Characterization of the murine Nramp1 promoter: requirements for transactivation by Miz-1.
Murine Nramp1 encodes a divalent cation transporter that is expressed in late endosomes/lysosomes of macrophages, and the transported cations facilitate intracellular pathogen growth control. The Nramp1 promoter is TATA box-deficient, has two initiator elements, and is repressed by c-Myc, in accordance with the notion that genes that deplete the iron content of the cell cytosol antagonize cell growth. Repression via c-Myc occurs at the initiator elements, whereas a c-Myc-interacting protein (Miz-1) stimulates transcription. Here we demonstrate that a non-canonical E box (CAACTG) inhibits basal promoter activity and activation by Miz-1. A consensus Sp1-binding site or GC box is also necessary for Miz-1-dependent transactivation, but not repression. Repression occurs by c-Myc competing with p300/CBP for binding Miz-1. Our results show that an Sp1 site mutant inhibits coactivation by p300 and that the murine Nramp1 promoter is preferentially expressed within macrophages (relative to a beta-actin control) compared with non-macrophage cells. The effect of the Sp1 site mutation on promoter function shows cell-type specificity: stimulation in COS-1 and inhibition in RAW264.7 cells. Miz-1-directed RNA interference confirms a stimulatory role for Miz-1 in Nramp1 promoter function. c-Myc, Miz-1, and Sp1 were identified as binding to the Nramp1 core promoter in control cells and following acute stimulation with interferon-gamma and lipopolysaccharide. These results provide a description of sites that modulate the activity of the initiator-binding protein Miz-1 and indicate a stimulatory role for GC box-binding factors in macrophages and a inhibitory role for E box elements in proliferating cells.
Bowen
Holly
H
Division of Biochemistry and Molecular Biology, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, United Kingdom.
Lapham
Abigail
A
Phillips
Emma
E
Yeung
Irene
I
Alter-Koltunoff
Michal
M
Levi
Ben-Zion
BZ
Perry
V Hugh
VH
Mann
Derek A
DA
Barton
C Howard
CH
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D027682:Cation Transport Proteins / Q000235:genetics*; D002412:Cations; D002455:Cell Division; D002843:Chromatin / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D017930:Genes, Reporter; D007371:Interferon-gamma / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008957:Models, Genetic; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D034622:RNA Interference; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D016335:Zinc Fingers; D000099173:Natural Resistance-associated Macrophage Protein 1
D027682:Cation Transport Proteins; D002412:Cations; D002843:Chromatin; D004268:DNA-Binding Proteins; D007371:Interferon-gamma; D007501:Iron; D008070:Lipopolysaccharides; D016271:Proto-Oncogene Proteins c-myc; D016329:Sp1 Transcription Factor; D000099173:Natural Resistance-associated Macrophage Protein 1