Nancy Bigley (Committee Member), Mauricio Di Fulvio (Committee Member), Michael Leffak (Committee Chair), Debra Mayes (Committee Member), Courtney Sulentic (Advisor)
Doctor of Philosophy (PhD)
The immunoglobulin heavy chain (IGH) locus is partially responsible for immunoglobulin (Ig) production in B cells. The human IGH locus contains two 3’ regulatory regions (3’IghRR) that each contain three enhancers, which are thought to help drive overall transcription of the locus and also influence class switching to alternative Ig isotypes. The hs1.2 enhancer within the 3’IghRR is polymorphic in humans, containing a 53 bp invariant sequence (IS) that can be repeated up to four times. In vitro, the human hs1.2 enhancer is a sensitive target of exogenous chemicals, particularly 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin), a potent inhibitor of Ig expression in animal models. The IS polymorphisms have also been associated with many immunological disorders in human patients. Therefore, understanding the role of the hs1.2 polymorphisms could be invaluable to human health. To investigate the function of the hs1.2 polymorphism, a mutational analysis was performed in a luciferase assay system to assess the contribution of each hs1.2 transcription factor binding site to overall hs1.2 activity. From this analysis, it is clear that each transcription factor binding site individually contributes to the changing activity of the hs1.2 enhancer with B-cell stimulation. Surprisingly, results also indicate that TCDD likely acts on the hs1.2 enhancer through NF1 binding sites rather than dioxin response elements. However, further studies using luciferase reporters containing additional 3’IghRR elements call into question the value of luciferase assays in assessing 3’IghRR function. In order to study the endogenous 3’IghRR directly, the CRISPR/Cas9 gene editing system was used to modify the hs1.2 enhancer in the human 3’IghRR. Using a single CRISPR/Cas9 plasmid targeting the polymorphic hs1.2 invariant sequence (IS) repeats in a human B-cell line has successfully resulted in clonal populations containing a reduced number of IS repeats. Experiments with these cells have revealed that changing the combination of hs1.2 polymorphisms decreases the expression of some Ig isotypes while increasing others. Significantly, the alpha1 hs1.2 enhancer has a surprisingly large effect on expression of e transcripts, suggesting this enhancer should be investigated as a target of small molecule therapy in allergy sufferers.
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