Dynamics of Nucleosome Assembly Characterized by Atomic Force Microscopy

Nucleosomes are the basic repeating unit defining the assembly and function of chromatin. Understanding the fundamental mechanisms of nucleosome structure and dynamics is critical to elucidating the chromatin assembly process. This dissertation describes my work in elucidating the role of different factors that drive the nucleosome dynamics. In my first study, we characterized, for the first time, the effect of sequence on nucleosome assembly. We then characterized the role of internucleosomal interactions, discovering a critical role internucleosomal interactions in the assembly of higher order structures. Based on the previous study and literature regarding histone tails, we hypothesized the histone H4 tail is critical in internucleosomal interactions. We characterized nucleosomes lacking the H4 tail on different sequences and discovered the H4 tail, thought to facilitate internucleosomal interaction, is responsible for nucleosome stability in a sequence dependent manner. We also discovered that internucleosomal interactions rescue nucleosome instability due to H4 truncation. To test the hypothesis that DNA sequence and internucleosomal interactions are the factors that determine nucleosomal compaction, we assembled tetranucleosomes on DNA substrates with different sequences and simulated random nucleosome placement. In a first of its kind study, our data, supported by a theoretical model, revealed that nucleosomes adopt a condensed conformation at a predictable rate significantly higher than seen in simulations, confirming nucleosomes communicate and interact to form heterogeneous higher order structures, as opposed to the theorized homogenous structure. In forward-looking studies, we addressed unanswered questions in complex nucleosome systems. In the first, we studied the nucleosome variant containing centromere protein A (CENP-A) on centromeric DNA, hypothesizing this unique region contains structures distinct from bulk chromatin due to these two features. Our results indicate that CENP-A nucleosomes assemble similarly to canonical (H3) nucleosomes on centromeric and other DNA, indicating assembly is dictated by interplay with other factors. In the second, we studied the interaction of transcription factor (TF) nuclear factor-κB (NF-κB) with nucleosomes, hypothesizing that it can alter nucleosome structure. For the first time, we discovered this TF induces nucleosome unraveling and identified it as a pioneer factor. We proposed two models explaining the interaction of NF-κB with nucleosomes. This dissertation also describes advances we have made in methodology for studying nucleosomal systems. We successfully designed a DNA nanoring to induce mechanical strain upon the DNA, which can be used to mimic the strain imposed by DNA wrapping around nucleosomes. Another structure we designed, a three-way junction as a DNA end-label, was applied to nucleosome assemblies, demonstrating its usefulness in the study of nucleosomal systems

The application of molecular biology techniques to analyse diversity in Theileria parva populations in Zambia

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.Theileria parva is a complex protozoan parasite causing East Coast fever in Eastern and Central Africa. Vaccination using live parasites is an effective control measure and has been used in Zambia based on locally isolated and introduced T. parva stocks. Diversity among T. parva populations was investigated in parasites from two Zambian provinces with different disease epidemiologies and control histories. Isolates from the pre-vaccination era, local and exotic stocks used for vaccination, and one recent field isolate were cloned and passaged in vitro to study genomic stability over time. The results of the data from three genome-wide probes indicate a marked homogeneity and stability among the Zambian isolates in contrast to East African isolates. Results from Southern blot profiles and the polymorphic immunodominant molecule (PIM) sequence analysis suggest a common origin for the Zambian isolates from the pre-vaccination era, except for one isolate (Zam5) from Southern Province. This isolate showed characteristics suggesting a buffalo origin. Assays for genotype characterisation were developed using five allelic markers. Multilocus characterisation revealed identical profiles in a recent Zambian isolate from Southern Province and two components of an exotic cocktail vaccine, indicating the escape of one of the vaccine stocks in the field. Characterisation of T. parva field populations by RFLP-PCR assays after immunisation revealed the presence of dominant genotypes from those that had been used for vaccination. Circumstantial evidence for the involvement of one of the exotic vaccine parasites in epidemics in Southern Province is presented and a hypothesis formulated for the rapid spread of this genotype. Analysis of the characterisation data suggested the existence of two groups of T. parva parasites of different origin. The classic T. parva group, characterised by a dimorphism of the p150, p104 and p32 loci and the absence of a p67 insert and a buffalo-derived group which showed a polymorphism of p150, p104 and p32 and the presence of a p67 insert. There is evidence that recombination occurs, resulting in parasites that have characteristics of both groups. The relevance of these recombinant parasites in the epidemiology of the disease seems low. Characterisation of larger samples from areas of regular buffalo-cattle contact is necessary to clarify this. Sequence analysis of the most discriminative locus (PIM) was undertaken and gene conversion could be the main mechanism generating diversity. A more appropriate nomenclature for T. parva is proposed based on the growing evidence of molecular differences among isolates and stocks.Belgian Administration for Development Cooperatio

