Role of specific protein binding motifs in TCDD-induced activation of the human polymorphic HS1,2 enhancer

Abstract

<p>The immunoglobulin heavy chain (lgH) gene is transcriptionally regulated in part by the 3'lgH regulatory region (3'lgHRR) which is located downstream of the IgH locus and in humans consists of three enhancers (hs3; hsl,2; hs4). Utilizing a well-characterized mouse B-cell line (CH12.LX), our previous results have demonstrated a sensitive inhibition of the mouse 3'lgHRR by 2,3, 7, 8-tetrachlorodibenzor-dioxin (TCDDL a known disrupter of B-cell differentiation, which correlated well with TCDD-induced inhibition of IgH expression and Ig secretion. Interestingly, in humans a polymorphism of the hsl,2 enhancer (resulting in a varying number of tandem repeats of a 53 bp sequence) has been correlated with several autoimmune diseases. The human hsl,2 enhancer is also sensitive to TCDD-induced modulation but in contrast to the mouse hsl,2 and 3'lgHRR, TCDD activated the human hsl,2 enhancer perhaps through altered binding to one or more transcription factor binding sites within the hsl,2 enhancer (Le., ORE, kB, AP-l, Oct, SP1). The purpose of the current study was to elucidate the transcriptional regulation of the human polymorphic hsl,2 enhancer following TCDD induction. Utilizing site-directed mutagenesis, mutated luciferase reporter plasm ids were designed containing a variety of binding site deletions (ORE, Oct, 53bp, Oct+53bp). These plasm ids were transiently transfected into the CH12.LX cells then treated with TCDD in the absence or presence of lipopolysaccharide (LPS) stimulation. Deletion of the ORE site showed a modest increase in TCDD-induced activity; whereas deletion of both the Oct site and the 53bp repeat resulted in a complete loss of TCDD-induced activation. These results suggest that there is a concerted activation of the polymorphic hsl,2 enhancer involving Oct and a site within the 53 bp repeat that may be independent of the ORE. Studies are ongoing to evaluate the role of the AP-l site in relation to these effects. (Supported by NIEHS R01ES014676)</p><p>This presentation occurred at the Wright State University Campus-Wide Celebration of Research, Scholarship and Creative Activities on April 16, 2010</p>