京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18179号 / 医博第3899号 / 新制||医||1004(附属図書館) / 31037 / 京都大学大学院医学研究科医学専攻 / (主査)教授 清水 章, 教授 岩井 一宏, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The processes of secondary antibody diversification are initiated by the mutagenic, B cell specific enzyme, Activation-Induced Deaminase (AID). AID deaminates deoxycytosine (dC) that is located in single-stranded DNA (ssDNA) in actively transcribed DNA to initiate the processes of somatic hypermutation (SHM), gene conversion (GCV) and class switch recombination (CSR) at the antibody gene loci. These processes lead to high affinity antibodies and antibodies of various effector functions that are required to efficiently neutralize invading pathogens. It is currently unclear how the antibody genes are specifically targeted by AID over other genes. I found that AID is able to mutate a non-immunoglobulin (Ig) transgene independent of its chromosomal integration site at rates that were above background mutation rates, but were ~10-fold lower than at the antibody variable (V) region. This result suggests that AID can mutate non-Ig genes at low rates, which may explain AID’s role in oncogenesis, but nevertheless shows that AID preferentially mutates the Ig locus over other loci. While it is understood that AID specifically deaminates dC bases in ssDNA, the size, distribution and origin of these ssDNA substrates is unknown. By utilizing a unique in situ sodium bisulfite assay to detect regions of ssDNA in intact nuclei, I characterized ssDNA regions and found that they are accurate predictors of AID activity during the processes of SHM and CSR in mammalian B cells and E.coli. Importantly, with the use of E.coli models, I show that these ssDNA substrates are the product of transcription-induced negative-supercoiled DNA that correlates strongly with the mutagenic activity of AID. While several underlying mechanisms exist to prevent the mistargeting of AID, my findings suggest that by simply gaining access to ssDNA that is produced by transcription-induced negative supercoiling, AID has the potential to mutate non-Ig genes, albeit at lower rates than the antibody V-region.

The processes of secondary antibody diversification are initiated by the mutagenic, B cell specific enzyme, Activation-Induced Deaminase (AID). AID deaminates deoxycytosine (dC) that is located in single-stranded DNA (ssDNA) in actively transcribed DNA to initiate the processes of somatic hypermutation (SHM), gene conversion (GCV) and class switch recombination (CSR) at the antibody gene loci. These processes lead to high affinity antibodies and antibodies of various effector functions that are required to efficiently neutralize invading pathogens. It is currently unclear how the antibody genes are specifically targeted by AID over other genes. I found that AID is able to mutate a non-immunoglobulin (Ig) transgene independent of its chromosomal integration site at rates that were above background mutation rates, but were ~10-fold lower than at the antibody variable (V) region. This result suggests that AID can mutate non-Ig genes at low rates, which may explain AID’s role in oncogenesis, but nevertheless shows that AID preferentially mutates the Ig locus over other loci. While it is understood that AID specifically deaminates dC bases in ssDNA, the size, distribution and origin of these ssDNA substrates is unknown. By utilizing a unique in situ sodium bisulfite assay to detect regions of ssDNA in intact nuclei, I characterized ssDNA regions and found that they are accurate predictors of AID activity during the processes of SHM and CSR in mammalian B cells and E.coli. Importantly, with the use of E.coli models, I show that these ssDNA substrates are the product of transcription-induced negative-supercoiled DNA that correlates strongly with the mutagenic activity of AID. While several underlying mechanisms exist to prevent the mistargeting of AID, my findings suggest that by simply gaining access to ssDNA that is produced by transcription-induced negative supercoiling, AID has the potential to mutate non-Ig genes, albeit at lower rates than the antibody V-region.

