Macrophage colony-stimulating factor (CSF-1) regulates the differentiation, proliferation and survival of cells of the mononuclear phagocyte system. The activity of CSF-1 is mediated by the CSF-1 receptor (CSF-1 R), that is encoded by c-fms proto-oncogene. The c-fms gene is exclusively expressed in macrophage and trophoblast cell lineages. Its proximal promoter displays characteristics of macrophage-specific gene promoter in which it lacks classical promoter elements including a TATA-Box, initiator, GC-rich sequences, Sp1-binding site and CAAT-Boxes commonly found in housekeeping genes. Instead, several purine-rich GGAA-containing sequences that are recognised by Ets family transcription factors are present, and indeed the myeloid-specific Ets family member, PU. 1 is a key regulator of c-fms transcription. Although data on c-fms transcriptional regulation are now accumulating,
further characterisation of cis-elements and trans-acting factors is required to obtain a more complete picture of c-fms gene regulation. In this PhD project, DNA-binding proteins have been shown to bind a region around the proposed myeloid-specific initiator. Mass spectrometry and peptide sequencing analyses identified these proteins as mFUS and mEWS, members of the Ewing's Sarcoma (EWS) family of proteins. It is proposed that mFUS and mEWS might be involved in the formation of pre-initiation complex, possibly by cooperating with the basal transcription factor TFIID, PU. 1, or other factors.
Previous studies have indicated that sequences in intron 2 control transcript elongation in tissue-specific and regulated expression of c-fms. In humans, an alternative promoter was implicated in
expression of the gene in trophoblasts. Results obtained in this study show that in mice, c-fms transcripts in trophoblasts initiate from multiple points within the 3.5 kb region flanking the first coding exon. A reporter gene construct containing 3.5 kb of 5' flanking sequence and the downstream intron 2 directed expression of enhanced green fluorescent protein (EGFP) to both trophoblasts and macrophages. EGFP was detected in trophoblasts from the earliest stage of implantation examined at embryonic day 7.5. During embryonic development, EGFP expression highlighted the large numbers of c-fms positive macrophages in most organs, including those that originate from the yolk sac. These embryonic macrophages contribute to organogenesis and tissue remodelling. In adult transgenic (TG) mice, which have been called the MacGreen mice, EGFP location was consistent with known macrophage populations, and permitted convenient detection
of tissues macrophages as well as their isolation from disaggregated tissues. EGFP was also expressed by Langerhans cells and CD11c+ splenic dendritic cells, which leads to the consideration that these cells are actually member of the Mononuclear Phagocyte System. Expression of EGFP in transgenic mice was dependent upon intron 2 as no lines with detectable EGFP expression were obtained when either all of intron 2, or a conserved enhancer element the Fms Intronic Regulatory Element (FIRE) was removed. This study has therefore both defined the elements required to generate myeloid and trophoblast-specific transgene expression and provided a model system for the study of mononuclear phagocyte development and function.