GATA-1 is a key haematopoietic transcription factor which plays a pivotal role in differentiation of the erythroid, megakaryocytic, eosinophilic, mast cell and dendritic cell lineages. Since its initial cloning and characterisation in 1989 a huge amount of information has been gathered on the molecular mechanisms of action of GATA-1. This knowledge has helped understanding of the processes by which cells enact differentiation programmes and suppress alternative lineage choices. GATA-1 produces at least two protein isoforms – the well characterised GATA-1 full-length (GATA-1FL) isoform and a truncated isoform – GATA-1 short (GATA-1s). GATA-1FL comprises two conserved Zinc fingers (which interact with DNA and essential co-factors), a C-terminal tail (of mostly unknown function) and an N-terminal domain (thought to confer activation properties to the molecule, but which may also be involved in transcriptional repression). GATA-1s lacks the N-terminal domain but is otherwise identical. The biological role of GATA-1s is unknown and this isoform received scant attention until the discovery that GATA-1FL mutations were linked to a rare, but highly informative, acute megakaryoblastic leukaemia seen in children with Down syndrome (constitutional trisomy 21). This discovery was particularly interesting, not only because the association between trisomy 21 and the X-linked GATA-1 mutation was extremely tight (being seen in 100% of the cases examined), but also because the GATA-1FL mutations were not randomly located, but rather clustered within the N-terminus, allowing unhindered production of the GATA-1s isoform. This finding led to interest in the pathological and physiological role of GATA-1s in haematopoiesis. Some insight has been gained into the pathological role of GATA-1s by creation of a GATA-1s knock-in transgenic mouse and by experiments looking at the ability of GATA-1s to rescue GATA-1 deficient embryonic stem (ES) cell lines. GATA-1s produces hyper-proliferation of fetal liver meg-erythroid progenitors but allows at least partial differentiation of these cells. However, a number of key questions remain. In particular what is the physiological role of GATA-1s and the reason for the tight association between trisomy 21 and GATA-1s mutations? Given this background, this thesis describes experiments designed to address the physiological role of GATA-1s, to establish whether additional GATA-1 isoforms exist, and to investigate the association between GATA-1 isoform expression and trisomy 21. Firstly a comprehensive expression analysis was performed in murine and human primary tissues and cell lines. This aimed to identify whether GATA-1s had a unique expression profile, either in particular lineages, or at distinct stages of haematological ontogeny. Reverse-transcriptase polymerase chain reaction (RT-PCR) and western blot analyses showed that the expression patterns of GATA-1s and GATA-1FL were virtually identical, with the possible exception of one human primary monocytic cell preparation which appeared to preferentially express GATA-1s. Before proceeding to further analysis of GATA-1s a search was made for additional GATA-1 isoforms using in silico analysis, RT-PCR and western blotting. This led to identification of a clone carrying a GATA-1 mutation involving the C-terminal tail, derived from a patient with chronic myeloid leukaemia. An analysis of the properties of this clone was performed, confirming its altered C-terminus and demonstrating that this conferred increased transactivation properties on the molecule as measured by luciferase assays. This observation suggests that the C-terminal tail may be an important, and previously under-recognised, functional region of the GATA-1 molecule. The discovery of this potentially hyper-functioning GATA-1 mutation led to investigation of whether GATA-1 mutations could be a widespread phenomenon in CML. However, GATA-1 mutational analysis in 21 patient samples from CML blast crisis did not reveal any additional coding mutations. To address the physiological role of GATA-1s, attempts were made to perform gene targeting in murine embryonic stem cells to produce isoform specific knock-out cells i.e. ES cells engineered so that they exclusively express the GATA-1FL isoform (a GATA-1s knock-out) or the GATA-1s isoform (a GATA-1FL knockout). These cells could then be used in in vitro haematopoietic differentiation assays and for transcriptional profiling. In this way it was hoped to establish whether GATA-1s fulfilled any unique roles in primitive or definitive haematopoiesis that could not be compensated for by the presence of the GATA-1FL isoform. Unfortunately, despite evidence of apparently successful targeting from PCR screening of ES cell clones, it was impossible to confirm the existence of endogenously targeted alleles on Southern blotting. Following exhaustive attempts at screening further clones and subclones (more than 1000 clones in total), this approach was abandoned in favour of transgenic expression of GATA-1 isoforms in cell lines. Transgenic expression studies in murine ES cells showed that whilst GATA-1FL expression led to an expansion in numbers and maturity of erythroid and non-erythroid haematopoietic colonies in vitro, GATA-1s was incapable of supporting colony formation in this assay. Studies then moved on to human cell lines. Two cell lines were identified, both capable of in vitro haematopoietic differentiation into megakaryocytic and erythroid cells, but one carrying trisomy 21 (Meg-01) and the other disomic for chromosome 21 (K562). GATA-1FL expression in these cells generally drove differentiation along the megakaryocytic or erythroid lineage as measured by DNA ploidy analysis, haemoglobinisation, upregulation of erythroid or megakaryocytic gene expression (by quantitative PCR) and suppression of alternative lineage genes (PU.1 and Ikaros) and genes associated with progenitor proliferation (cyclin D2 and c-myb). GATA-1s, in contrast, produced less evidence of differentiation with lower DNA ploidy, less up-regulation of erythroid genes and failure to repress other lineage and haematopoietic progenitor associated genes. Examination of the link with trisomy 21 confirmed that that the chromosome 21 candidate gene Erg3 was upregulated in trisomic cells and that expression of GATA-1s appeared to confer a selective advantage in the presence of trisomy 21. However, no clear mechanistic reasons for the selective advantage could be identified. Overall, these studies show widespread GATA-1s expression in haematopoietic cells, confirm the association with inadequate repression of genes associated with primitive progenitors, and suggest that the C-terminal tail of GATA-1 may be an important functional part of the molecule. Finally, these observations have generated a number of testable hypotheses which could form the basis for future work.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:513351 |
| Date | January 2009 |
| Creators | Halsey, Christina |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/870/ |
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