The family of Nucleoplasmin (NPM) proteins play an important role in a number of chromatin remodelling processes. The first NPM protein discovered in the eggs and oocytes of Xenopus laevis was NPM2, a tissue specific histone chaperone. In Xenopus, NPM2 has been linked to paternal chromatin decondensation following fertilization through the removal of sperm proteins, nucleosome assembly through the storage and addition of H2A-H2B dimers and apoptosis. In mammals, NPM2 correlates strongly with nucleolus-like bodies, and has been suggested by various groups to differ in its roles when compared to the X. laevis homologue. However, the exact roles of NPM2 in mammals remain to be fully elucidated. In this dissertation, attempts are made to determine the physical interaction sites between mouse NPM2 and core histone proteins, H2A, H2B, H3 and H4, as well as physical interactions between mouse NPM2 and protamines (sperm proteins) P1 and P2. Interaction sites between mouse NPM2 and various chromosomal proteins were investigated using a number of different techniques. First, NPM2: chromosomal protein binding assays were attempted to determine the ratio of NPM2 to both core histones and protamines. When visualized through 12% Native gels, NPM2 was determined to interact with histone octamers at a molar ratio of 1-1.5 mol NPM2/mol histone octamer. Mouse sperm protamines were determined to form complexes with mouse NPM2 at a molar ratio of 2.5 mol protamine/mol NPM2 (or mol protamine/0.4 mol NPM2). Analytical Ultracentrifuge (AUC) analysis was conducted on NPM2 and chromosomal proteins separately and in complex formation. Although determining that isolated, full length mouse NPM2 exists in a pentamer form, attempts with AUC were unsuccessful in determining specific NPM2:chromosomal protein binding affinity and complex formation. Specific physical interaction sites between NPM2 and chromosomal proteins were investigated using Cross Linking Mass Spectrometry. Here, a number of new interaction sites as well as sites previously identified by other groups were determined. In combination, our results present likely interaction sites between NPM2 and chromosomal proteins and represent an interesting point of reference for future work. / Graduate

Recent studies have uncovered de novo mutations of the gene encoding the chromatin remodelling protein Snf2l in patients with schizophrenia, Rett-like syndrome and intellectual disability. Snf2l and its closely related protein, Snf2h, play a critical role in embryonic and post-natal brain development. Murine models lacking functional Snf2h or Snf2l point to complementary activities of these remodelers; Snf2h cKO mice present with a significantly reduced cerebellum, while Snf2l Ex6DEL (exon 6 deleted) cerebella are larger than their wild-type counterparts. Granule neuron progenitors (GNPs) isolated from Ex6DEL cerebella display delayed cell cycle exit and hindered terminal differentiation compared to wild-type controls. Moreover, loss of Snf2l activity results in widespread transcriptome shifts which underlie the Ex6DEL GNP differentiation phenotype. In particular, key transcription factors are differentially expressed without Snf2l remodelling activity. We confirm that ERK pathway activation is misregulated in Ex6DEL GNPs, possibly in response to elevated fibroblast growth factor 8 (Fgf8) expression in these cultures. We find that Snf2l activity maintains the chromatin landscape throughout GNP differentiation, as Ex6DEL cultures have a global increase in chromatin accessibility. We suggest that Snf2l-mediated chromatin condensation is responsible for proper regulation of gene expression programs in GNP differentiation.

Regulation of gene expression is required for embryogenesis and maintenance of the highly specialized and diverse neuron populations of the retina. Chromatin remodelling proteins control gene expression by modifying chromatin structure and are essential for many biological processes including mammalian development. The ATP-dependent chromatin remodelling protein Snf2h is highly expressed in the central nervous system, and pathogenic variants that cause neurodevelopmental abnormalities in the human population have recently been identified. This work aims to characterize the effects of Snf2h loss in the retina. Snf2h retinal conditional knockout (cKO) mice were generated using Snf2h-floxed mice and Chx10-Cre retina-specific Cre driver lines to ablate the Snf2h protein from the retina at embryonic day 10.5. Visual function was assessed via optomotor response-based testing and full-field scotopic electroretinography, and histological changes were examined via immunohistochemistry. Disease progression was tracked at one, two, three, and six months of age. Snf2h cKO mice showed a significant decline in visual function and exhibited retinal neuron loss compared to wildtype control littermates at all time points assessed. This work shows that the chromatin remodelling protein Snf2h plays an essential role in the structure and function of the retina.

