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* 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; and
Department of Molecular and Cellular Biochemistry and the Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210
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.
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1 This work was supported by the Cooperative Research Centre for Chronic Inflammatory Diseases, the Special Research Centre for Functional and Applied Genomics, and by National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant R01-AR-0447129 (to M.C.O.). S.R.H. and T.S. were funded by the National Health and Medical Research Council. M.C. was supported by a Commonwealth Postgraduate Research Award.
2 Address correspondence and reprint requests to Dr. D.A. Hume at the current address: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin EH25 9PS, Scotland, U.K. E-mail address: David.Hume{at}bbsrc.ac.uk
3 Abbreviations used in this paper: BMM, bone marrow-derived macrophage; CAGE, cap analysis of gene expression; ChIP, chromatin immunoprecipitation; EWS, Ewing sarcoma protein; FANTOM, functional annotation of mouse transcriptome project; Inr, initiator; MS/MS, tandem mass spectrometry; RT, room temperature; TSS, transcriptional start site.
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