The regulation of megakaryocyte-specific genes byFli-1 and GATA-1

The successive activation of tissue-specific genes during cellular differentiationis orchestrated by the formation of transcriptional complexes consisting of cellspecificand ubiquitous transcription factors. Understanding the molecularevents associated with normal megakaryocyte (Mk) differentiation is an issue ofcentral importance to haematology. The aims of this study were therefore to: (i)define the transcription factors responsible for regulating the expression of Mkspecificgenes such as Glycoprotein IX, (ii) identify the protein partners of suchimportant Mk-regulatory transcription factors and (iii) examine themechanisms utilised by these factors to regulate gene expression.First, the regulatory elements in the GPIX promoter required for basal andinducible expression were examined in megakaryoblastic Dami cells stimulatedto undergo differentiation. The resulting data suggested that an Ets site in theGPIX promoter binding the Ets-family member Fli-1 was crucial in regulatingboth constitutive and inducible GPIX expression. Second, a two-hybrid screenof a K-562 cDNA library was used to identify transcription factors thatinteracted with Fli-1 and were potential regulators of Mk development. Resultsof this screen identified a novel protein-protein interaction with GATA-1, apreviously well-characterised zinc finger transcription factor also implicated inerythroid and Mk development. Mapping of the domains required for theinteraction show that the zinc fingers of GATA-1 interact with the Ets domainof Fli-1.The biological significance of the Fli-1/GATA-1 interaction was demonstratedin transient transfection assays, which resulted in synergistic activation of Mkspecificpromoters. Analysis of Fli-1 and GATA-1 expression in a series oferythroleukaemic and megakaryoblastic cell lines demonstrated that the Fli-1/GATA-1 combination correlates with a Mk-phenotype. Moreover, expressionof Fli-1 in K-562 cells (a line rich in GATA-1 but normally lacking Fli-1) inducesendogenous 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 throughprotein-protein interaction and cooperative DNA-binding to activate theexpression of genes associated with the terminal differentiation of Mks