Student Theses and Dissertations

Date of Award

2014

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Papavasiliou Laboratory

Abstract

Activation-induced cytidine deaminase (AID) is essential for two processes of immunoglobulin diversification in germinal center B cells: somatic hypermutation (SHM), in which mutations are introduced into immunoglobulin (Ig) genes, and class-switch recombination (CSR), in which genomic constant regions are recombined to encode antibodies of different isotypes. Both of these processes require AID-catalyzed C-to-U lesions at the Ig loci, which are resolved to generate point mutations or double-stranded DNA breaks in the cases of SHM and CSR, respectively. Despite over a decade of intense study, a number of open issues remain surrounding AID. The diversity of findings regarding AID’s role in DNA demethylation raises the question of the scope of its involvement in this process. Additionally, while it is clear that AID-mediated damage occurs, the effects of this damage on the average B cell have not been characterized. Finally, the issue of whether AID is able to edit RNA in vivo has never been rigorously addressed in the literature. In each of these cases, the advent of high-throughput sequencing provides methods for genome-wide characterization of AID’s effects. This thesis presents the application of a number of genome-scale, sequencing-based methods to characterize the effects of AID deficiency and overexpression on the activated B cell: mRNA-Seq and miRNA-Seq allow for measurements of RNA expression and editing, while reduced-representation bisulfite sequencing (RRBS) assays DNA methylation. These analyses confirmed AID’s known role in immunoglobulin isotype switching, while also demonstrating that it has little other effect on gene expression. Additionally, no evidence of AID-dependent mRNA or miRNA editing could be detected. Finally, RRBS data failed to support a role for AID in the regulation of DNA methylation. Thus, despite evidence of its additional activities in other systems, antibody diversification appears to be AID’s sole physiological function in activated B cells. Following the conclusion of my studies of AID’s effects in B cells, I applied similar genomics tools to two amenable topics in nucleic acid modifications. First, I used mRNA-Seq to attempt to determine the substrate of the orphan cytidine deaminase Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 2 (APOBEC2). Next, I used whole-genome bisulfite sequencing to explore the distribution of 5-methylcytosine in Trypanosoma brucei. In both of these cases, results were inconclusive but suggest future directions for investigation.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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