Identification and characterization of genes deregulated by DNA methylation in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is one of the most common leukemias in adults in the Western world characterized by the accumulation of mature-appearing B-cells in the peripheral blood, lymphoid tissues and bone marrow. The use of novel inhibitors targeting BCR- and PI3K-signaling in CLL as well as the application of CD20-antibodies in the clinic have led to improved outcome for the patients. Nevertheless, CLL remains an incurable disease and in order to develop new treatment strategies, there is still a strong need to elucidate the pathomechanism of CLL further. CLL is amongst other aspects a disease of the epigenome, displaying genome-wide hypomethylation while specific regions are hypermethylated, resulting in the deregulation of tumor suppressors, non-coding RNAs and oncogenes. Interestingly, DNA methylation in CLL patients was shown to be relatively stable over time suggesting that genes which are deregulated by this DNA modification are affected permanently, independent of disease stage and course. In this project, it was therefore aimed to identify and characterize genes deregulated in CLL by DNA methylation, which are likely involved in the pathomechanism of the disease. To this end, expression and methylation array profiling was conducted in CD19+ sorted CLL cells and B-cells of healthy donors. Out of the 1,866 genes identified as differentially expressed between cancer cells and their non-malignant counterpart, 33 showed significantly differentially methylated regions (DMR) that displayed negative correlation with gene expression. Technical validation using mass spectrometry based quantification of methylation confirmed the presence of DMRs in 17 of these genes including the transcription and stem cell factor KLF4. KLF4, of which repression and hypermethylation could be confirmed in a larger patient subset, was previously shown to play an important role in B-cell differentiation and maturation and further to act as tumor suppressor in other B-cell malignancies including classical- and non-Hodgkin lymphoma. Moreover, KLF4 was shown to be a downstream target of NOTCH1 which is recurrently mutated and constitutively active in CLL. In fact, treatment of six different lymphoblastoid and leukemia cell lines and of primary peripheral blood mononuclear cells (PBMCs) from eight CLL patients with γ-secretase inhibitor resulted in the inhibition of NOTCH1 activity and in the re-expression of KLF4 on RNA and protein level, suggesting transcriptional regulation of KLF4 by NOTCH1. Furthermore, expression of KLF4 was correlated with levels of BCL-2 family members BAK, BAX and BCL-2 as well as the cell cycle regulator CCND1 in primary CLL and B-cells. Although, overexpression of KLF4 in three leukemia cell lines did not induce apoptosis in these cells, it resulted in the deregulation of a number of genes involved in prominent signaling pathways which are highly relevant for the pathogenesis of CLL, including BCR- and PI3K-signaling. In summary, these findings suggest the repression of KLF4 by DNA promoter methylation and NOTCH signaling in CLL. Overexpression of KLF4 results in the deregulation of genes involved in signaling pathways important for B-cells which indicates that KLF4 is in part involved in keeping the CLL cells in a highly activated state

Purine– and pyrimidine–triple-helix-forming oligonucleotides recognize qualitatively different target sites at the ribosomal DNA locus

Triplexes are noncanonical DNA structures, which are functionally associated with regulation of gene expression through ncRNA targeting to chromatin. Based on the rules of Hoogsteen base-pairing, polypurine sequences of a duplex can potentially form triplex structures with single-stranded oligonucleotides. Prediction of triplex-forming sequences by bioinformatics analyses have revealed enrichment of potential triplex targeting sites (TTS) at regulatory elements, mainly in promoters and enhancers, suggesting a potential function of RNA – DNA triplexes in transcriptional regulation. Here, we have quantitatively evaluated the potential of different sequences of human and mouse ribosomal RNA genes (rDNA) to form triplexes at different salt and pH conditions. We show by biochemical and biophysical approaches that some of these predicted sequences form triplexes with high affinity, following the canonical rules for triplex formation. We further show that RNA triplex-forming oligos (TFOs) are more stable than their DNA counterpart, and point mutations strongly affect triplex formation. We further show differential sequence requirements of pyrimidine and purine TFO sequences for efficient binding, depending on the G–C content of the TTS. The unexpected sequence specificity, revealing distinct sequence requirements for purine and pyrimidine TFOs, shows that in addition to the Hoogsteen pairing rules, a sequence code and mutations have to be taken into account to predict genomic TTS

