Publication Date

2010

Document Type

Thesis

Committee Members

Nancy Bigley (Committee Member), Michael Leffak (Committee Member), Courtney E Sulentic (Advisor)

Degree Name

Master of Science (MS)

Abstract

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a disrupter, of B-cell differentiation, induces binding of the aryl hydrocarbon receptor (AhR) nuclear complex to dioxin responsive elements (DRE) within the mouse immunoglobulin heavy chain regulatory region (3'IgHRR), and produces a marked inhibition of 3'IgHRR activation, IgH expression, and antibody secretion in a well-characterized mouse B-cell line (CH12.LX). The mouse 3'IgHRR consists of at least four enhancers (hs3a; hs1,2; hs3b; hs4), and is highly homologous with the three enhancers (hs3; hs1,2; hs4) of the human 3'IgHRR. A polymorphism of the human hs1,2 enhancer (resulting in varying numbers of tandem repeats containing a DRE and κB site) has been correlated with several autoimmune diseases. Although the human and mouse hs1,2 enhancers are share a ~90% identity, luciferase reporter studies in mouse CH12.LX B-cells showed that TCDD inhibited LPS stimulation of the mouse hs1,2 enhancer but co-treatment with TCDD and LPS synergistically activated human hs1,2 enhancer activity. To evaluate transcriptional differences between the human and mouse hs1,2 enhancers, our objectives were to characterize the IM-9 cells as a potential human B-cell model, and to evaluate TCDD-induced transcriptional regulation of the polymorphic human hs1,2 enhancer in a human cell line. We confirmed AhR expression and TCDD-induced CYP1A1 induction in IM-9 cells. Then we transiently transfected IM-9 cells with the human hs1,2 reporters and determined that TCDD activates the human hs1,2 enhancer in IM-9 B-cells, as seen in CH12.LX B-cells. However, the TCDD-induced fold-activation in human IM-9 cells appeared less compared to results in mouse CH12.LX B-cells perhaps due to differences between the mouse and human AhR. Our data suggests that the TCDD-induced inhibition of the mouse hs1,2 enhancer versus the activation of the human hs1,2 enhancer may be related to an inhibitory BSAP site located on the mouse hs1,2 enhancer that is absent from the human hs1,2 enhancer. Our results support the use of IM-9 cells as a model for studies evaluating mechanistic differences between the mouse and human hs1,2 enhancers.

Page Count

106

Department or Program

Microbiology and Immunology

Year Degree Awarded

2010

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