Abstract
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.