Pediatric Rheumatology Online Journal → June 2003 → Epidemiology, Classification, Immunology and Immunogenetics → Abstract #7

INCREASED AFFINITY OF A TRANSCRIPTION FACTOR COMPLEX FOR THE -174G ALLELE OF THE IL-6 GENE AUGMENTS ITS TRANSCRIPTIONAL ACTIVITY: RATIONALE FOR SUSCEPTIBILITY TO SYSTEMIC JIA

M. S. Fife,¹ R. Jeffery,¹ E. M. Ogilvie,¹ P. Woo.¹

¹Paediatric Rheumatology Unit, Dept Immunology and Molecular Pathology, University College London, London, United Kingdom

Background: The 174GC variant of the IL-6 promoter has been associated with numerous inflammatory diseases. We have previously reported this association with juvenile systemic arthritis (SA), and recently confirmed this in a multi-centre family based study. Additionally we demonstrated a lower transcriptional activity of the C allele in HeLa transfection experiments. In this study electrophoretic mobility shift assays (EMSAs) are used to investigate differences in transcription factors binding to the -174 alleles of the IL-6 promoter that might account for the observed variation in IL-6 expression.
Methods: Radio-labelled EMSA probes of the IL-6 promoter were synthesised with either the G- or the C-allele at position -174. Reactions were carried out by combining the probe with Hela nuclear extracts. Cold competition studies used unlabelled probes to confirm specificity. Antibody competition EMSAs were performed using antibodies specific for transcription factors under investigation.
Results: EMSAs revealed a difference in the banding pattern between G- and C- alleles, the G-allele producing a more intense band. The dominant band was confirmed as specific by cold-competition assay, the unlabelled G probe was a stronger competitor for the nuclear factors. Addition of antibodies specific for C/EBP, CREB1, NF-1 and CBP to the binding reaction resulted in the complete removal of the shifted band.
Conclusion: Hela nuclear factors bind differentially to the IL6 promoter -174G and C alleles. We have identified four transcription factors that interact with the probe. The removal of any single factor by antibody competition obliterates the interaction of all proteins with the probe, implying that they may bind in a co-operative manner. Thus, the G/C interchange at -174 appears to affect the net interactions between transcription factors in this region, which may explain the observed differences in transcriptional activity of the IL-6 promoter alleles.