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Thesis Submitted in Conformity with the Requirements for the Degree of Doctor of Philosophy Department of Immunology University of Toronto Molecular Requirements of Class Switch Recombination by Alexanda Ka-Shing Ling A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Immunology University of Toronto © Copyright by Alexanda Ka-Shing Ling 2020 Molecular Requirements of Class Switch Recombination Alexanda Ka-Shing Ling Doctor of Philosophy Department of Immunology University of Toronto 2020 Abstract To meet the depth and breadth of antigen space brought to bear by potential pathogens, adaptive immunity requires somatic diversification of antigen receptors through programmed DNA damage and concomitant recombination and repair of antigen receptor loci. This biological fact is conserved in all vertebrate lineages and is elaborated upon with secondary phenomena such as class switch recombination (CSR). Together, these recombinatorial adaptive immune processes are recapitulated billions of times over in each organism, mostly without negative consequences such as undesirable mutagenesis and genomic instability. This juxtaposition of damage and repair in a physiological model has been felicitous to the understanding of DNA repair more generally, and insights into the basis of adaptive immunity and DNA repair have played off together symbiotically. In that tradition, this manuscript endeavours to examine the molecular requirements of class switch recombination (CSR) along two complementary axes. Firstly, although it is known that double-stranded breaks (DSB) are substrates necessary for CSR, it is unknown how the structure and polarity of DSBs affects downstream DNA repair. Using the Cas9 enzyme and related variants, I generated DSBs of defined structures near the switch regions of the immunoglobulin heavy chain locus (Igh) and examined the resultant CSR frequency and repair junction characteristics. I found that single-stranded breaks upwards of 250 bp apart on opposite strands could resolve as DSBs in CSR. Moreover, 5′ DSBs were more preferred CSR substrates ii than 3′ DSBs, although both types of staggered DSBs had a higher level of resection and microhomology usage at the repair junction than blunt DSBs. Taken together, these observations suggest that DSB structure influences the type of repair effected as well as the frequency of productive CSR. I also investigated the effect of the newly-identified shieldin complex downstream of 53BP1 on concluding CSR. Shld2–/– B cells have profoundly impaired CSR, although B cell development and V(D)J recombination were not grossly affected. Additionally, I observed an increased Iglo population in shieldin-deficient switching B cells that was the result of extreme resection impairing Ig expression, rather than increased inversional recombination, and congruent with the proposed function of shieldin. iii Acknowledgments Dulce et Decorum est pro Scientia laborat, autem nihil est fructum plus quam familia et amici convivens. iv Table of Contents Acknowledgments.......................................................................................................................... iv Table of Contents .............................................................................................................................v List of Tables ................................................................................................................................ vii List of Figures .............................................................................................................................. viii List of Abbreviations .......................................................................................................................x Chapter 1 INTRODUCTION ...........................................................................................................1 Origins of Adaptive Immunity ....................................................................................................1 1.1 Humoral immunity ...............................................................................................................1 1.2 Antigen receptor diversification ..........................................................................................2 1.2.1 V(D)J recombination ...............................................................................................3 1.2.2 Lymphocyte development ........................................................................................5 1.2.3 Gene conversion.......................................................................................................7 1.2.4 Somatic hypermutation ............................................................................................8 1.2.5 Class switch recombination .....................................................................................9 1.3 DNA Damage Response and Repair ..................................................................................13 1.3.1 DNA damage detection and signaling ...................................................................13 1.3.2 Base excision repair ...............................................................................................15 1.3.3 Mismatch repair .....................................................................................................15 1.3.4 Non-homologous end joining ................................................................................16 1.3.5 Alternative end joining ..........................................................................................19 1.3.6 Homologous recombination ...................................................................................19 1.4 Prokaryotic adaptive immunity ..........................................................................................20 1.5 CSR and NHEJ repair as mutually reinforcing paradigms of inquiry ...............................20 Chapter 2 DOUBLE-STRANDED DNA BREAK POLARITY SKEWS REPAIR PATHWAY CHOICE DURING CLASS-SWITCH RECOMBINATION ..............................21 v Abstract .....................................................................................................................................22 2.1 Introduction ........................................................................................................................22 2.2 Results ................................................................................................................................23 2.2.1 Cas9- and nickase-mediated DSBs induce CSR ....................................................23 2.2.2 CSR has a preference for 5′ DSBs over 3′ DSBs ...................................................27 2.2.3 Staggered DSBs promote alternative end-joining during CSR..............................27 2.3 Discussion ..........................................................................................................................31 2.4 Experimental Methods .......................................................................................................33 Chapter 3 THE SHIELDIN COMPLEX PROMOTES CLASS SWITCH RECOMBINATION .................................................................................................................36 Abstract .....................................................................................................................................37 3.1 Introduction ........................................................................................................................37 3.2 Results ................................................................................................................................39 3.2.1 Lymphocyte development and B cell populations are largely unaffected by Shld2 deficiency .....................................................................................................39 3.2.2 SHLD2 is necessary for CSR .................................................................................42 3.2.3 The shieldin complex and other NHEJ factors exhibit an Iglo population upon CSR ........................................................................................................................45 3.2.4 The Iglo population is not the result of increased inversional recombination ........49 3.2.5 CSR in 53bp1–/– and Shld2–/–/– CH12 cells leads to aberrant recombination involving deletions in the acceptor constant region ...............................................51 3.3 Discussion ..........................................................................................................................57 3.4 Experimental Methods .......................................................................................................58 Conclusions ...............................................................................................................................61 References ......................................................................................................................................63 Appendices .....................................................................................................................................85 vi List of Tables Appendix Table 1. Oligonucleotides used in Chapter 2 for Cas9-mediated CSR and gene perturbation. .................................................................................................................................. 90 Appendix Table 2. Primers used in Chapter 3. ........................................................................... 104 Appendix Table 3. Cas9 sgRNA used in Chapter 3...................................................................
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