Characterization of a Pfemp1 variant from a P. falciparum field is isolate and the development of anti-Pfemp1 antibodies

Rosetting (binding of infected erythrocytes to uninfected ones) is a parasite adhesion phenotype thought to be mediated by Plasmodium falciparum membrane protein 1 (PfEMP1). Despite the association between rosetting and severe malaria in Africa, rosette-mediating PfEMP1 variants from African-derived strains are yet to be characterized. I initially attempted to detect the predominantly expressed var genes in five recently culture-adapted Kenyan P. falciparum isolates selected for rosetting. Unfortunately no clear rosetting-associated genes could be detected, possibly because of incomplete selection and lack of a clear phenotype in the selected parasites. I then went on to study another Kenyan parasite line, SA075, whose predominant var gene tag had interesting sequence features previously linked to rosetting. Using PCR walking, cloning and sequencing, I identified the full-length var gene corresponding to this tag, called SA075var1. Subsequent experiments failed to confirm a role for the PfEMP1 variant encoded by SA075var1 in rosetting. However, SA075var1 had domain cassette DC 8-like features which have lately been linked to severe disease. Antibodies raised in rabbits against recombinant proteins from domains of SA075var1 revealed functional antibodies with an ability to recognize the surface of parasite infected erthrocytes and to mediate phagocytosis. Although mainly variant-specific, the antibodies showed limited cross-reactivity with one other parasite strain. Using plasma pairs from Kenyan malaria patients, I examined antibody responses against SA075 and four other rosetting P. falciparum strains selected to enrich for single rosette-mediating variants. Antibody responses were seen against particular rosette-elected strains suggesting that some variants may be important for particular severe malaria syndromes. Overall, the data described in this thesis demonstrates the need for continued research into functional and immunological characterization of PfEMP1 variants, especially in field isolates. Antibody data however showed clinical relevance of some rosette-mediating variants that could become important targets for malaria intervention studies

Characterization of Human Endogenous Retrovirus sequences identifed in the human genome using the RetroTector software

Human Endogenous Retroviruses (HERVs) represent the inheritance of ancient germ-line cell infections by exogenous retroviruses and the subsequent transmission of the proviral integrated elements to the descendants. Actually, no replication-competent HERV sequence is recognizable in the human genome. However, some HERVs retain one or several intact retroviral genes and may express protein products that could interfere with the human immune system. The number and classification of HERVs vary according to method of enumeration. The focus of this project is to perform a systematic analysis and a classification of the most intact HERV sequences in order to better understand their evolution and their involvement in shaping the human genome. The human genome assembly GRCh 37/hg19 was analyzed with RetroTector software and a total of 3290 HERV proviral sequences were identified. The complex genetic structure of the 3290 proviruses was resolved through a multi-step classification procedure that involved a novel type of similarity image analysis (Simage). The 3290 HERV were classified in 40 unique clades (groups) which could be placed into class I (Gamma- and Epsilon-like), II (Beta-like) and III (Spuma-like). Simage analysis contributed to define the presence of a high number (around 40%) of mosaic forms, with heterogenous sequence content. A finest characterization of the HERV sequences was achieved with the investigation of a broad panel of structural markers that contributed to confirm and extend the previously performed classification. Finally, the HERV background of integration was also studied. Integration patterns analysis showed a tendency for proviruses from the same clade to occur together, within 100000 bases, maybe due to local duplications. Representatives from some gammaretroviral clades (HERVH and HERVE) integrated more frequently than expected by chance into the 5´end of transcriptional units, mostly in antisense orientation. A few lncRNAs were also found to contain HERV sequences. Thus, cis-effects from HERVs are to be expected. In conclusion, this study represents an advance in the state-of-the art of HERV characterization within the human genome and a starting point for upcoming studies on HERVs

