Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Microbiology and Immunology

Collaborative Specialization

Developmental Biology

Supervisor

DeKoter, Rodney

Abstract

Activation-induced cytidine deaminase (AID, encoded by Aicda) plays a primary role in producing mutations during somatic hypermutation and DNA breaks during class switch recombination, producing antibody diversity. Our laboratory generated a murine B cell-specific PU.1/Spi-B knockout, and whole-exome sequencing of their leukemias revealed C>T transition mutations compatible with being induced by AID. Therefore, we hypothesized that PU.1 negatively regulates Aicda during B cell development. Using ChIP-seq, two regulatory regions (R1 & R2) within the first intron of Aicda were identified to bind PU.1, and R1 was targeted with CRISPR-Cas9 mutagenesis. ChIP-qPCR and sequencing experiments revealed that the mutations created were heterozygous and dampened PU.1’s ability to bind. In addition, larger deletions in the R1 region resulted in an up-regulation of Aicda expression in response to lipopolysaccharide. However, alteration of the PU.1 binding site resulted in no change in expression, suggesting that PU.1 does not act in a silencer fashion.

Summary for Lay Audience

Activation-induced cytidine deaminase (AID, encoded by the gene Aicda) plays a primary role in producing mutations that enhance antibody specificity, thus making the immune system more specialized. AID mutations target immunoglobin genes; however, AID may mutate off-target genes, creating cancerous mutations, leading to B cell acute lymphoblastic leukemia (B-ALL). To explore the role of transcription factors PU.1 and Spi-B in B cell development, our laboratory generated a B cell-specific PU.1 and Spi-B knockout mouse model. Sequencing of the tumors from these mice revealed C>T transition mutations compatible with being induced by AID. Therefore, we hypothesized that PU.1 functions as a negative regulator of Aicda during B cell development. Antibody sequencing revealed two regulatory regions (R1 & R2) within the inhibitory region of Aicda capable of binding PU.1. CRISPR mutagenesis was then used to disrupt the predicted PU.1 binding site in the R1 region of Aicda in a pre-B cell line. Sequencing of the mutated cells revealed that only half of the Aicda gene was mutated, dampening PU.1’s ability to bind to this region. Additional results showed that large sequence deletions in the R1 region led to an up-regulation of Aicda gene expression in response to inflammation. However, direct alteration of the PU.1 binding site in R1 resulted in no change in Aicda gene expression in response to inflammation, which suggests that PU.1 does not act in a silencer fashion. These findings will contribute to a deeper understanding of the Aicda gene and the role of AID in the development of childhood leukemia.

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Available for download on Thursday, August 28, 2025

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