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Identification and Characterization of Novel Class Switch Recombination Factors

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Identification and Characterization of Novel Class Switch Recombination Factors Identification and characterization of novel class switch recombination factors D I S S E R T A T I O N zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) Im Fach Biologie/Molekularbiologie eingereicht an der Lebenswissenschaftlichen Fakultät der Humboldt-Universität zu Berlin von M.Sc. Verónica Delgado Benito Präsidentin der Humboldt-Universität zu Berlin Prof. Dr.-Ing. Dr. Sabine Kunst Dekan der Lebenswissenschaftlichen Fakultät der Humboldt-Universität zu Berlin Prof. Dr. Bernhard Grimm Gutachter/innen 1. Prof. Dr. Michela Di Virgilio 2. Prof. Dr. Achim Leutz 3. Prof. Dr. Chiara Romagnani Tag der mündlichen Prüfung: 23.06.2020 Table of contents Abstract…………………………………………………………………………............. 1 Zusammensfassung………………………………………………………………..…. 2 1. Introduction……..…………………………………………………………………... 4 1.1. The immune system…………………………………………………………… 4 1.2. B-lymphocytes and antibody structure………………………………………. 4 1.2.1. Murine immunoglobulin heavy chain….………………………………. 6 1.2.2. Murine immunoglobulin light chain……………………………………. 7 1.3. Murine B cell development……………………………………………………. 8 1.4. Somatic hypermutation (SHM)……………………………………………….. 11 1.5. Class switch recombination (CSR)…………………………………………… 12 1.5.1. Germline transcription (GLT)…………………………………………... 13 1.5.2. AID function in CSR…………………………………………………….. 14 1.5.3. AID targeting to S regions……………………………………………… 15 1.5.4. DSB repair during CSR ………………………………………………… 16 1.5.5. Cell cycle progression………………………………………………….. 17 1.5.6. Chromatin organization………………………………………………… 18 1.6. Igh 3’ regulatory region (3’RR)……………………………………………….. 18 1.6.1. 3’RR role in B cell development……………………………………….. 19 1.6.2. 3’RR function during SHM……………………………………………… 20 1.6.3. 3’RR function during CSR……………………………………………… 20 1.7. 53BP1 and RIF1 role in CSR…………………………………………………. 20 1.7.1. 53BP1…………………………………………………………………….. 21 1.7.2. RIF1………………………………………………………………………. 22 1.8. Physiological relevance of Ig diversification………………………………… 22 1.8.1. Primary antibody deficiencies caused by defects in CSR/SHM……. 23 1.8.2. Regulation of AID activity……………………………………………… 24 1.8.3. Implications of AID off-targeting and dysregulation………………… 26 2. Goal and specific aims……………………………………………………………. 28 3. Materials and methods……………………………………………………………. 29 3.1. Materials………………………………………………………………………… 29 3.1.1. Mouse lines…………………………………………………………….… 29 3.1.2. Cell lines………………………………………………………………….. 29 3.1.3. Antibodies………………………………………………………………... 30 3.1.4. Chemical, peptides recombinant proteins and commercial kits…… 31 3.1.5. Materials and equipment……………………………………………….. 34 3.1.6. Plasmids…………………………………………………………………. 35 3.1.7. Softwares and algorithms………………………………………………. 35 3.2. Methods…………………………………………………………………………. 36 3.2.1. Mice………………………………………………………………………. 36 3.2.2. Cell lines…………………………………………………………………. 36 3.2.3. Genotyping………………………………………………………………. 37 3.2.4. CRISPR/Cas9 loss-of-CSR screen……………………………………. 37 3.2.5. CRISPR/Cas9-mediated generation of KO clonal derivatives……... 38 3.2.6. CRISPR/Cas9-mediated CSR…………………………………………. 39 3.2.7. Primary B cell isolation and culture……………………………………. 39 3.2.8. CSR ex vivo and in vitro………………………………………………... 40 3.2.9. Cell proliferation analysis and cell counting………………………….. 40 3.2.10. Immunization and blood serum collection…………………………... 41 3.2.11. ELISA…………………………………………………………………… 41 3.2.12. Retroviral infection and AID overexpression……………………….. 42 3.2.13. Analysis of B cell development………………………………………. 43 3.2.14. SHM analysis…………………………………………………………... 43 3.2.15. Assessment of GC B cells and CSR in vivo………………………… 44 3.2.16. Metaphase analysis…………………………………………………… 44 3.2.17. RT-qPCR……………………………………………………………….. 45 3.2.18. Aicda mRNA decay analysis…………………………………………. 46 3.2.19. RNA-seq and splicing analysis………………………………………. 46 3.2.20. Mutational analysis (MutPE-seq)…………………………………….. 47 3.2.21. Clonogenic survival assay……………………………………………. 47 3.2.22. Switch junction analysis………………………………………………. 48 3.2.23. Cell lysis and immunoblotting………………………………………… 48 3.2.24. I-DIRT and immunoisolation of RIF1FH complexes………………… 49 3.2.25. Immunofluorescence………………………………………………….. 50 3.2.26. ChIP-seq………………………………………………………………... 50 4. Results……………………………………………………………………………….. 52 4.1. Identification of novel CSR factors…………………………………………… 52 4.1.1. Functional loss-of-CSR screen………………………………………… 52 4.1.2. Identification of potential factors involved in CSR…………………… 53 4.2. The chromatin reader ZMYND8 regulates the 3’ Igh enhancer to promote CSR…………………………………………………………………… 57 4.2.1. ZMYND8 is essential for efficient CSR……………………………….. 57 4.2.2. ZMYND8 is dispensable for B cell proliferation……………………… 62 4.2.3. ZMYND8 does not regulate the repair of DSBs……………………… 63 4.2.4. ZMYND8 is not essential for AID expression and function…………. 