TRANSCRIPTIONAL REGULATION OF THE c-fms PROMOTER BY THE ETS FAMILY OF TRANSCRIPTION FACTORS
University of Queensland
Professor David Hume
CSF-1, and its cognate CSF-1 receptor encoded by the fms proto-oncogene, controls the proliferation, differentiation and survival of macrophages. The c-fms gene has two promoters, separately directing expression in macrophage and trophoblast cell lineages. The macrophage c-fms proximal promoter is an archetypal myeloid-specific promoter. It lacks elements that normally determine the site of transcriptional initiation. Instead, the promoter structure consists of multiple purine-rich motifs near the multiple transcriptional start sites, which contribute to the different functions of transcriptional initiation. This study focuses on the role of the ETS family transcription factors as regulators, which bind purine-rich motifs as regulators of gene expression in macrophages. The first section of this thesis was concerned with the identification of ETS family transcription factors expressed in mouse macrophages. Quantitative real-time PCR demonstrated that 17 of 24 ETS factors were expressed in macrophages grown from marrow in CSF-1. The macrophage-expressed ETS factors were tested for their ability to regulate the c-fms proximal promoter, via transient transfection analysis. In addition to known factors (PU.1 and ETS-2), ERG and FLI-1 were shown to transactivate the c-fms promoter; conversely NET and TEL trans-repressed activity. This study provides evidence towards ERG as the key factor regulating c-fms expression in embryonic macrophages before other ETS factors are expressed, namely PU.1 The second section of this thesis aimed at identifying the ETS protein-protein interaction network within the macrophage cell. This network was discerned from multiple data sets, including ETS protein partners identified from the modified version of the mammalian two-hybrid system, together with the integration of literature and public database searches. Furthermore, the mammalian two-hybrid system generated previously uncharacterized ETS protein partners. Parameter analysis of the macrophage-ETS-transcriptional network demonstrated the networks scalefree topology, in accordance with previously characterised protein-protein networks. ETS-2 was an example of a predicted hub within the macrophage-ETS-transcriptional network. ETS-2 is known to be phosphorylated on threonine 72 by CSF-1 regulated MAP kinase activation. These findings demonstrated that ablation of ETS-2 phosphorylation did not have a significant impact on the macrophage-ETStranscriptional network. In addition, ETS-2 phosphorylation was shown to be redundant in c-fms promoter signalling. An alternative ETS factor that may substitute for ETS-2 trans-activation of the c-fms promoter was identified to be NET. The results indicate that ETS factors constitute a transcriptional network within the macrophage cell, with the c-fms gene as one target for regulation.