ORCID

http://orcid.org/0000-0002-6738-813X

Date of Award

Spring 5-15-2022

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Cellular DNA is regularly threatened by both physiologic (normal cellular mechanisms) and genotoxic (external exposures, such as irradiation and chemotherapy) insults. DNA double- stranded breaks (DSBs) are the greatest risk to the genome as they present opportunities for errant repair as mutations or translocations. To minimize these risks, DSBs activate highly conserved signaling pathways to coordinate cell cycle arrest, cell death and DNA repair. During normal development, B cells intentionally create site-specific chromosomal DSBs to assemble the immunoglobulin receptor (Ig) genes necessary for specific responses to pathogens. These programmed DNA breaks are generated by the recombinase activating gene (RAG) complex, a specific endonuclease that is selectively expressed in immune cells at discrete developmental stages. While essential for immune development, these RAG DSBs pose a significant risk as errors in the localization or response to these DSBs are a principal driver of oncogenic mutations and translocations in pediatric pre-B cell acute lymphoblastic leukemia (ALL). The cellular mechanisms that limit the extent of DNA damage and prevent oncogenic errors are unknown.Previous work has established that RAG-mediated DSBs activate a B-cell specific developmental program, known as the non-canonical DNA damage response (ncDDR). Surprisingly, we find that exposure of B cells to ionizing radiation or chemotherapy, which generate DNA damage randomly throughout the genome, does not activate this ncDDR, but instead, promptly triggers rapid activation of cell death pathways, known as the canonical DNA damage response (cDDR). Thus, developing B cells respond to DSBs through distinct signaling pathways depending on the mechanism of DNA injury, as RAG DSBs induce both cDDR and ncDDR, whereas genotoxic DSBs selectively induce only the cDDR. I hypothesize that the specific response to RAG DSBs is determined by either: 1) the unique landscape of the Ig locus (i.e., genomic location of the DSB) or 2) distinct domains of the RAG endonuclease itself. To examine the mechanisms downstream of DNA injury, I designed a novel CRISPR-Cas9 system using a tetracycline-inducible Cas9 in pre-B cells to generate targeted DSBs at precise locations throughout the genome. This approach was used to examine if all DSBs in pre-B cells trigger identical cellular responses. Pre-B cells were transduced with gRNAs to target Cas9 to regions outside of Ig loci. These Cas9-mediated DSBs in non-Ig genes activated the cDDR but did not trigger ncDDR. Thus, ncDDR signals are not a conserved component of pre-B cell response to all DSBs. Next, Cas9 DSBs were targeted to Ig loci to directly compare to a RAG DSB. Cas9-mediated DSBs at Ig also only activate cDDR. Therefore, while both Cas9 and RAG DSBs activate cDDR, the ncDDR program is uniquely activated by RAG DSBs. To investigate the underlying mechanisms of this specific response to RAG DSBs, mutagenesis studies of RAG1, a component of the RAG complex, were conducted. These experiments identified that non-core domains of RAG1 are critical for activation of the ncDDR developmental program. Thus, RAG DSBs induce distinct DDR programs in pre-B cells through novel nuclease-independent activities of RAG1. Ongoing studies are further characterizing the functions of RAG1 in DDR signaling using RAG1-Cas9 fusion constructs to recruit RAG1 to non-RAG-mediated DSBs. Additionally, immunoprecipitation studies will determine the RAG1-binding partners at DSBs that regulate DDR signals, which have not been previously defined. Overall, my dissertation characterizes the role of RAG1 in dictating DNA damage responses and demonstrates that B cells initiate distinct cellular responses to DNA breaks based on the mechanism of DNA injury. These studies provide key insights into a novel paradigm that directs cellular responses to DNA injury in pre-B cells. Understanding how RAG DSBs activate cellular programs to promote cellular survival and differentiation while maintaining genomic integrity has important implications for lymphocyte development. Selective activation of ncDDR is likely beneficial to developing pre-B cells as it promotes maturation of cells with recombined Ig genes. In contrast, non-RAG-mediated DSBs do not activate this process which may function to restrict abnormal B cell maturation and limit autoreactivity or malignant transformation. Further, defining these DSB-specific mechanisms that dictate cellular response may reveal previously unrecognized opportunities to optimize therapies for pre-B cell leukemia.

Language

English (en)

Chair and Committee

Jeffrey J. Bednarski

Committee Members

Nima Mosammaparast

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