Characterization of Human Endogenous Retrovirus sequences identifed in the human genome using the RetroTector software

Human Endogenous Retroviruses (HERVs) represent the inheritance of ancient germ-line cell infections by exogenous retroviruses and the subsequent transmission of the proviral integrated elements to the descendants. Actually, no replication-competent HERV sequence is recognizable in the human genome. However, some HERVs retain one or several intact retroviral genes and may express protein products that could interfere with the human immune system. The number and classification of HERVs vary according to method of enumeration. The focus of this project is to perform a systematic analysis and a classification of the most intact HERV sequences in order to better understand their evolution and their involvement in shaping the human genome. The human genome assembly GRCh 37/hg19 was analyzed with RetroTector software and a total of 3290 HERV proviral sequences were identified. The complex genetic structure of the 3290 proviruses was resolved through a multi-step classification procedure that involved a novel type of similarity image analysis (Simage). The 3290 HERV were classified in 40 unique clades (groups) which could be placed into class I (Gamma- and Epsilon-like), II (Beta-like) and III (Spuma-like). Simage analysis contributed to define the presence of a high number (around 40%) of mosaic forms, with heterogenous sequence content. A finest characterization of the HERV sequences was achieved with the investigation of a broad panel of structural markers that contributed to confirm and extend the previously performed classification. Finally, the HERV background of integration was also studied. Integration patterns analysis showed a tendency for proviruses from the same clade to occur together, within 100000 bases, maybe due to local duplications. Representatives from some gammaretroviral clades (HERVH and HERVE) integrated more frequently than expected by chance into the 5´end of transcriptional units, mostly in antisense orientation. A few lncRNAs were also found to contain HERV sequences. Thus, cis-effects from HERVs are to be expected. In conclusion, this study represents an advance in the state-of-the art of HERV characterization within the human genome and a starting point for upcoming studies on HERVs

Biofunctional Nanodot Arrays in Living Cells Uncover Synergistic Co-Condensation of Wnt Signalodroplets

Qualitative and quantitative analysis of transient signaling platforms in the plasma membrane has remained a key experimental challenge. Here, biofunctional nanodot arrays (bNDAs) are developed to spatially control dimerization and clustering of cell surface receptors at the nanoscale. High-contrast bNDAs with spot diameters of ≈300 nm are obtained by capillary nanostamping of bovine serum albumin bioconjugates, which are subsequently biofunctionalized by reaction with tandem anti-green fluorescence protein (GFP) clamp fusions. Spatially controlled assembly of active Wnt signalosomes is achieved at the nanoscale in the plasma membrane of live cells by capturing the co-receptor Lrp6 into bNDAs via an extracellular GFP tag. Strikingly, co-recruitment is observed of co-receptor Frizzled-8 as well as the cytosolic scaffold proteins Axin-1 and Disheveled-2 into Lrp6 nanodots in the absence of ligand. Density variation and the high dynamics of effector proteins uncover highly cooperative liquid-liquid phase separation (LLPS)-driven assembly of Wnt "signalodroplets" at the plasma membrane, pinpointing the synergistic effects of LLPS for Wnt signaling amplification. These insights highlight the potential of bNDAs for systematically interrogating nanoscale signaling platforms and condensation at the plasma membrane of live cells

Organization of the Centromeric Satellite I Cluster and D21z1 Short Arm Junction Region of Human Chromosome 21

