Functional analysis of the murine Emr1 promoter identifies a novel purine-rich regulatory motif required for high-level gene expression in macrophages Dawn O’Reilly, Mark Addley, Carmel Quinn, Alison J. MacFarlane, Siamon Gordon, Andrew J. McKnight 1 , David R. Greaves * Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK Received 5 August 2004; accepted 21 August 2004 Available online 5 October 2001 Abstract This study has investigated the transcriptional regulation of the Emr1 gene in murine macrophages and defined an enhancer element within the proximal promoter that is necessary for Emr1 expression in myeloid cells. This element consists of an extended purine-rich sequence (PuRS) of 83 consecutive purine residues containing 9 GGAA sequences, the core binding sequence for members of the Ets family of transcription factors. The Ets factor PU.1 associates with this PuRS element both in vitro and in vivo. Using a standard BLAST search we identified similar PuRS elements in other myeloid and nonmyeloid genes. All PuRS elements tested confer enhancer activity onto a heterologous promoter and chromatin immunoprecipitation experiments revealed that PU.1 associates in vivo with the PuRS elements from the genes expressed in myeloid cells. Our results provide evidence that extended purine-rich sequence elements may constitute a new transcription regulatory motif and that PU.1 association is a prerequisite for macrophage-specific expression. D 2004 Elsevier Inc. All rights reserved. Keywords: Macrophages; Promoter analysis; PU.1; Ets transcription factors; F4/80; Emr1 gene; Macrophage-specific gene expression; Chromatin immunoprecipitation Introduction Austyn and Gordon [1] first described the F4/80 monoclonal antibody in 1981. McKnight and colleagues [2] and Hume and colleagues [3,4] demonstrated that it recognized a macrophage-restricted 160-kDa cell-surface glycoprotein found exclusively in tissue resident macro- phages including Kupffer cells of the liver, splenic red pulp macrophages, Langerhans cells of the skin, and microglia of the central nervous system. Consequently, the Emr1 gene product (F4/80) has proved to be a useful marker for macrophage populations in a variety of immunohistological studies [5]. The isolation of the Emr1 cDNA by McKnight and colleagues [2] led to the characterization of its unusual structure encompassing multiple extracellular EGF repeats linked by a mucin stalk to seven transmembrane spanning domains. The murine gene (and the human ortholog EMR1 ) helped to define a small family of cell-surface TM7 proteins, some of which are expressed on leukocytes, including CD97, EMR2, and EMR3. EMR4 and others are expressed on nonhematopoietic cell types, reviewed by Kwakkenbos and co-workers [6] and McKnight and colleagues [7]. To date, the function of the Emr1 gene product (F4/80) has not yet been determined. Schaller and colleagues [8] showed that F4/80-deficient mice were healthy and macrophage populations in tissues remained 0888-7543/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2004.08.016 * Corresponding author. Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK. Fax: +44 1865 275515. E-mail address: david.greaves@path.ox.ac.uk (D.R. Greaves). 1 Current address: Celltech R & D Ltd, 216 Bath Road, Slough, SL1 4EN, UK. Genomics 84 (2004) 1030 – 1040 www.elsevier.com/locate/ygeno