Access Type
Open Access Embargo
Date of Award
January 2024
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Chemistry
First Advisor
Ashok Bhagwat
Abstract
Activation-induced cytosine deaminase (AID) is required for both somatic hypermutation (SHM) and class switch recombination (CSR) within mammalian antibody genes. Uracils created by AID in the Ig loci are recognized and processed by base-excision and mismatch repair pathways leading to antibody diversification. This involves a hypermutation process restricted to the V(D)J segments of the immunoglobulin genes, SHM, and an error-prone end-joining process that causes isotype switching, CSR, but the molecular details of these processes have remained obscure for the past 20+ years. Most studies of SHM and CSR have used AID-promoted mutations as a proxy for the uracils, but their results are distorted by error-prone and error-free repair of uracils. To directly map uracils created by cytosine deaminase family enzymes, Bhagwat lab developed a technique where the uracils are excised by a uracil-DNA glycosylase, and a biotinylated alkoxyamine is used to covalently tag and sequence uracil-containing DNA fragments (UPD-Seq). Previously, this method has been used to map uracils created by human AID in the E. coli genome and created a uracilation map, i.e., uracilome. Here we report the first uracilome of a mammalian genome undergoing CSR. The murine CH12F3 cell line undergoes robust isotype switching from IgM to IgA upon stimulation with cytokines but does not undergo SHM. Using UPD-seq, we determined the uracilome of AID in the entire murine genome. An analysis of the uracilome showed uracilation peaks in the switch μ (Sμ) and switch α (Sα) regions of the IgH gene. No peaks were observed in any other switch regions or in the V(D)J exons. This shows that the creation of uracils by AID is a prerequisite for isotype switching and the lack of SHM in CH12F3 cells is caused by the lack of creation of uracils by AID in the V(D)J regions. Furthermore, the uracilation peaks in Sμ and Sα coincide with previously reported switch junctions suggesting that most, perhaps all, uracils created in the switch regions have the potential of being processed into strand breaks. There is a direct relationship between the occurrence of potential R-loop forming and G-quadruplex forming sequences and uracil accumulation in Sμ, but not in Sα. In contrast there is no direct relationship between the occurrence of WRCY sequences and uracil accumulation. Interestingly, the uracilation peak within Sμ and Sα regions have a correlation with the transcription stalling. This correlation is consistent with the cytosine to uracil conversion by AID occurring concurrently with transcription elongation and stalling. Intriguingly, the uracilation peak within Sμ has a sharp boundary on the 3' side. These and other observations based on results of UPD-seq clarify the mechanism of CSR in mammalian cells.
Recommended Citation
Mohamad Ramshan, Fathima Rukshana, "Uracil Landscape Of Mouse B Cell Genome Undergoing Class-Switch Recombination" (2024). Wayne State University Dissertations. 3959.
https://digitalcommons.wayne.edu/oa_dissertations/3959