To study chromosomal segregation errors causing Down syndrome one needs a chromosome 21 (HC21) specific centromeric marker, which presently does not exist. Alphoid DNA is the only repetitive sequence at all human centromeres. The current map of HC21 has a gap in the p-arm alphoid (D21Z1) junction region and the centromeric satellite I (satI) sequence. This satellite I cluster was shown not to be a specific centromeric marker since it is also on HC13. There are actually multiple satI families on both HC13 and HC21. This project also filled the gap in the HC21map and characterized the D21Z1 p- arm junction region. A complete HC21 centromere physical map now exists that identified a novel Y-satellite I cluster and established clear linkage relations between D21Z1, satellite I and Y-satellite I. The D21Z1 p-arm junction region\u27s structure is not consistent with the unequal crossover model for the evolution of this region

The development and application of a ChIP and MeDIP assay to determine a link between epigenetics and metabolism in ovarian cancer progression

Epithelial ovarian cancer (EOC) has the highest mortality rate of all gynaecological cancers globally. High-grade serous ovarian carcinomas (HGSOC) comprise 75% of EOCs and are characterised by diagnosis at advanced stages due to the late manifestation of non-specific symptoms. The majority of HGSOC cells are found to metastasise to the omentum, an adipocyte-rich membrane which can metabolise chemotherapeutic drugs. EOC patients develop malignant ascites, characterised by the high concentration of cytokines and the presence of multicellular aggregates of EOC cells known as spheroids. Both characteristics can facilitate EOC metastasis. EOC patients initially respond well to platinum-based chemotherapy, but the vast majority will relapse and eventually develop chemotherapy-resistant disease. Long-term survival for OC has shown no recent improvement which highlights the needs for new strategies that improve EOC prognosis. The dysregulation of the epigenetic landscape is a hallmark of EOC leading to the activation of oncogenes and suppression of anti-tumour genes. Ten-eleven translocation (TET) 2 is involved in DNA demethylation, playing a role in transcriptional regulation and gene expression. Oncometabolites present in EOC can inhibit TET2 which leads to aberrant gene regulation, contributing to malignant transformation and progression. Additionally, TET2 can be stabilised by metformin through activation of AMPK, a key regulator of cellular energy homeostasis. Metformin, a biguanide drug widely prescribed as first line treatment for diabetes, has shown promise as an anti-cancer drug. In EOC, metformin has been linked with an increased survival rate in addition to being able to decrease cytokine production and tumour formation in ovarian tumour cells. However, here metformin was found to increase cytokine (IL-6 and IL-8) production in chemosensitive EOC cells, under conditions of a physiological EOC tumour microenvironment (i.e., restricted glucose conditions), but not in chemoresistant EOC cells. This may be unfavourable if metformin was to be repurposed as treatment for EOC and may account for its inconsistent efficacy as a potential therapeutic drug in other cancers. Pathways including AMPK/TET2, and JAK/STAT were explored using techniques such as western Blot, Chromatin immunoprecipitation (ChIP) and methylated DNA immunoprecipitation (MeDIP) as possible mechanisms for this response. Cell viability and proliferation assays were used to explore the effect of oncometabolites on EOC cells. The results will provide insights into how metabolites affect EOC progression in addition to identifying potential therapeutic targets

Host-pathogen-drug interactions in the context of antibiotic resistance: How host xenobiotic metabolism can affect antibiotic efficacy in a Methicillin-Resistant Staphylococcus aureus infection

Our arsenal of weapons to fight against bacterial infections is weakening: bacteria are gaining resistance to the common antibiotics, while industries are struggling to develop new effective ones. To avoid triggering de-novo antibiotic resistance, we need the right antibiotic for the specific bacteria, at a dose adapted to the patient genetics. Genes driving the degradation of antibiotics have indeed known genetic variants that can dramatically affect the kinetics of antibiotic metabolism from one patient to another. This could lead to treatment failure, excessive side effects or emergence of resistance. I first investigated the clinical relevance of the vancomycin-rifampicin combination to treat Methicillin-Resistant Staphylococcus aureus infections (Chapter 3). I showed in various experimental settings that these two antibiotics may promote an environment prone for antibiotic resistance. Their interaction might be unstable in vitro because of environmental factors, one could wonder how the host environment might generate such instability. I then explored how interactions between antibiotics and host xenobiotic genetics could influence antibiotic concentrations, potentially triggering increased treatment failure, side-effects and antibiotic resistance in patients carrying particular variants. In silico, I estimated the effects of genetic variants of the Cytochrome P450 3A4 gene to its enzyme, and, as they are unequally distributed in the world, their global relevance (Chapter 4). In vivo, I focused on the Carboxylesterase 2 gene and I found two of its variants, rs11075646 and rs8192925, capable of significantly altering the degradation of various drugs, including rifampicin and mycophenolate mofetil. A clinical study was designed, to explore possible correlations between genotype for these variants and treatment response in patients (Chapter 5). Altogether, this body of work highlights the prescribing importance of considering not only the strain in bacterial infections, but also the genetics of the human host. This raises a need to make sure the right antibiotics are used in practices, at doses adapted to the patients. As part of personalised medicine, checking their genotype for these biomarkers could tailor their therapy, improving recovery while avoiding antibiotic resistance