During IgH class switch recombination (CSR) in mature B lymphocytes, activation-induced cytidine deaminase (AID) initiates DNA double strand breaks (DSBs) within switch (S) regions flanking different sets of the IgH locus (IgH) constant \((C_H)\) region exons. End-Joining of DSBs in the upstream donor S region (Sm) to DSBs in a downstream acceptor S region \((S_{acc})\) replaces the initial set of \(C_H\) exons, Cm, with a set of downstream \(C_H\) exons, leading to Ig class switching from IgM to another IgH class (e.g., IgG, IgE, or IgA). In addition to joining to DSBs within another S region, AID-induced DSBs within a given S region are often rejoined or joined to other DSBs in the same S region to form internal switch deletions (ISDs). ISDs were frequently observed in Sm but rarely in \(S_{acc}s\), suggesting that AID targeting to \(S_{acc}s\) requires prior recruitment to Sm. To test this hypothesis, we assessed CSR and ISDs in B cells lacking Sm and found that AID frequently targets downstream \(S_{acc}s\) independently of Sm. These studies also led us to propose an alternative pathway of "downstream" IgE class switching that involves joining of DSBs within the downstream \(S\gamma1\) and \(S\epsilon\) regions as a first step before joining of \(S\mu\) to the hybrid downstream S region. To further elucidate the CSR mechanism, we addressed the long-standing question of whether S region DSBs during CSR involves a direction-specific mechanism similar to joining of RAG1/2 endonuclease-generated DSBs during V(D)J recombination. We used an unbiased high throughput method to isolate junctions between I-SceI meganuclease-generated DSBs at a target site that replaces the IgH \(S\gamma1\) region and other genomic DSBs of endogenous origin. Remarkably, we found that the I-SceI-generated DSBs were joined to both upstream DSBs in \(S\mu\) and downstream DSBs in \(S\epsilon\) predominantly in orientations associated with joining during productive CSR. This process required the DSB response factor 53BP1 to maintain the orientation-dependence, but not the overall levels, of joining between these widely separated IgH breaks. We propose that CSR exploits a mechanism involving 53BP1 to enhance directional joining of DSBs within IgH in an orientation that leads to productive CSR.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19228号 / 医博第4027号 / 新制||医||1011(附属図書館) / 32227 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武田 俊一, 教授 竹内 理, 教授 髙田 穣 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

DNA encodes the genetic instructions for the development and function of organisms and hence maintaining genomic integrity is essential for the propagation of life. However, DNA molecules are under constant threat of metabolic and environmental insults resulting in DNA damages including DNA double strand breaks (DSB), which are considered as a serious threat to cell survival. The majority of these DSB are repaired by Non-homologous end joining (NHEJ). Unrepaired DSB can lead to genomic instability resulting in cell cycle arrest, apoptosis, and mutations. Thus, delineating this DNA repair process is important in understanding the molecular mechanisms of aging and malignant progression. B lymphocytes undergo physiological DNA breaks and NHEJ-mediated DNA repair during their bone marrow differentiation and peripheral class switch recombination (CSR), thus lending them as a good model system in which to delineate the DNA repair mechanisms. To determine the effect of aging on NHEJ, B lymphocytes from old mice were analyzed. The results showed compromised DNA repair in cells from old mice compared to cells from adult mice. These results suggest that NHEJ is compromised during aging and might play critical roles in the aging process and age-associated conditions. To delineate the role of a CT in regulating the immune system, transgenic mice expressing NUP98-HOXD13 (NHD13) were analyzed for B lymphocyte differentiation, peripheral development, CSR, and antibody production. The results showed impaired B cell development and antibody production, which worsened with antigenic stimulation, suggesting the role of NHD13 in immune regulation. These studies explored the possibility of altered NHEJ-mediated DNA repair as a contributing reason for aging process and age-associated conditions. Also, the results from NHD13 study suggested that a primary CT can result in impaired NHEJ and regulate immune cell development and function. Furthermore, the results pointed to the possibility that a primary CT may lead to secondary mutations through altered NHEJ. Thus, these studies shed insight into the molecular mechanisms of altered NHEJ and may help in developing preventive or therapeutic strategies against accumulation of DNA damage, aging process and secondary mutations. / Ph. D.