Immunoglobulins (Ig) are essential for maintaining immunity against a wide variety of pathogens. However, little is known regarding the impact of chemicals or disease states on Ig gene expression. We have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a well-known suppressor of B cell differentiation, potently inhibits activation of the transcriptional regulatory region found downstream of the IgH locus (3'lgH RR). In addition to its proposed role in Ig gene expression, the 3'lgH RR has also been associated with specific human pathologies including Burkitt's lymphoma, IgA nephropathy and Celiac disease. Many of the toxic effects of TCDD and related chemicals have been attributed to changes in gene expression resulting from the activation of the aryl hydrocarbon receptor (AhR) which subsequently binds to dioxin-responsive elements (DRE) in the affected genes. We have detected binding of AhR to DRE sites within 2 enhancers of the 3'lgH RR, hs1,2 and hs4, and find that these DRE sites are closely associated with NF-kB binding motifs. We hypothesize that TCDD represses 3'lgH RR activation through an AhR-dependent shift in the NF-kB/Rel protein complexes binding to kB motifs within the hs1,2 and hs4 enhancers. The following specific aims (SA) will test this hypothesis. SA#1: Determine which element(s) within the 3'lgHH RR are required for activation and for TCDD-induced repression utilizing an IgH mini-locus regulated by the 3'lgH RR and CRE-loxP technology. SA#2: Determine if TCDD's inhibition of 3'lgH RR activation is dependent on the AhR by inhibiting AhR expression with siRNA targeted to the AhR gene. SA#3: Determine the TCDD and LPS-induced binding profile of NF-kB hypothesize that TCDD represses 3'lgH RR activation through an AhR-dependent shift in the NF-kB/Rel proteins within the hs4 and hs1,2 enhancers by EMSA-Western and ChIP analyses. SA#4: Determine whether NF-kB/Rel proteins mediate TCDD's repressive effect on 3'lgH RR activation by repression of these proteins with an IkBa super represser protein or by over-expression of specific NF-kB/Rel fusion proteins. SA#5: Determine if the polymorphic human hs1,2 enhancer is sensitive to TCDD-induced inhibition with luciferase reporter genes regulated by the human hs1,2 enhancer. The proposed studies will provide the foundation for our long-term goal of elucidating the physiological and pathological (induced by AhR ligands) roles of the AhR and NF-?B/Rel proteins in the regulation of the human 3'lgH RR and its enhancers and their relation to human disease. Results of these studies will also be applicable to the hazard evaluation of other AhR agonists (and antagonists) which include a wide array of chemicals from environmental, dietary and pharmaceutical origin. ? ? ? ?
