveal the challenges that still lay ahead on the path to stop cancer.

The last two decades were marked by discoveries and breakthroughs in different biological disciplines, and stem cell biology is an example of the quickly developing field. Discovery of stem cell niches and successful reprogramming of somatic cells into pluripotent stem state are only a few step stones that recently changed our understanding of stem cells. All the recent achievements in stem cell biology also made clear the technical limitations that scientists working in the field are facing every day, limitations that become harder and harder to overcome. One of the key technical problems is a shortage of models that allow studying pluripotent stem cell in vivo, within its microenvironment. It is hard to overestimate the importance of traditional model systems, such as mice, fishes or flies, for the stem cell research, yet the need for new complementary models becomes obvious. Marine flatworm Macrostomum lignano is a recently emerged model organism. It possesses a population of adult somatic stem cells known as neoblasts, that make it an attractive model for stem cell studies. Exploring genomic resources for this organism and, more generally, developing it as a reliable model are the main goals of this thesis that we pursue by expanding the toolkit for the model, testing its suitability in different scientific fields and studying the functioning of stem cell system in the organism. To the existing toolkit available for M. lignano we add knowledge of its genomic resources. By that we mean de novo sequencing, assembling and annotation of genome and transcriptome and miRNAs profiling, but also generating massive data on gene expression in M. lignano at different developmental stages and under different conditions. Most of this data are already publicly available, and soon we plan to introduce a new online database on M. lignano gene expression that would allow everyone to benefit from the results of our work. To the list of tools for studying M. lignano we also add reliable miRNA in situ hybridization method. We tested the potential of M. lignano, as a model for different kinds of studies outside the stem cell field. First, it is an interesting organism from the evolutionary point of view, and both its genome and presumably ancient miRNA system could add a lot to our understanding of evolution of many physiological processes. Second, M. lignano can serve as a model for chemical compound screens. Studying of bioelectrical processes presents a third field where advantages of this worm model can be exploited. We also made a step forward towards understanding the functioning of stem cells in M. lignano. We identified a set of genes involved in stem cell regulation during homeostasis and regeneration, and even larger group of transcripts, expression of which is enriched in neoblasts. We also made a candidate list of miRNAs potentially involved in stem cell regulation and addressed the role of bioelectric signaling in regeneration. Finally, we initiated the studies of the mechanism involved in rapid gonad degradation following exposure to the low salinity.

The basic helix-loop-helix (bHLH) transcription factors oligodendrocyte transcription factor 1 (Olig1) and Olig2 are structurally similar and, to a first approximation, coordinately expressed in the developing CNS and postnatal brain. Notwithstanding these similarities, it was apparent from early on after their discovery that Olig1 and Olig2 have non-overlapping developmental functions in patterning, neuron subtype specification and the formation of oligodendrocytes. Early in development, Olig2 functions as an anti-neural bHLH transcription factor and maintains the pool of proliferating neural progenitor cells. Later on, Olig2 switches to pro-neural functions and is important for the generation of motor neurons and oligodendrocytes. Olig1 is specifically important for the maturation of oligodendrocytes. The unique biological functions of Olig1 and Olig2 reflect their distinct genetic targets, co-regulator proteins and post-translational modifications. We show here that the proliferative function of Olig2 is controlled by developmentally regulated phosphorylation of a conserved triple serine motif within the amino terminal domain. In the phosphorylated state, Olig2 maintains anti-neural (i.e. pro-mitotic) functions that are reflected in human glioma cells and in a genetically defined murine model of primary glioma. Furthermore, the pro-mitogenic functions of phospho-Olig2 reflect, at least in part, an oppositional relationship with p53 functions. We have used genome scale ChIP-seq, RNA-seq and mass spectroscopy proteomic methods to define the molecular mechanisms whereby phosphorylation of Olig2 promotes mitosis and opposes p53-mediated responses to genotoxic damage. We show that target gene binding is not affected by Olig2 phosphorylation. However, Olig2 phosphorylation confers global gene expression changes and association with co-regulator proteins. Towards these ends, phosphorylation regulates intranuclear compartmentalization of Olig2. Finally, Olig2 meets the requirements for a suitable therapeutic target to inhibit brain tumor growth, because it (i) is essential for brain tumor growth, (ii) specific to the central nervous system and (iii) dispensable for mature brain function. Generally speaking, bHLH transcription factors require dimerization to carry out their downstream functions. Here, we describe a set of stabilized alpha-helical (SAH) mimetics that we developed to interfere with Olig2 dimerization, and thus Olig2 function. Hydrocarbon stapling successfully produced a number of alpha-helical peptides mimicking a single helix or the entire HLH domain of Olig2. However, none of the SAH OLIGopeptides specifically and potently disrupted Olig2 dimerization and subsequent DNA binding. We conclude that the HLH domain is required, but perhaps not sufficient for Olig2 dimerization. Our studies indicate that specification and differentiation of oligodendrocytes are completely independent of Olig2’s triple serine phosphorylation function in proliferation. The ability to uncouple these functions one from the other suggests an avenue to specifically target tumors within the brain while sparing normal white matter. In the fullness of time, small molecule inhibitors of Olig2 protein may thus have practical applications for glioma medicine.

