The successive activation of tissue-specific genes during cellular differentiation is orchestrated by the formation of transcriptional complexes consisting of cellspecific and ubiquitous transcription factors. Understanding the molecular events associated with normal megakaryocyte (Mk) differentiation is an issue of central importance to haematology. The aims of this study were therefore to: (i) define the transcription factors responsible for regulating the expression of Mkspecific genes such as Glycoprotein IX, (ii) identify the protein partners of such important Mk-regulatory transcription factors and (iii) examine the mechanisms utilised by these factors to regulate gene expression. First, the regulatory elements in the GPIX promoter required for basal and inducible expression were examined in megakaryoblastic Dami cells stimulated to undergo differentiation. The resulting data suggested that an Ets site in the GPIX promoter binding the Ets-family member Fli-1 was crucial in regulating both constitutive and inducible GPIX expression. Second, a two-hybrid screen of a K-562 cDNA library was used to identify transcription factors that interacted with Fli-1 and were potential regulators of Mk development. Results of this screen identified a novel protein-protein interaction with GATA-1, a previously well-characterised zinc finger transcription factor also implicated in erythroid and Mk development. Mapping of the domains required for the interaction show that the zinc fingers of GATA-1 interact with the Ets domain of Fli-1. The biological significance of the Fli-1/GATA-1 interaction was demonstrated in transient transfection assays, which resulted in synergistic activation of Mkspecific promoters. Analysis of Fli-1 and GATA-1 expression in a series of erythroleukaemic and megakaryoblastic cell lines demonstrated that the Fli- 1/GATA-1 combination correlates with a Mk-phenotype. Moreover, expression of Fli-1 in K-562 cells (a line rich in GATA-1 but normally lacking Fli-1) induces endogenous GPIX expression. Quantitative mobility shift assays reveal that Fli- 1 and GATA-1 exhibit cooperative DNA-binding in which the binding of GATA-1 to DNA is increased approximately 26 fold in the presence of Fli-1. This data provides a mechanism for the observed transcriptional synergy. In conclusion, this work suggests that Fli-1 and GATA-1 work together through protein-protein interaction and cooperative DNA-binding to activate the expression of genes associated with the terminal differentiation of Mks.
| Identifer | oai:union.ndltd.org:ADTP/282211 |
| Date | January 2003 |
| Creators | Eisbacher, Michael, School of Medical Science, UNSW |
| Publisher | Awarded by:University of New South Wales. School of Medical Science |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Michael Eisbacher, http://unsworks.unsw.edu.au/copyright |
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