Oncogene (2005) 24, 5278–5286 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc The Runx1 transcription factor controls CSF-1-dependent and -independent growth and survival of macrophages Stewart R Himes1, Stephen Cronau1, Christine Mulford1 and David A Hume*,1 1CRC for Chronic Inflammatory Disease, Institute for Molecular Biosciences, Queensland Biosciences Precinct, Bldg. 80, Services Rd., University of Queensland, Brisbane, Queensland 4072, Australia Gene translocations that repress the function of the Runx1 (Tracey and Speck, 2000). Runx1 has been shown to transcription factor play a critical role in the development be involved in expression of numerous tissue-specific of myeloid leukemia. In this report, we demonstrate that genes and can cooperatively activate transcription in Runx1 precisely regulates c-fms (CSF-1 receptor) gene concert with tissue-specific transcription factors such as expression. Runx1 controlled expression by binding to C/EBPa, PU.1 and c-Myb (Zhang et al., 1996a, b; multiple sites within the mouse c-fms gene, allowing Lutterbach and Hiebert, 2000). The importance of interaction between promoter and downstream enhancer Runx1 and CBFb in normal and malignant hematopoi- elements. The runx1 and c-fms genes showed an identical esis is evident from frequent translocations in both genes pattern of expression in mature macrophages. Runx1 in leukemia and a complete loss of fetal hematopoiesis expression was repressed in CSF-1 stimulated, proliferat- by targeted disruption of either gene (Miyoshi et al., ing bone marrow-derived macrophages (BMM) and 1993; Okuda et al., 1996; Sasaki et al., 1996; Wang et al., significantly increased in quiescent, CSF-1 starved cells. 1996). The RAW264.7 and Mono-Mac-6, macrophage-like cell Translocations in CBF genes result in fusion proteins lines expressed low levels of Runx1 and both showed that act as dominant inhibitors of native CBF function. growth arrest and cell death with ectopic expression of The t(8;21)translocation fuses nearly all of the Runx1. The EM-3 cell line, which represents an early transcriptional repressor ETO to the N-terminus of myeloid progenitor cell line, showed growth arrest with Runx1, resulting in replacement of the C-terminal trans- Runx1 expression in the absence of any detectable activation domain of Runx1 with the histone deacety- changes in cell differentiation. These findings suggest that lase domain of ETO (Runx1-ETO)(Downing et al., Runx1 regulates growth and survival of myeloid cells and 1993; Meyers et al., 1995). The inv(16) translocation provide a novel insight into the role of Runx family gene fuses MYH11, a smooth muscle myosin heavy chain translocations in leukemogenesis. gene, to CBFb(CBFb-SMMC). The resulting fusion Oncogene (2005) 24, 5278–5286. doi:10.1038/sj.onc.1208657; protein interferes with nuclear localization of the CBF published online 27 June 2005 protein complex (Thompson et al., 1991; Adya et al., 1998). Ectopic expression in transgenic mice showed Keywords: Runx1; macrophage; CSF-1; survival that although these fusion proteins can disrupt normal hematopoiesis, they are not sufficient for the develop- ment of leukemia (Castilla et al., 1999; Heibert et al., 2001). The link between loss of CBF function and leukemia Introduction suggests that CBF proteins act as strong tumor suppressors. Many genes that reside at breakpoint The AML-1/Runx1 transcription factor is a common regions in leukemogenic translocations and numerous target of gene translocation in leukemia (Maruyama tumor suppressor genes are important regulators of cell et al., 1993; Heibert et al., 2001). The DNA-binding proliferation and survival. Loss of Runx1 function was activity of this protein was originally characterized as believed to impact primarily on cell differentiation core binding factor (CBF)based upon the ability to bind resulting in abnormal accumulation of progenitor cells. the core enhancer sequence of the Moloney Leukemia A recent report used Cre/Lox inducible disruption of Virus LTR. CBF was subsequently found to comprise a exon 5 to show that myeloid differentiation in adult complex of Runx1 and a non-DNA-binding subunit, mice was not affected by loss of Runx1 expression CBFb (Wang and Speck, 1992). Runx1 and CBFb are (Ichikawa et al., 2004). Normal myeloid development in normally expressed in all hematopoietic tissues, during the absence of Runx1 expression is not consistent with myeloid differentiation and in mature macrophages lineage progression being the primary role of Runx1 in suppression of myeloid leukemia. *Correspondence: DA Hume; E-mail: [email protected] Several studies have indicated that Runx1 activity Received 19 October 2004; revised 18 January 2005; accepted 3 February may extend beyond regulation of cell differentiation. 