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Environmental toxicants and human B cells: Insights from CRISPR editing and genomic sequencing

Allex-Buckner, Clayton
http://rave.ohiolink.edu/etdc/view?acc_num=wright1685007053669299

Abstract Details

2023, Master of Science (MS), Wright State University, Microbiology and Immunology.
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.
Courtney Sulentic, Ph.D. (Committee Co-Chair)
Michael Raymer, Ph.D. (Committee Co-Chair)
Michael Markey, Ph.D. (Committee Member)
84 p.
Bioinformatics; Biology; Immunology; Toxicology
IGH; immunology; immunotoxicology; CRISPR editing

Recommended Citations

Citations

  • Allex-Buckner, C. (2023). Environmental toxicants and human B cells: Insights from CRISPR editing and genomic sequencing [Master's thesis, Wright State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=wright1685007053669299

    APA Style (7th edition)

  • Allex-Buckner, Clayton. Environmental toxicants and human B cells: Insights from CRISPR editing and genomic sequencing. 2023. Wright State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=wright1685007053669299.

    MLA Style (8th edition)

  • Allex-Buckner, Clayton. "Environmental toxicants and human B cells: Insights from CRISPR editing and genomic sequencing." Master's thesis, Wright State University, 2023. http://rave.ohiolink.edu/etdc/view?acc_num=wright1685007053669299

    Chicago Manual of Style (17th edition)

wright1685007053669299
184
© 2023, some rights reserved.Creative Commons License Environmental toxicants and human B cells: Insights from CRISPR editing and genomic sequencing by Clayton Allex-Buckner is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by Wright State University and OhioLINK.
Release 3.2.12