Identification of a novel cell type-specific intronic enhancer of macrophage migration inhibitory factor (MIF) and its regulation by mithramycin
Open Access
- 19 November 2010
- journal article
- research article
- Published by Oxford University Press (OUP) in Clinical and Experimental Immunology
- Vol. 163 (2), 178-188
- https://doi.org/10.1111/j.1365-2249.2010.04289.x
Abstract
The aim of this study was to determine the genetic regulation of macrophage migration inhibitory factor (MIF). DNase I hypersensitivity was used to identify potential hypersensitive sites (HS) across the MIF gene locus. Reporter gene assays were performed in different human cell lines with constructs containing the native or mutated HS element. Following phylogenetic and transcription factor binding profiling, electrophoretic mobility shift assay (EMSA) and RNA interference were performed and the effects of incubation with mithramycin, an antibiotic that binds GC boxes, were also studied. An HS centred on the first intron of MIF was identified. The HS acted as an enhancer in human T lymphoblasts (CEMC7A), human embryonic kidney cells (HEK293T) and human monocytic cells (THP-1), but not in a fibroblast-like synoviocyte (FLS) cell line (SW982) or cultured FLS derived from rheumatoid arthritis (RA) patients. Two cis-elements within the first intron were found to be responsible for the enhancer activity. Mutation of the consensus Sp1 GC box on each cis-element abrogated enhancer activity and EMSA indicated Sp1 binding to one of the cis-elements contained in the intron. SiRNA knock-down of Sp1 alone or Sp1 and Sp3 together was incomplete and did not alter the enhancer activity. Mithramycin inhibited expression of MIF in CEMC7A cells. This effect was specific to the intronic enhancer and was not seen on the MIF promoter. These results identify a novel, cell type-specific enhancer of MIF. The enhancer appears to be driven by Sp1 or related Sp family members and is highly sensitive to inhibition via mithramycin.Keywords
This publication has 51 references indexed in Scilit:
- Sp1: Emerging roles—Beyond constitutive activation of TATA-less housekeeping genesBiochemical and Biophysical Research Communications, 2008
- Reduced arthritis in MIF deficient mice is associated with reduced T cell activation: down-regulation of ERK MAP kinase phosphorylationClinical and Experimental Immunology, 2008
- HIF1α delays premature senescence through the activation of MIFGenes & Development, 2006
- Macrophage migration inhibitory factor and glucocorticoid sensitivityRheumatology, 2006
- Novel GC-rich DNA-binding compound produced by a genetically engineered mutant of the mithramycin producer Streptomyces argillaceus exhibits improved transcriptional repressor activity: implications for cancer therapyNucleic Acids Research, 2006
- Sp transcription factor family and its role in cancerEuropean Journal Of Cancer, 2005
- Hypoxia-induced Activation of the Retinoic Acid Receptor-related Orphan Receptor α4 Gene by an Interaction between Hypoxia-inducible Factor-1 and Sp1Journal of Biological Chemistry, 2004
- Inhibition of c-src Transcription by Mithramycin: Structure−Activity Relationships of Biosynthetically Produced Mithramycin Analogues Using the c-src Promoter as TargetBiochemistry, 2003
- Preliminary Observations on the Therapy of the Myeloid Blast Phase of Chronic Granulocytic Leukemia with Plicamycin and HydroxyureaNew England Journal of Medicine, 1986
- Effect of mithramycin on boneβ-glucuronidase and resorptionCalcified Tissue International, 1978