Identification of a novel cell type-specific intronic enhancer of macrophage migration inhibitory factor (MIF) and its regulation by mithramycin.

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.

Functional evaluation of TNFAIP3 (A20) in rheumatoid arthritis.

OBJECTIVES: To determine the protein expression of TNFAIP3 in synovium and to show the capability of 6q23 intergenic SNPs, associated with rheumatoid arthritis (RA) susceptibility, to influence TNFAIP3 gene transcription. METHODS: Immunohistochemistry for TNFAIP3, NF-kB p65 and phosphorylated NF-kB p65 protein expression was performed in 6 RA knee joint synovium samples compared to 9 osteoarthritis (OA) samples. Luciferase reporter gene assays were used to examine the regulatory ability of RA associated SNP variants on TNFAIP3 promoter activity. Sense and antisense constructs were prepared for rs6920220 alleles, together with each of the 4 SNPs in r2=1 with it (rs6933404, rs2327832, rs6927172 and rs17264332), coupled to the TNFAIP3 promoter. Transient transfections were performed in a human T lymphoblastoid (CEMC7A) cell line. Bioinformatic software was utilised to prioritise SNPs for further investigation. Electrophoretic mobility shift assays (EMSA), using CEMC7A nuclear extracts, were conducted for the rs6927172 SNP alleles. RESULTS: TNFAIP3 protein expression was seen in the synovium samples and differential TNFAIP3 protein expression between RA vs. OA synoviocytes observed. Within RA synoviocytes TNFAIP3 expression is predominately cytoplasmic, whereas in OA its expression is strongly nuclear and cytoplasmic. For 3 of the 5 SNPs investigated (rs6920220, rs6933404, rs6927172) evidence of repressor activity of TNFAIP3 transcription was seen and EMSA data showed evidence of differential transcription factor binding to rs6927172 alleles. CONCLUSIONS: This is the first observation of TNFAIP3 protein expression in RA and OA synovium. In vitro analysis of 6q23 intergenic SNPs supports the possibility of the functional regulation of TNFAIP3.