The function of the conserved regulatory element within the second intron of the mammalian Csf1r locus.

Kristin A Sauter,Constanze Bonifer,Richard M Ingram,David P Sester,Clare Pridans,David A Hume,Julie O’Neal,Hiromi Tagoh,M Amine Bouhlel,Thomas Langmann

doi:10.1371/journal.pone.0054935

Abstract

The gene encoding the receptor for macrophage colony-stimulating factor (CSF-1R) is expressed exclusively in cells of the myeloid lineages as well as trophoblasts. A conserved element in the second intron, Fms-Intronic Regulatory Element (FIRE), is essential for macrophage-specific transcription of the gene. However, the molecular details of how FIRE activity is regulated and how it impacts the Csf1r promoter have not been characterised. Here we show that agents that down-modulate Csf1r mRNA transcription regulated promoter activity altered the occupancy of key FIRE cis-acting elements including RUNX1, AP1, and Sp1 binding sites. We demonstrate that FIRE acts as an anti-sense promoter in macrophages and reversal of FIRE orientation within its native context greatly reduced enhancer activity in macrophages. Mutation of transcription initiation sites within FIRE also reduced transcription. These results demonstrate that FIRE is an orientation-specific transcribed enhancer element.

Highlights

Macrophage colony-stimulating factor (CSF-1) controls the proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage [1,2]
Since RAW264.7 cells express relatively low levels of receptor on the cell surface and do not respond to CSF-1 treatment [22], we generated a stable cell line over-expressing CSF-1 receptor (CSF-1R). This cell line was transfected with the Csf1r promoter or the Fms-Intronic Regulatory Element (FIRE) enhancer cloned in the antisense orientation, and treated with CSF-1, PMA, or both
Since we found that the FIRE AP-1 site was occupied in our dimethyl sulfate (DMS) footprint analysis in cells stimulated with CSF-1 and LPS, we sought to determine if a macrophage nuclear protein complex bound to the site

Summary

Introduction

Macrophage colony-stimulating factor (CSF-1) controls the proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage [1,2]. The proximal promoter of Csf1r is not sufficient to generate maximal expression but requires the enhancer activity of a highlyconserved ,330 bp sequence in the second intron downstream of the macrophage promoter, which we named the Fms-Intronic Regulatory Element (FIRE; [7,8]). This element is functionally conserved between human and mouse [9] and the chicken Csf1r locus contains a conserved element in an equivalent location within the first intron [10]. Antisense promoter activity of FIRE was activated in B cells by PAX5, and was associated with repression of Csf1r expression [11]

Methods

Results

Conclusion