MicroRNAs (miRNAs) are involved in a wide range of biological processes, such as development, cell proliferation and death and oncogenesis, thus identification and characterization of miRNAs has become a rapidly growing area of research. Mir-34 is one of a few deeply conserved miRNAs, found in worms, flies and human. Mir-34 function was mostly studied in mammals and linked to diverse developmental processes, such as cell-cycle arrest, apoptosis. In this thesis study, I have used C. elegans as a model organism to further advance our understanding of mir-34 gene.

For many years bone research has been mainly performed in mice, chicken, cell culture systems, or human material from the clinic. In this thesis, we make use of the zebrafish (Danio rerio), a relatively new model system in this field. This small teleost offers possibilities which makes it a great complement to the mouse: forward genetic screens are possible in fish due to extra-uterine development and large brood size, and the recent generation of osteoblast-specific reporter lines allows visualization of osteoblasts in vivo. As key regulators of bone formation are highly conserved between mammals and teleosts, findings in fish likely apply to mammalian osteogenesis and tissue mineralization. Out of a forward genetic screen we performed, we identified 27 specific bone mutants which can be categorized in three different groups: (1) less or no bone, (2) ectopic over-ossification, and (3) more perichondral bone, but normal dermal bone. Two mutants out of the second class were analyzed in detail. The stocksteif mutant is characterized by over-ossification of the notochord resulting in fused vertebrae. stocksteif encodes cyp26b1, a retinoic acid metabolizing enzyme. We show that cyp26b1 is expressed in osteoblasts and that -upon retinoic acid treatment- osteoblasts enhance their activity. Consequently, the over-production of bone causes the fusion of the vertebrae. Thus, regulation of retinoic acid levels and the tight control of cyp26b1 activity are essential for regulation of skeletogenesis in zebrafish. The second mutant, dragonfish, is also characterized by over-ossification of the notochord and the subsequent fusion of the vertebrae. However, dragonfish functions in a pathway which is clearly distinct of that of retinoic acid. Additionally, ectopic mineralizations in the brain and neural tube can be observed in these mutants and, interestingly, at these spots the typical osteoblast-specific genes are not detected. Future research has to unravel the molecular lesion in these mutants and will provide novel insight into the regulation of bone development and disease.

RNAi is widely used as a genetic tool in a range of model organisms, however still relatively little is known about the endogenous functions of RNAi. In this thesis we have studied several aspects of the endogenous role of RNAi in order to enhance our understanding of the capabilities of this mechanism. We investigated an RNAi-related silencing mechanism (cosuppression) which is triggered by repetitive sequences resulting in silencing of endogenous genes in trans. We found both chromatin remodeling factors and RNAi factors to be involved in cosuppression, indicating a transcriptional branch to the RNAi mechanism. We also identified three members of a relatively new family of RNA-metabolising enzymes in C. elegans: the TRF-like nucleotidyltransferases. Analysing the family of TRF-like nucleotidyltransferases we found that all members are expressed in the germline of C. elegans and that three of them (cde-1, cde-2 and rde-3) are required in different combinations of RNAi-related silencing processes. Nucleotidyltransferase mutants cde-1 and rde-3 showed defects in germline stability and were unable to maintain germline function over time, suggesting that the RNAi silencing pathway and developmental pathways of the germline intersect at the level of the TRF-like nucleotidyltransferases. Next we created tagged constructs of RDE-3 to further analyse its function, and found that RDE-3 is localized to the germline cytoplasm, and to a subset of neurons. The protein was detected in two complexes: a large complex of 600-700kD, and a smaller less abundant 400kD complex. We used mass spectrometry to identify potential binding partners of RDE-3. We also studied the function of CDE-1 in RNAi and chromosome segregation. CDE-1 localises specifically to mitotic chromosomes in embryos, and requires the RdRP EGO-1, and the Argonaute protein CSR-1 for this localisation. We found that CDE-1 is required for the uridylation of CSR-1 bound siRNAs, and that in the absence of CDE-1 these siRNAs accumulate to inappropriate levels, accompanied by defects in both meiotic and mitotic chromosome segregation. Elevated siRNA levels are associated with erroneous gene silencing, most likely through the inappropriate loading of CSR-1 siRNAs into other Argonaute proteins. We propose a model in which CDE-1 restricts specific EGO-1-generated siRNAs to the CSR-1 mediated, chromosome associated RNAi pathway, thus separating it from other endogenous RNAi pathways. The conserved nature of CDE-1 suggests that similar sorting mechanisms may operate in other animals, including mammals. Finally, we identified a homologue of the small RNA methylating enzyme HEN-1, named REM-1. We showed that rem-1 mRNA is present in the germline, and that REM-1 is responsible for the methylation of the class of 21U RNAs confirming the notion that 21U RNAs are the worm counterpart of piRNAs. Methylation of 21U RNAs, however seems to be not essential for overall 21U RNA function. Taken together we identified two different chromatin-related branches to the RNAi mechanism, which changes our view of RNAi from a posttranscriptional silencing mechanism to a mechanism with a wider range of effects. In addition we reveal a regulatory mechanism involving nucleotidyltransferases and methylases, determining the stability and Argonaute loading preferences of small RNAs.