Identification of novel inner membrane complex and apical annuli proteins of the malaria parasite Plasmodium falciparum

The inner membrane complex (IMC) is a defining feature of apicomplexan parasites, which confers stability and shape to the cell, functions as a scaffolding compartment during the formation of daughter cells and plays an important role in motility and invasion during different life cycle stages of these single-celled organisms. To explore the IMC proteome of the malaria parasite Plasmodium falciparum we applied a proximity-dependent biotin identification (BioID)-based proteomics approach, using the established IMC marker protein Photosensitized INA-Labelled protein 1 (PhIL1) as bait in asexual blood-stage parasites. Subsequent mass spectrometry-based peptide identification revealed enrichment of 12 known IMC proteins and several uncharacterized candidate proteins. We validated nine of these previously uncharacterized proteins by endogenous GFP-tagging. Six of these represent new IMC proteins, while three proteins have a distinct apical localization that most likely represents structures described as apical annuli in Toxoplasma gondii. Additionally, various Kelch13 interacting candidates were identified, suggesting an association of the Kelch13 compartment and the IMC in schizont and merozoite stages. This work extends the number of validated IMC proteins in the malaria parasite and reveals for the first time the existence of apical annuli proteins in P. falciparum. Additionally, it provides evidence for a spatial association between the Kelch13 compartment and the IMC in late blood-stage parasites

Comparative Heterochromatin Profiling Reveals Conserved and Unique Epigenome Signatures Linked to Adaptation and Development of Malaria Parasites.

Heterochromatin-dependent gene silencing is central to the adaptation and survival of Plasmodium falciparum malaria parasites, allowing clonally variant gene expression during blood infection in humans. By assessing genome-wide heterochromatin protein 1 (HP1) occupancy, we present a comprehensive analysis of heterochromatin landscapes across different Plasmodium species, strains, and life cycle stages. Common targets of epigenetic silencing include fast-evolving multi-gene families encoding surface antigens and a small set of conserved HP1-associated genes with regulatory potential. Many P. falciparum heterochromatic genes are marked in a strain-specific manner, increasing the parasite's adaptive capacity. Whereas heterochromatin is strictly maintained during mitotic proliferation of asexual blood stage parasites, substantial heterochromatin reorganization occurs in differentiating gametocytes and appears crucial for the activation of key gametocyte-specific genes and adaptation of erythrocyte remodeling machinery. Collectively, these findings provide a catalog of heterochromatic genes and reveal conserved and specialized features of epigenetic control across the genus Plasmodium

Transcriptional profiling defines histone acetylation as a regulator of gene expression during human-to-mosquito transmission of the malaria parasite Plasmodium falciparum

Transmission of the malaria parasite Plasmodium falciparum from the human to the mosquito is mediated by the intraerythrocytic gametocytes, which, once taken up during a blood meal, become activated to initiate sexual reproduction. Because gametocytes are the only parasite stages able to establish an infection in the mosquito, they are crucial for spreading the tropical disease. During gametocyte maturation, different repertoires of genes are switched on and off in a well-coordinated sequence, pointing to regulatory mechanisms of gene expression. While epigenetic gene control has been studied during erythrocytic schizogony of P. falciparum, little is known about this process during human-to-mosquito transmission of the parasite. To unveil the potential role of histone acetylation during gene expression in gametocytes, we carried out a microarray-based transcriptome analysis on gametocytes treated with the histone deacetylase inhibitor trichostatin A (TSA). TSA-treatment impaired gametocyte maturation and lead to histone hyper-acetylation in these stages. Comparative transcriptomics identified 294 transcripts, which were more than 2-fold up-regulated during gametocytogenesis following TSA-treatment. In activated gametocytes, which were less sensitive to TSA, the transcript levels of 48 genes were increased. TSA-treatment further led to repression of ~145 genes in immature and mature gametocytes and 7 genes in activated gametocytes. Up-regulated genes are mainly associated with functions in invasion, cytoadherence, and protein export, while down-regulated genes could particularly be assigned to transcription and translation. Chromatin immunoprecipitation demonstrated a link between gene activation and histone acetylation for selected genes. Among the genes up-regulated in TSA-treated mature gametocytes was a gene encoding the ring finger (RING)-domain protein PfRNF1, a putative E3 ligase of the ubiquitin-mediated signaling pathway. Immunochemistry demonstrated PfRNF1 expression mainly in the sexual stages of P. falciparum with peak expression in stage II gametocytes, where the protein localized to the nucleus and cytoplasm. Pfrnf1 promoter and coding regions associated with acetylated histones, and TSA-treatment resulted in increased PfRNF1 levels. Our combined data point to an essential role of histone acetylation for gene regulation in gametocytes, which can be exploited for malaria transmission-blocking interventions