Exploring optimal Taxol® CYP725A4 activity in Saccharomyces cerevisiae

Background: CYP725A4 catalyses the conversion of the first Taxol® precursor, taxadiene, to taxadiene-5α-ol (T5α-ol) and a range of other mono- and di-hydroxylated side products (oxygenated taxanes). Initially known to undergo a radical rebound mechanism, the recent studies have revealed that an intermediate epoxide mediates the formation of the main characterised products of the enzyme, being T5α-ol, 5(12)-oxa-3(11)-cyclotaxane (OCT) and its isomer, 5(11)-oxa-3(11)-cyclotaxane (iso-OCT) as well as taxadienediols. Besides the high side product: main product ratio and the low main product titre, CYP725A4 is also known for its slow enzymatic activity, massively hindering further progress in heterologous production of Taxol® precursors. Therefore, this study aimed to systematically explore the key parameters for improving the regioselectivity and activity of eukaryotic CYP725A4 enzyme in a whole-cell eukaryotic biocatalyst, Saccharomyces cerevisiae. / Results: Investigating the impact of CYP725A4 and reductase gene dosages along with construction of self-sufficient proteins with strong prokaryotic reductases showed that a potential uncoupling event accelerates the formation of oxygenated taxane products of this enzyme, particularly the side products OCT and iso-OCT. Due to the harmful effect of uncoupling products and the reactive metabolites on the enzyme, the impact of flavins and irons, existing as prosthetic groups in CYP725A4 and reductase, were examined in both their precursor and ready forms, and to investigate the changes in product distribution. We observed that the flavin adenine dinucleotide improved the diterpenoids titres and biomass accumulation. Hemin was found to decrease the titre of iso-OCT and T5α-ol, without impacting the side product OCT, suggesting the latter being the major product of CYP725A4. The interaction between this iron and the iron precursor, δ-Aminolevulinic acid, seemed to improve the production of these diterpenoids, further denoting that iso-OCT and T5α-ol were the later products. While no direct correlation between cellular-level oxidative stress and oxygenated taxanes was observed, investigating the impact of salt and antioxidant on CYP725A4 further showed the significant drop in OCT titre, highlighting the possibility of enzymatic-level uncoupling event and reactivity as the major mechanism behind the enzyme activity. To characterise the product spectrum and production capacity of CYP725A4 in the absence of cell growth, resting cell assays with optimal neutral pH revealed an array of novel diterpenoids along with higher quantities of characterised diterpenoids and independence of the oxygenated product spectra from the acidity effect. Besides reporting on the full product ranges of CYP725A4 in yeast for the first time, the highest total taxanes of around 361.4 ± 52.4 mg/L including 38.1 ± 8.4 mg/L of T5α-ol was produced herein at a small, 10-mL scale by resting cell assay, where the formation of some novel diterpenoids relied on the prior existence of other diterpenes/diterpenoids as shown by statistical analyses. / Conclusions: This study shows how rational strain engineering combined with an efficient design of experiment approach systematically uncovered the promoting effect of uncoupling for optimising the formation of the early oxygenated taxane precursors of Taxol®. The provided strategies can effectively accelerate the design of more efficient Taxol®-producing yeast strains