Master of Medicine / Non-melanoma skin cancer (NMSC) is the most common human cancer worldwide. Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) make up almost all NMSC. SCC usually arises from actinic keratosis (AK) as a result of exposure to sunlight. SCC and AK provide a useful clinical model to investigate changes involved in the progression of NMSC. This project examines the expression of Brm, Brg-1, Snail 1 and Snail 2 in the progression of NMSC. Brm and Brg-1 are subunits of the SWI/SNF chromatin-remodelling complex which is involved in regulating the access of cell machinery to DNA by altering the structure of chromatin. It has been suggested that loss of this function is involved in carcinogenesis as the cell is unable to access to DNA normally in order to repair mutations or activate apoptosis. The loss of Brm or Brg-1 has been described in several human cancers. Snail 1 and Snail 2 are zinc-finger transcription factors that are known for their role in epithelial to mesenchymal transition (EMT), a process vital to embryological development. Increased expression of these factors leads to a loss of cell-cell adhesion and a migratory phenotype and has been described in some human cancers. In this project, double-label immunohistochemistry was used to determine the relative expression of these proteins in human SCC, BCC, AK and normal skin. The expression of Snail was unable to be determined due to poor specificity of the antibodies used. The expression of both Brm and Brg-1 proteins was found to be dramatically and consistently decreased in SCC and BCC when compared to normal skin and AK. This loss of Brm and Brg-1 occured as the tumour progressed from benign AK to malignant SCC. This finding suggests that the loss of either Brm or Brg-1 constitutes a key step in carcinogenesis. The results of this study identify Brm and Brg-1 as putative tumour suppressors involved in the progression of non-melanoma skin cancer from benign to malignant.

Cytokinins are plant hormones that affect the primary growth of shoots and roots. Application of the cytokinin N6-benzylaminopurine (BAP) to the shoot apical meristem of <i>Arabidopsis thaliana Landsberg erecta </i>(L.) Heynh induces aberrant flower development and a significant genetic response, and some of these phenotypes and expression patterns were carried to the next generation. Analysis of altered transcript levels with Affymetrix GeneChips® indicated significant changes in transcript levels of genes associated with shoot meristem activity, circadian rhythms, cytokinin metabolism, two-component systems, stress and defense responses, auxin regulation, ethylene and salicylic acid biosynthesis, and signal transduction. Specific genes were also mined from the data as potentially responsible for the BAP-induced aberrant floral phenotypes, increased floral organ number, buds in axils of sepals, and mosaic floral organs. Of particular note was a decrease in the transcript levels of CLAVATA1 (CLV1), a gene encoding a receptor kinase involved in organ differentiation and maintenance of shoot and floral meristems. Time course analysis by RT-PCR showed a decline and subsequent recovery of transcript levels of CLV1 and a coincident increase in WUSCHEL (WUS) transcript, consistent with the known suppression of WUS by CLV. WUS encodes a homeodomain protein associated with shoot meristem proliferation. The temporal coincidence of an increased floral organ phenotype with changes in transcript levels of CLV1 and WUS suggests that cytokinins regulate flower development by affecting the activity of genes controlling shoot meristem activity. Aberrant floral phenotypes in subsequent non-treated generations suggest epigenetic inheritance of some BAP-altered transcript patterns. Repressed expression of the majority of significant genes in the untreated T1 population suggests a mechanism of gene silencing, such as methylation, was involved in this epigenetic inheritance. Also, transcript levels of time-keeping genes, including CIRCADIAN CLOCK ASSOCIATED 1 / ELONGATED HYPOCOTYL, and associated genes with oscillating expression patterns, such as COLD-RESPONSIVE, were affected by BAP in treated plants and the subsequent generation, suggesting the capacity of cytokinins to affect the phase of the circadian clock. Hormonal regulation of heritably altered diurnal periodicity and environmental responses may provide a developmental and, therefore, evolutionary advantage to plants.

Cytokinins are plant hormones that affect the primary growth of shoots and roots. Application of the cytokinin N6-benzylaminopurine (BAP) to the shoot apical meristem of <i>Arabidopsis thaliana Landsberg erecta </i>(L.) Heynh induces aberrant flower development and a significant genetic response, and some of these phenotypes and expression patterns were carried to the next generation. Analysis of altered transcript levels with Affymetrix GeneChips® indicated significant changes in transcript levels of genes associated with shoot meristem activity, circadian rhythms, cytokinin metabolism, two-component systems, stress and defense responses, auxin regulation, ethylene and salicylic acid biosynthesis, and signal transduction. Specific genes were also mined from the data as potentially responsible for the BAP-induced aberrant floral phenotypes, increased floral organ number, buds in axils of sepals, and mosaic floral organs. Of particular note was a decrease in the transcript levels of CLAVATA1 (CLV1), a gene encoding a receptor kinase involved in organ differentiation and maintenance of shoot and floral meristems. Time course analysis by RT-PCR showed a decline and subsequent recovery of transcript levels of CLV1 and a coincident increase in WUSCHEL (WUS) transcript, consistent with the known suppression of WUS by CLV. WUS encodes a homeodomain protein associated with shoot meristem proliferation. The temporal coincidence of an increased floral organ phenotype with changes in transcript levels of CLV1 and WUS suggests that cytokinins regulate flower development by affecting the activity of genes controlling shoot meristem activity. Aberrant floral phenotypes in subsequent non-treated generations suggest epigenetic inheritance of some BAP-altered transcript patterns. Repressed expression of the majority of significant genes in the untreated T1 population suggests a mechanism of gene silencing, such as methylation, was involved in this epigenetic inheritance. Also, transcript levels of time-keeping genes, including CIRCADIAN CLOCK ASSOCIATED 1 / ELONGATED HYPOCOTYL, and associated genes with oscillating expression patterns, such as COLD-RESPONSIVE, were affected by BAP in treated plants and the subsequent generation, suggesting the capacity of cytokinins to affect the phase of the circadian clock. Hormonal regulation of heritably altered diurnal periodicity and environmental responses may provide a developmental and, therefore, evolutionary advantage to plants.

