title: The Ewing sarcoma protein (EWS) binds directly to the proximal elements of the macrophage-specific promoter of the CSF-1 receptor (csf1r) gene.
abstract: Many macrophage-specific promoters lack classical transcriptional start site elements such as TATA boxes and Sp1 sites. One example is the CSF-1 receptor (CSF-1R, CD115, c-fms), which is used as a model of the transcriptional regulation of macrophage genes. To understand the molecular basis of start site recognition in this gene, we identified cellular proteins binding specifically to the transcriptional start site (TSS) region. The mouse and human csf1r TSS were identified using cap analysis gene expression (CAGE) data. Conserved elements flanking the TSS cluster were analyzed using EMSAs to identify discrete DNA-binding factors in primary bone marrow macrophages as candidate transcriptional regulators. Two complexes were identified that bind in a highly sequence-specific manner to the mouse and human TSS proximal region and also to high-affinity sites recognized by myeloid zinc finger protein 1 (Mzf1). The murine proteins were purified by DNA affinity isolation from the RAW264.7 macrophage cell line and identified by mass spectrometry as EWS and FUS/TLS, closely related DNA and RNA-binding proteins. Chromatin immunoprecipitation experiments in bone marrow macrophages confirmed that EWS, but not FUS/TLS, was present in vivo on the CSF-1R proximal promoter in unstimulated primary macrophages. Transfection assays suggest that EWS does not act as a conventional transcriptional activator or repressor. We hypothesize that EWS contributes to start site recognition in TATA-less mammalian promoters.
authors.affiliation: Australian Research Council Special Research Centre for Functional and Applied Genomics and the Cooperative Research Centre for Chronic Inflammatory Diseases, University of Queensland, Brisbane, Queensland, Australia. David.Hume@bbsrc.ac.uk
authors.forename: David A
authors.initials: DA
authors.lastname: Hume
authors.forename: Tedjo
authors.initials: T
authors.lastname: Sasmono
authors.forename: S Roy
authors.initials: SR
authors.lastname: Himes
authors.forename: Sudarshana M
authors.initials: SM
authors.lastname: Sharma
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Bronisz
authors.forename: Myrna
authors.initials: M
authors.lastname: Constantin
authors.forename: Michael C
authors.initials: MC
authors.lastname: Ostrowski
authors.forename: Ian L
authors.initials: IL
authors.lastname: Ross
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression; D006801:Humans; D047468:Immunoprecipitation; D008264:Macrophages / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D010802:Phylogeny; D011401:Promoter Regions, Genetic; D034802:RNA-Binding Protein EWS / Q000378:metabolism*; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D034802:RNA-Binding Protein EWS; D016186:Receptor, Macrophage Colony-Stimulating Factor