2005; published online 27 June 2005 Runx1-ETO expression in mice resulted in the presence Runx1 regulation of macrophage growth and survival SR Himes et al 5279 of hematopoietic progenitors that showed aberrant from a highly conserved intronic enhancer, termed the proliferation in vitro with an increased capacity for Fms intronic regulatory element (FIRE)(Himes et al., self-renewal (Okuda et al., 1998). Similar results were 2001), which has additional potential Runx1 binding obtained from ectopic expression of Runx1-ETO in sites (Tagoh et al., 2002; Follows et al., 2003). Since hematopoietic stem and progenitor cells using retroviral CSF-1R is similarly expressed in human and mouse transduction. Runx1-ETO expression caused a strong macrophages, we anticipated that the key control enhancement in self-renewal of hematopoietic stem cells mechanisms would also be conserved across species. but inhibited colony formation by committed progeni- We therefore carried out experiments to determine tors in response to cytokine treatment (Mulloy et al., whether Runx1 regulates expression of the mouse gene 2002). and to determine whether activation involves putative Part of the reason why studies of Runx1-associated conserved binding sites in the FIRE enahncer. Towards leukemogenesis have focused on its role in differentia- this end, we produced expression constructs for both tion is that the protein is a transcriptional regulator of full-length Runx1 (PEBP2aB)and CBF b proteins based known differentiation associated genes. For example, on the pEF6 expression plasmid that uses the EF-1a Runx1 may control macrophage development by promoter for stable expression of genes. Stable transfec- regulating CSF-1 receptor (CSF-1R)expression (Zhang tion of the macrophage-like RAW264.7 cell line with et al., 1996a). CSF-1 (also termed macrophage-CSF) is either the pEFRunx1 or pEFCBFb expression con- required for macrophage development as determined by structs was performed and cells were analysed for CSF-1R the phenotype of mice with a targeted deletion of the expression by antibody staining and flow cytometry. CSF-1R or a natural mutation in the CSF-1 gene The level of CSF-1R expression in RAW264.7 cells was (Cecchini et al., 1997; Dai et al., 2002). Previous work low, but increased significantly with expression of either showed that Runx1 can synergistically activate the Runx1 or CBFb. Runx1 expression alone resulted in a human c-fms (CSF-1R)gene promoter with PU.1 and greater than 10-fold increase in surface expression of C/EBP proteins (Zhang et al., 1996a; Petrovick et al., CSF-1R when compared to the empty vector (pEF6) 1998). The binding site in the human c-fms promoter, control (Figure 1). Expression of the CBFb cofactor also however, is not conserved between human and mouse. enhanced CSF-1R expression but to a lesser extent than In this report, we characterize multiple binding sites Runx1. This finding represents the first direct evidence within the human and mouse genes and demonstrate that expression of the CSF-1R gene product, as opposed that Runx1 is also an important regulator of the mouse to promoter activity, can be controlled by Runx1. c-fms gene. c-fms Although gene expression is a differentiation Mouse and human c-fms genes contain multiple CBF et al marker for the macrophage lineage (Tagoh ., 2002; binding sites Sasmono et al., 2003), signaling from CSF-1 receptor encoded by this gene is also involved in macrophage Sequence analysis of regulatory regions revealed that the survival and proliferation. Removal of CSF-1 from location of CBF binding sites within the mouse and macrophage cultures leads to cessation of proliferation, human c-fms genes may not be conserved (Himes et al., and eventually to programmed cell death. An early 2001). Electrophoretic mobility shift assays (EMSA) event in this pathway is rapid upregulation of the CSF-1 were used to identify binding sites in either the promoter receptor. We report here that expression of the Runx1 or enhancer of the mouse and human genes. Specific transcription factor is itself acutely regulated by CSF-1 antibody to the Runx1 protein was used to identify and may contribute to both CSF-1 receptor upregula- Runx1 containing protein complexes by antibody super- tion and the control of cell survival and proliferation at shift. This analysis showed that both the human and later stages of macrophage development. We further mouse genes contain two sites that bind CBF proteins show that ectopic Runx1 expression resulted in growth with high affinity. In the mouse gene, both CBF binding arrest of macrophage-like and early myeloid progenitor sites were present in the enhancer. There was an cell lines. Our data suggest a dual role for Runx1 as an upstream site whose sequence showed no conservation important factor for macrophage development through with the human gene enhancer by alignment (MRxE) CSF-1R expression and as an inhibitor of cell prolifera- and a second downstream element which was completely tion or survival by restricting cytokine-independent conserved (HMRxE)(Figure 2a).The sequence within proliferation. the mouse promoter that corresponds to the previously characterized human element contains a single base change from the TGTGGT core consensus and showed weak CBF protein binding (MRxP).