Publication Date

2023

Document Type

Thesis

Committee Members

Courtney Sulentic, Ph.D. (Committee Co-Chair); Michael Raymer, Ph.D. (Committee Co-Chair); Michael Markey, Ph.D. (Committee Member)

Degree Name

Master of Science (MS)

Abstract

The human immunoglobulin heavy chain gene locus (IGH) has two 3 prime regulatory regions (3’IGHRR), each containing three enhancers (hs3, hs1.2, hs4). In animal models, the 3’IghRR regulates IgH expression and class switch recombination (CSR) to different Ig isotypes. The 3’IGHRR hs1.2 enhancer in humans is polymorphic in that an invariant sequence (IS) can be repeated one to four times in tandem. The hs1.2 polymorphism is of interest due to its association with several human autoimmune disorders and its potential sensitivity to exogenous substances such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin). In mouse models, TCDD inhibits the hs1.2 enhancer and 3’IghRR activation through the aryl hydrocarbon receptor (AHR), which correlates with Ig inhibition. However, in humans, TCDD activates the hs1.2 enhancer, decreases IgG secretion and increases IgE secretion, suggesting species differences in hs1.2 activity and overall IGH regulation. Furthermore, in the human CL01 B-cell line, that can be induced to secrete antibodies and undergo CSR, loss of the AHR via siRNA and CRISP/Cas9 gene editing results in a significant decrease in IgG secretion. We utilized CRISPR/Cas9 gene editing to target the hs1.2 IS repeats in the CL01 cell line. Several clones exhibited a functional effect on IGH expression with a reduction in the number of hs1.2 IS repeats within one or both of the 3’IGHRRs. Treatment with TCDD resulted in modulated IgG secretion profiles dependent on the hs1.2 IS genotype changes. We also developed a bioinformatics pipeline to characterize the genomes of our parent cell line, CL-01, and an AHR CRISPR/Cas9-edited clone that only expresses a functional transactivation domain (TAD) due to the editing. Finally, to further assess the sensitivity of the 3’IGHRR to environmental chemicals we exposed our CL01 cell line model to two additional environmental toxicants, perfluorooctanoic sulfonic acid (PFOS) and naphthalene. Decreased IgA was observed with these exposures. This work further implies that the IGH is a target of a variety of chemicals which may be mediated through the number of signaling pathways that converge at the 3’IGHRR.

Page Count

84

Department or Program

Microbiology and Immunology

Year Degree Awarded

2023

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