67 4.2.5. ZMYND8 promotes efficient transcription of acceptor S regions…………………………………………………………............... 68 4.2.6. ZMYND8 associates to the 3’ Igh enhancer and regulates its transcriptional activity…………………………………………………… 70 4.2.7. ZMYND8 regulates SHM of the heavy chain locus………………….. 72 4.3. PDAP1 is a novel CSR factor required for efficient AID expression and suppression of the integrated stress response………………….. 75 4.3.1. PDAP1 is required for CSR…………………………………………….. 75 4.3.2. PDAP1 is largely dispensable for B cell development………………. 80 4.3.3. PDAP1 is required for efficient AID expression……………………… 81 4.3.4. PDAP1 supports efficient SHM of Ig loci……………………………... 84 4.3.5. PDAP1 is dispensable for the post-transcriptional regulation of Aicda mRNA……………………………………………………. 86 4.3.6. PDAP1 deficiency leads to activation of the integrated stress response in B cells………………………………………………………. 88 5. Discussion…………………………………………………………………………... 91 5.1. Advantages and caveats of the loss-of-CSR assay………………………... 91 5.2. ZMYND8 function in Igh gene diversification………………………………... 92 5.2.1. ZMYND8-RIF1 interaction……………………………………………… 92 5.2.2. ZMYND8 role in DNA repair……………………………………………. 93 5.2.3. Transcriptional regulation of the Igh 3’RR……………………………. 94 5.2.4. ZMYND8 as a transcriptional repressor and activator………………. 96 5.2.5. Molecular mechanism of ZMYND8 function in CSR………………… 97 5.3. PDAP1 role in B cell physiology………………………………………………. 98 5.3.1. PDAP1 regulation of Aicda expression and GLT…………………….. 98 5.3.2. PDAP1 and the integrated stress response………………………….. 99 Conclusion and future outlook………………..…..………………………………... 102 Supplementary figures……………………………………………………………….. 103 Tables……………………………………………………………………………………. 106 References………………………………………………………………………………. 113 Abbreviations…………………………………………………………….…………….. 132 Statement of contribution…………………...……………………………………….. 137 Acknowledgements…………………….……………………………………………… 139 Erklärung………………………..………………………………………………………. 141 Publications………….…………………………………………………………………. 142 ABSTRACT Class Switch Recombination (CSR) is the mechanism responsible for antibody isotype differentiation in mature B-lymphocytes and it is required for the establishment of an efficient immune response. CSR is a deletional somatic reaction that replaces the gene encoding for the constant region (donor) of the immunoglobulin heavy chain (Igh) with one of the several downstream constant genes (acceptor) within the same locus. Consequently, a B cell switches from expressing an antibody molecule of the IgM class to one of the other isotypes (IgG, IgE or IgA), which have the same antigen specificity but different effector function. At the molecular level, CSR occurs between donor and acceptor repetitive DNA elements, the so-called switch (S) regions that precede each constant gene. Upon antigen encountering, B cells proliferate and undergo cytokine stimulation that induces germ-line transcription (GLT) at the Igh locus. This process leads to non-coding transcripts and the exposure of single strand stretches of DNA, which are the substrate for the B cell specific enzyme AID (activation-induced cytidine deaminase). AID enzymatic activity generates U:G mismatches that are eventually converted into DNA double-strand breaks (DSBs). DSBs in donor and acceptor S regions are then directly ligated by the classical non-homologous end-joining pathway (c-NHEJ), leading to antibody isotype differentiation. Structurally, the Igh locus comprises 250 kb and contains two enhancers, E and the 3’RR (3’ Regulatory Region). While E is essential during early stages of B cell development, the 3’RR controls antibody isotype differentiation. Altogether, CSR is a complex physiological process that involves the formation and repair of DSBs through different molecular mechanisms that have not been fully elucidated yet. For example, it has been shown that the 3’RR is required for GLT and therefore CSR but how the enhancer activity is regulated is still unknown. Therefore, the aim of this study was to identify and characterize novel CSR factors that play a role in any of these mechanisms, contributing to the establishment of an efficient immune response. To do so, a functional loss-of-CSR screen was set-up and performed in the CH12 lymphoma B cell line that can undergo antibody isotype differentiation to IgA in vitro with high efficiency. Specifically, potential 53BP1 and RIF1 interactors were somatically targeted by CRISPR/Cas9, since these two factors play a key role during CSR. In this study, the chromatin reader ZMYND8 was found to regulate GLT and CSR by binding and modulating the transcriptional activity of the 3’RR at the Igh locus. Moreover, PDAP1 was independently identified as a novel factor required for efficient CSR by modulating Aicda (AID) mRNA levels. Conclusively,
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