Master of Medicine / Non-melanoma skin cancer (NMSC) is the most common human cancer worldwide. Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) make up almost all NMSC. SCC usually arises from actinic keratosis (AK) as a result of exposure to sunlight. SCC and AK provide a useful clinical model to investigate changes involved in the progression of NMSC. This project examines the expression of Brm, Brg-1, Snail 1 and Snail 2 in the progression of NMSC. Brm and Brg-1 are subunits of the SWI/SNF chromatin-remodelling complex which is involved in regulating the access of cell machinery to DNA by altering the structure of chromatin. It has been suggested that loss of this function is involved in carcinogenesis as the cell is unable to access to DNA normally in order to repair mutations or activate apoptosis. The loss of Brm or Brg-1 has been described in several human cancers. Snail 1 and Snail 2 are zinc-finger transcription factors that are known for their role in epithelial to mesenchymal transition (EMT), a process vital to embryological development. Increased expression of these factors leads to a loss of cell-cell adhesion and a migratory phenotype and has been described in some human cancers. In this project, double-label immunohistochemistry was used to determine the relative expression of these proteins in human SCC, BCC, AK and normal skin. The expression of Snail was unable to be determined due to poor specificity of the antibodies used. The expression of both Brm and Brg-1 proteins was found to be dramatically and consistently decreased in SCC and BCC when compared to normal skin and AK. This loss of Brm and Brg-1 occured as the tumour progressed from benign AK to malignant SCC. This finding suggests that the loss of either Brm or Brg-1 constitutes a key step in carcinogenesis. The results of this study identify Brm and Brg-1 as putative tumour suppressors involved in the progression of non-melanoma skin cancer from benign to malignant.

Der pRB/E2F-Signalweg ist ein zentraler Regulator der Proliferationskontrolle in Säugerzellen, der in fast allen auftretenden Tumoren dereguliert ist. Durch unterschiedliche Mutationen in Komponenten dieses Signalwegs kommt es letzten Endes zu einer erhöhten Aktivität der E2F-Transkriptionsfaktoren und somit zu einer verstärkten Transkription von E2F-Zielgenen in diesen Tumoren. Um die molekularen Mechanismen der Rolle von E2F3 in der Zellzykluskontrolle und der Tumorigenese besser zu verstehen, wurden in dieser Arbeit per GST-Pulldown mit anschließender Massenspektrometrie neue potenzielle Interaktions-partner von E2F3 identifiziert. Ein identifizierter Interaktionspartner war die SNF2-ähnliche Helikase HELLS. HELLS interagiert in vitro und in vivo spezifisch mit der Marked Box-Domäne von E2F3, aber nicht mit anderen untersuchten E2F-Transkriptionsfaktoren, wie durch GST-Interaktionsstudien und Ko-Immunpräzipi-tationsexperimente demonstriert werden konnte. Durch Chromatin-Immunpräzipitation konnte zusätzlich gezeigt werden, dass E2F3 für die Rekrutierung von HELLS an E2F-regulierte Promotoren wie z. B. CDC6 oder p107 verantwortlich ist. Die shRNA-vermittelte Depletion von HELLS führte zu einer stark verminderten Induktion von allen untersuchten E2F-Zielgenen nach Serumstimulation und einem verspäteten Eintritt in die S-Phase der HELLS-depletierten Zellen, was zeigt, dass HELLS essenziell für die Induktion von E2F-Zielgenen ist. Bei der immunhistochemischen Untersuchung der E2F3- und HELLS-Expression in humanen Prostatakarzinomen zeigte sich, dass sowohl E2F3 als auch HELLS in späten aggressiven Stadien dieser Tumore sehr stark exprimiert sind, jedoch nur sehr schwach in den weniger aggressiven Tumoren. Diese Versuche zeigen, dass es sich bei HELLS um einen neuen Bestandteil des pRB/E2F-Signalwegs handelt, der eventuell in der Entstehung gewisser Tumorarten eine Rolle spielt und somit ein neues potenzielles Ziel für neuartige Krebstherapien darstellt. / The pRB/E2F pathway is a key regulator of proliferation in mammalian cells and is commonly mutated in human tumors. These mutations in the components of the pRB/E2F pathway lead to deregulated activity of the E2F transcription factors resulting in increased expression of E2F target genes. To further understand the molecular mechanisms of E2F3 in cell cycle control and its role in tumorigenesis new interaction partners for E2F3 were identified in the course of this thesis with the help of a GST-Pulldown approach coupled to mass spectrometric analysis. One of the identified interaction partners was the SNF2-like helicase HELLS. With the help of GST-interaction studies and Co-Immunoprecipitation assays it could be demonstrated that HELLS interacts specifically with E2F3 via its Marked Box domain but does not bind to the other investigated E2F transcription factors. HELLS could be detected at E2F target genes like p107 and CDC6 in vivo with the help of Chromatin-Immunoprecipitation assays. Furthermore, the forced recruitment of E2F3 to E2F target genes led to an enhanced binding of HELLS to these promotors suggesting that HELLS is recruited to E2F target genes via protein-protein interaction with E2F3. The shRNA-mediated depletion of HELLS led to a strongly reduced induction of E2F target genes and a delay in S-phase entry, showing that HELLS is essential for the induction of E2F target genes. During the immunohistochemical analysis of human prostate cancer specimens it became evident that both E2F3 and HELLS are strongly expressed in the more aggressive late stages but only weakly expressed in the early stages of this tumor type. These findings demonstrate that HELLS is a new component of the E2F/pRB pathway which might play a role in the development of certain tumors and might represent a new target for novel cancer therapies.