An HDAC9-MALAT1-BRG1 complex mediates smooth muscle dysfunction in thoracic aortic aneurysm

Thoracic aortic aneurysm (TAA) has been associated with mutations affecting members of the TGF-β signaling pathway, or components and regulators of the vascular smooth muscle cell (VSMC) actomyosin cytoskeleton. Although both clinical groups present similar phenotypes, the existence of potential common mechanisms of pathogenesis remain obscure. Here we show that mutations affecting TGF-β signaling and VSMC cytoskeleton both lead to the formation of a ternary complex comprising the histone deacetylase HDAC9, the chromatin-remodeling enzyme BRG1, and the long noncoding RNA MALAT1. The HDAC9–MALAT1–BRG1 complex binds chromatin and represses contractile protein gene expression in association with gain of histone H3-lysine 27 trimethylation modifications. Disruption of Malat1 or Hdac9 restores contractile protein expression, improves aortic mural architecture, and inhibits experimental aneurysm growth. Thus, we highlight a shared epigenetic pathway responsible for VSMC dysfunction in both forms of TAA, with potential therapeutic implication for other known HDAC9-associated vascular diseases

The private life of malaria parasites:Strategies for sexual reproduction

Malaria parasites exhibit a complex lifecycle, requiring extensive asexual replication in the liver and blood of the vertebrate host, and in the haemocoel of the insect vector. Yet, they must also undergo a single round of sexual reproduction, which occurs in the vector’s midgut upon uptake of a blood meal. Sexual reproduction is obligate for infection of the vector and thus, is essential for onwards transmission to new hosts. Sex in malaria parasites involves several bottlenecks in parasite number, making the stages involved attractive targets for blocking disease transmission. Malaria parasites have evolved a suite of adaptations (“strategies”) to maximise the success of sexual reproduction and transmission, which could undermine transmission-blocking interventions. Yet, understanding parasite strategies may also reveal novel opportunities for such interventions. Here, we outline how evolutionary and ecological theories, developed to explain reproductive strategies in multicellular taxa, can be applied to explain two reproductive strategies (conversion rate and sex ratio) expressed by malaria parasites within the vertebrate host

Inducible developmental reprogramming redefines commitment to sexual development in the malaria parasite <i>Plasmodium berghei</i>

During malaria infection, Plasmodium spp. parasites cyclically invade red blood cells and can follow two different developmental pathways. They can either replicate asexually to sustain the infection, or differentiate into gametocytes, the sexual stage that can be taken up by mosquitoes, ultimately leading to disease transmission. Despite its importance for malaria control, the process of gametocytogenesis remains poorly understood, partially due to the difficulty of generating high numbers of sexually committed parasites in laboratory conditions1. Recently, an apicomplexa-specific transcription factor (AP2-G) was identified as necessary for gametocyte production in multiple Plasmodium species2,3, and suggested to be an epigenetically regulated master switch that initiates gametocytogenesis4,5. Here we show that in a rodent malaria parasite, Plasmodium berghei, conditional overexpression of AP2-G can be used to synchronously convert the great majority of the population into fertile gametocytes. This discovery allowed us to redefine the time frame of sexual commitment, identify a number of putative AP2-G targets and chart the sequence of transcriptional changes through gametocyte development, including the observation that gender-specific transcription occurred within 6 h of induction. These data provide entry points for further detailed characterization of the key process required for malaria transmission

Exploring the role of structured DNA elements and their specific binders in genome regulation