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24833号 / 医博第5001号 / 新制||医||1067(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田, 宏一, 教授 上野, 英樹, 教授 濵﨑, 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18899号 / 医博第4010号 / 新制||医||1009(附属図書館) / 31850 / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 三森 経世, 教授 岩井 一宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Klassenwechsel (CSR, class switch recombination) bezeichnet eine B-Zellen spezifische, somatische Rekombination. Sie ersetzt den konstanten Abschnitt der immunoglobulin schweren Kette (Igh), wenn die Zelle auf ein Antikörper trifft oder durch in vitro Aktivierung. Dadurch wechseln B-Zellen die exprimierten IgM Antikörpermoleküle zu anderen Isotypen (IgG, IgE oder IgA), welche die selbe Antikörperaffinität besitzen, aber eine andere Effektorfunktion. Dieser Vorgang ist elementar im Aufbau einer effektiven Immunantwort, da Defekte bei der CSR zu erhöhten IgM-Leveln führen und primären Anitkörperdefizit. Diese wurden bereits mit Autoimmunität, Autoinflamations-Syndrome und erhöhte Anfälligkeit für Infektionen und Krebs in Verbindung gebracht. CSR ist ein komplexer, physiologischer, vielgliedriger Prozess, welcher die Bildung und Reparatur von Doppelstrangbrüchen durch verschiedene molekulare Mechanismen leitet und welcher noch nicht vollständig verstanden ist. Das Ziel dieser Studie war daher die Identifikation neuer CSR-Faktoren und die Charakterisierung ihrer Rolle(n) innerhalb dieser Reaktion. Dafür wurde eine CH12-Lymphoma B-Zelllinie mit einem robusten Funktionsverlust der CSR erstellt. Diese wurden in vitro aktiviert um Antikörperisotypdifferenzierung zu IgA mit hoher Effizienz vorzunehmen. Ein Ergebnis dieser Arbeit ist die Identifikation von ZMYND8 als Notwendig für den CSR. Dieser Faktor bindet und reguliert die Transkription der regulatorischen 3´ Region, welcher ein super-enhancer am 3´ Ende der Igh Region ist, und die Antikörperisotypdifferenzierung reguliert. Davon unabhängig wurde PDAP1 als neuer Faktor für effiziente CSR identifiziert. Abschließend tragen die Ergebnisse dieser Arbeit zum weiteren Verständniss der Regulierung der Antikörperisotypdifferenzierung sowie der Aktivität von B-Zellen während der Immunantwort. / Class Switch Recombination (CSR) is a B-cell specific somatic recombination reaction that replaces the constant region of the immunoglobulin heavy chain (Igh) locus upon antigen encountering or in vitro cell activation. As a consequence, B cells switch from expressing IgM antibody molecules to another isotype (IgG, IgE or IgA), which harbor the same antigen affinity but different effector function. This process is essential for the establishment of an effective immune response since defects in CSR lead to increased serum IgM levels and primary antibody deficiencies, which are associated with autoimmunity, auto-inflammatory syndromes, increased sensitivity to infections and cancer. CSR is a complex physiological multistep process that involves the formation and repair of double strand breaks through different molecular mechanisms that have not been fully elucidated yet. Therefore, the aim of this study was to identify novel CSR factors, and characterize their role(s) in the reaction. To do so, a robust functional loss of CSR screen was set-up and performed in the CH12 lymphoma B cell line. These cells can be activated in vitro to undergo antibody isotype differentiation to IgA with high efficiency. As a result of this screen, the chromatin reader ZMYND8 was found to be required for CSR. Specifically, this factor binds and modulates the transcriptional activity of the 3’ regulatory region, which is a super-enhancer located at the 3’ end of the Igh locus that controls antibody isotype differentiation. Furthermore, PDAP1 was independently identified as a novel factor necessary for efficient CSR. Conclusively, the results of this thesis contributed to further understand the processes regulating antibody isotype differentiation and B cell activity during an immune response.