ISWI is a member of a versatile family of ATP-dependent chromatin remodelling complexes involved not only in transcription regulation (initiation, elongation and termination), but also in other cellular functions like maintenance of higher order chromatin structure and DNA replication. TbISWI, a novel ATPase of the ISWI family in Trypanosoma brucei, is involved in the transcriptional repression of silent VSG expression sites (ESs) in both bloodstream form (BF) and procyclic form (PF) life cycle stages of the parasite. Using in silico analysis, I have found that TbISWI is well conserved across the eukaryotic lineage, including those members of the order Kinetoplastida that do not exhibit antigenic variation. Compared to the ISWIs of higher eukaryotes, TbISWI has greater representation of random coils within its structure, an indicator of more structural fluidity and flexibility of interaction with multiple protein partners. Using an eGFP reporter based assay, I have studied the role of TbISWI in transcriptional repression of silent areas of the T. brucei genome. TbISWI was found to be involved in preventing inappropriate transcription of the silent VSG repertoires. TbISWI was also found to downregulate transcription in RNA pol I, but not pol II, transcription units. These results argue for the presence of at least two functionally distinct TbISWI complexes in T. brucei. Using DNA staining and fluorescence in situ hybridisation (FISH), I have investigated the potential effect of TbISWI depletion on cell cycle progression and minichromosome segregation. I did not find any evidence for the role of TbISWI in the maintenance of centromeric heterochromatin in T. brucei.

ATP-dependent chromatin remodellers represent a class of proteins that restructure chromatin through the action of a conserved helicase-like ATPase domain. Remodellers typically have several accessory binding domains alongside the ATPase. These confer target specificity and most commonly recognise histone post-translational modifications. SMARCAD1 is a ubiquitous chromatin remodeller involved with DNA replication and re- pair. It binds directly to PCNA at the site of DNA replication and recruits co-repressor KAP1 in order to silence newly produced chromatin. In contrast to most other chromatin remodellers, SMARCAD1 does not contain several different types of accessory domains. Only two CUE do- mains have been identified in addition to the SMARCAD1 core ATPase domain. CUE domains are a type of helical ubiquitin-binding domain. This thesis presents the findings of an investigation into the structure and function of the SMARCAD1 double CUE domain. The solution NMR structure is presented with results from NMR binding experiments mapped onto the structure. Each CUE domain was found to be an independent helix bundle connected by a dynamic flexible linker. The N-terminal CUE domain, CUE-1, binds ubiquitin and has an adjacent SUMO (a ubiquitin-like protein) binding motif on a protruding extended helix. The C-terminal CUE domain, CUE-2, has a very similar structure to several published CUE domains but does not bind ubiquitin due to a charged substitution at a highly conserved CUE consensus position. The SMARCAD1 double CUE domain binds KAP1 from nuclear extract and is likely to mediate the interaction between SMARCAD1 and KAP1. SMARCAD1 double CUE domain is not involved with PCNA binding.