After the description of the double helix structure by Watson and Crick, the function of DNA in the cell was seen as rather static, a long stretched out database of information, which was read and interpreted by the protein machinery doing all the interesting work. That notion however, quickly changed with the discovery of the DNA’s potential to adopt various alternative conformations, like Z-DNA, quadruplex DNA or triplexes. Although initially characterized in vitro, these structures were soon implicated in the development of certain diseases and suggested to have possible roles in gene regulation and genome organization. The major focus thereby lay on the triplexes, three stranded structures comprising the DNA duplex and a single stranded RNA or DNA that binds in the major groove of the double helix. Strikingly, the recent advancements in high-throughput sequencing technology revealed the enrichment of putative triplex forming sequences within the regulatory regions of the genome. These observations combined with the discovery of an ever-growing number of functional ncRNAs, led to the assumption that triplex structures could present an interface between these ncRNAs and the chromatin. The data in this thesis reveal that the intergenic spacer region of the mammalian rRNA genes contains a cluster of possible triplex forming sequences in its regulatory enhancer and terminator elements. The triplex forming potential of these motifs was predicted by in-silico analysis of the rDNA sequence and validated using EMSA experiments. Microscale thermophoresis (MST) assays were established to investigate the formation of triplex structures in various conditions in vitro. These experiments presented new insights on the characteristics of the triplex formation process and suggest the complex formation to be readily possible in physiological conditions. Thus the results provide the basis for further investigations on a possible role for enhancer-derived triplex forming ncRNAs in the regulation of the rRNA genes. Besides their presence in regulatory gene regions, sequences prone to form non-canonical DNA structures were also shown to be part of matrix attachments regions (MARs) throughout the genome. These MAR sites are tethered to the nuclear matrix and mediate the anchoring of chromatin loops on this putative protein scaffold within the nucleus. The MAR elements are implicated to play various roles in transcriptional regulation and chromatin organization. Interestingly, MARs have also been shown to interact with specific MAR binding proteins and were implicated to form triplex structures with ncRNAs at the nuclear matrix. However, the exact nature of these processes and their implications for gene regulation and chromatin organization are so far poorly understood. In the second part of this thesis Tip5, a subunit of NoRC, the key player in epigenetic rRNA gene repression, is revealed as nuclear matrix binding protein. The AT-hook and TAM domains of the protein are identified as motifs mediating the localization of Tip5 to the nuclear matrix. The results furthermore show a Tip5 dependent recruitment of rDNA repeats to the nuclear matrix upon rDNA repression. This suggests an additional role for the NoRC complex in the large-scale chromatin organization of the rRNA genes to the nuclear matrix. Thus the insights provided by these results are a good example for the interplay between MAR elements and their specific binders in large-scale chromatin organization. In the course of these experiments the new extended AT-hook motif was discovered, which displays a DNA binding activity similar to canonical AT-hooks and moreover could be revealed as strong RNA binder. Initial in vivo experiments validated the importance of the eAT-hook domain for proper cellular localization of a candidate protein. Together these results suggest a role for the eAT-hook domain in chromatin organization, nuclear matrix binding and anchoring of proteins to structural DNA and RNA elements, similar to the canonical AT-hooks. In summary the data presented in this thesis provides new insights on the characteristics and various roles for structured DNA elements and their specific binders in transcriptional gene regulation and nuclear matrix mediated chromatin organization

Quaternion-Based Aircraft Attitude Estimation

Aircraft attitude estimation requires fusing several sensors in order to recover both high and low frequency information in an observable manner. This thesis explores the fusion of gyroscope integration, gravity vector estimation, and magnetic field vector estimation using a complementary filter and an extended Kalman filter (EKF), both of which use a unit quaternion to represent the attitude portion of the state. First, a set of models, which contain bias, scale factor errors, alignment errors, and Gaussian white noise, is introduced to govern the available sensors. The gyroscope bias is modeled as a random walk. A calibration routine is then established to minimize scale factor and bias errors. After some definitions and derivations for quaternion algebra are established, the attitude solution is then estimated using the complementary filter. Then the EKF is introduced and used to estimate both the quaternion state and gyroscope bias. The thesis is concluded with a Monte Carlo run to compare the complementary filter with the EKF. Due in large part to the estimation of gyroscope bias in the EKF, this filter is shown to give a significantly more accurate state estimate. The robustness is also evaluated, with both filters initialized with the incorrect initial quaternion and gyroscope bias estimate. The EKF is shown to converge relatively quickly, while the complementary filter does not reliably converge due to the lack of gyroscope bias estimation