Nature and modulation of the higher order chromatin fibre

The bulk of eukaryotic cellular DNA is compacted into chromatin, a nucleoprotein complex that is responsible for packaging DNA into the nucleus and regulating gene transcription. The chromatin fibre is a dynamic structure which is able to accommodate the many complex processes which occur simultaneously in a living cell. The fundamental building blocks of the lower order chromatin fibre have been studied extensively, providing us with a detailed understanding of the structures present; much is known about how these structures are modulated to allow processes like gene transcription and replication to occur in an organised fashion. In contrast to our detailed knowledge of the fundamental building blocks of chromatin, little is known about the conformation of the higher order chromatin fibre. This lack of understanding is due, predominantly, to the inaccessibility of the higher order fibre for study, and that much of the research to date has considered the conformation of the higher order fibre to be uniform. In this project I have analysed and modulated the higher order chromatin fibre to assess the role this fibre plays in the regulation of cellular processes.The conformation of the higher order chromatin fibre is often thought to change during the differentiation of cells. To study this alteration in conformation I have undertaken a detailed analysis of the higher order chromatin fibre from cells with different differentiation potentials and during their differentiation process. Using a hydrodynamic sedimentation approach to assess the conformation of the chromatin fibre I was unable to find any differences in its conformation. However, I have found that these chromatin fibres do have inherent differences in their nuclease sensitivities, suggesting that although the overall conformation of the fibres are similar, there are chromatin- related differences between cells with various differentiation potentials.To study the uniformity of the higher order chromatin fibre at different chromosomal locations I have analysed the chromatin structure found at centromeres to determine whether there is an alteration in the conformation of the chromatin fibre, which might affect the function of centromeres. My results clearly show that in mouse and human cells the chromatin fibre found at inner centromeric regions is more compact than the chromatin fibre present at outer centromeric regions, and in turn this is more compact than the bulk chromatin fibre. To determine the molecular basis for this, I have analysed acetylated centromeric heterochromatin from embryonic stem (ES) cells and heterochromatin associated with undermethylated centromeric DNA from F9 cells. My results demonstrate that this special chromatin architecture found at centromeres appears to be independent of histone acetylation and DNA methylation.To establish whether an alteration in the chromatin conformation will alter a cell's differentiation potential I have expressed histone H5, a replacement linker histone normally found in nucleated erythrocytes, in pluripotential ES cells. My results show that the constitutive expression of H5 in ES cells causes substantial cell death. I have therefore constructed a regulated, tetracycline based, histone H5 expression system in ES cells, but I was unable to express H5 in a controlled manner to investigate the underlying chromatin structure of these cells. In addition, I expressed histone H5 DNA -binding mutants in ES cells which also caused substantial cell death. I was therefore unable to determine whether the cellular phenotype obtained from expressing H5 in ES cells was due to an alteration in chromatin structure or a non- specific effect from expressing a positively charged molecule. As a first step towards studying the expression of linker histones in living cells and during development, I constructed and analysed a green fluorescent protein (GFP)- histone H5 fusion. As for histone H5, the GFP -H5 fusion protein is correctly expressed in a variety of cell types, but is lethal to cells when expressed at high levels for longer periods of time

Topoisomerase-II mediated biochemical mapping of centromeres and nitroreductase mediated drug metabolism in Trypanosomatids

PhDThe protozoan parasites Trypanosoma cruzi and Trypanosoma brucei are the causative agents of Chagas disease and Human African sleeping sickness respectively. Existing therapies are toxic and ineffective against the later stages of the two diseases, consequently safer, improved therapies are urgently required. Here, two areas of trypanosome biology are explored. In the first section, the process of cell division is approached from a fundamental biology perspective. Centromeres are the region of DNA where kinetochore structures form, allowing the attachment of microtubules to facilitate chromosome segregation. In T. brucei we have characterized the nature and location of centromeres by exploiting the localized activity of topoisomerase-II, a cancer chemotherapy target, at the centromere. Etoposide mediated DNA cleavage mapping revealed the presence of signature AT-rich repeat regions coupled with adjacent retrotransposons at the centromere. Further experiments demonstrate that of the two nuclear T. brucei topoisomerase-II isoforms, only topoisomerase-IIα is essential and active at the centromere. The second section centres on pro-drug development against a trypanosome type I nitroreductase. This enzyme has previously been implicated in activation of nifurtimox and benznidazole, the two therapies in clinical use against Chagas disease. Initially we have developed a luciferase based drug assay system in the clinically relevant intracellular T. cruzi stage and rapidly screened a range of nitroaromatic based compounds for trypanocidal activity. A series of derived nitrofuryl compounds previously developed against Chagas disease were also screened against T. brucei, where most demonstrate trypanocidal activities of less than 1 μM. Further we show that these compounds are active substrates of nitroreductase, and act as pro-drugs within the parasite by specific activation of nitroreductase to generate cytotoxic moieties