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Laboratory for Immune Diversity Team leader Ji-yang Wang Research Scientists : Rika Ouchida Keiji Masuda Technical Staff Assistant : Akiko Ukai Hiromi Mori : Kanae Fukui Germinal center (GC) B cells undergo somatic hypermutation (SHM) and class switch recombination of the immunoglobulin (Ig) genes and can ultimately differentiate into antibody-producing plasma cells or memory B cells. Dysregulation of this terminal differentiation pathway can lead to immunodeficiency, autoimmune diseases and B cell malignancies. The goal of the current research is to understand the mechanism of Ig gene SHM and to obtain new insights into the molecular basis of B cell terminal differentiation. SHM is initiated by the activation-induced cytidine deaminase (AID); however, the activity of multiple DNA polymerases is required to ultimately introduce mutations. For the past several years, we have been analyzing the roles of a number of low fidelity DNA polymerases, including POLQ and POLH, in the generation of different types of base substitutions during SHM of Ig genes. In parallel, by using a lacZtransgenic system where there is no positive or negative selection of mutations, we have found that among normal tissues/cell types, the GC B cells are a unique cell population that has an intrinsic property to generate A:T mutations. We aim to clarify the mechanism of A:T mutations and to isolate potential factors that are required to generate these mutations. The differentiation of GC B cells into antibody-producing plasma cells or memory B cells is a highly regulated complex process but the molecular mechanisms still remain poorly understood. Taking advantage of the large scale microarray experiments carried out at RCAI, we have conducted extensive analysis of gene expression profiles in over 100 different immune cells and have identified a number of uncharacterized genes that are selectively expressed in GC B cells. Although GC B-specific expression of these genes does not necessarily guarantee that they will have specific functions in GC B cells, we hope to obtain new insights into the molecular events that control GC B cell differentiation by elucidating their physiological roles. Role of the low-fidelity DNA polymerases in the somatic hypermutation of Ig genes We found that the absence of POLQ resulted in ~20% reduction of both C:G and A:T mutations. Others have shown that POLH deficiency caused an ~80% reduction of A:T mutations. To investigate whether the residual 46
Recent publications Masuda, K., Ouchida, R., Hikida, M., Kurosaki, T., Yokoi, M., Masutani, C., Seki, M., Wood, R. D., Hanaoka, F., O-Wang, J. DNA polymerases η and θ function in the same genetic pathway to generate mutations at A/T during somatic hypermutation of Ig genes. J. Biol. Chem. 282: 17387-17394 (2007) . Masuda, K., Ouchida, R., Hikida, M., Nakayama, M., Ohara, O., Kurusaki, T., and O-Wang, J. Absence of DNA polymerase θ results in decreased somatic hypermutation frequency and altered mutation patterns in Ig genes. DNA Repair 5: 1384-1391 (2006). Figure GC B cells have an intrisic property to generate A:T mutations in Ig and non-Ig genes. In Ig genes, AID deaminates cytosine (C) to uracil (U) and generates a U:G lesion. Under normal situations, this U:G lesion is correctly repaired by the error-free base excision repair (BER) pathway. During Ig gene SHM, however, the intermidiates of the BER are resolved by error-prone repair pathways, leading to the mutations at nondamaged A:T pairs. Using a lacZ-trangenic system, we found that GC B cells also efficiently generate A:T mutations in non-Ig genes in an AID-independent but POLH-dependent manner. A:T mutations observed in the absence of POLH are generated by POLQ and how these two polymerases might cooperate or compete with each other to generate A:T mutations, we have established and analyzed mice deficient for both POLH and POLQ. Polq -/- Polh -/- mice, however, did not show a further decrease of A:T mutations as compared to Polh -/- mice, suggesting that POLH and POLQ function in the same genetic pathway in the generation of these mutations. Frequent misincorporation of nucleotides, in particular opposite a template T, is a known feature of POLH, but the efficiency of extension beyond the misincorporation differs significantly depending on the nature of the mispairing. Remarkably, we found that POLQ catalyzed extension more efficiently than POLH from all types of mispaired termini opposite A or T. Moreover, POLQ was able to extend mispaired termini generated by POLH albeit at relatively low efficiency. These results reveal genetic and biochemical interactions between POLH and POLQ and suggest that POLQ might cooperate with POLH to generate some of the A:T mutations during SHM of Ig genes. Mechanism of increased A:T mutations in GC B cells To understand why AID-triggered U:G lesions lead to mutations at non-damaged A:T pairs in GC B cells, we decided to compare mutation patterns in non-Ig genes in GC B cells with those in other cells/tissues. For this purpose, we utilized a sensitive lacZ-transgenic (lacZ-Tg) system in which genome mutations can be detected in an unbiased way since there is no selective pressure to obscure the lacZ mutations. We found that approximately half of the mutations in the lacZ gene occurred at A:T pairs in GC B cells, which was in striking contrast to naïve B and non-GC B cells where mutations occurred predominantly at C:G pairs. To examine whether the increase of A:T mutations is a specific feature of GC B cells, we further analyzed mutation patterns in different tissues. Mutations occurred exclusively at C:G pairs in the brain, heart and liver, and only a small fraction of the mutations occurred at A:T in the small intestine. Importantly, the increased A:T mutations in GC B cells were also observed in mice lacking AID, indicating that the induction of A:T mutations is a process independent of AIDmediated Ig gene SHM. These results suggest that GC B cells have an intrinsic propensity to mutate A:T pairs in both Ig and non-Ig genes (Figure). The GC B cells likely express a factor(s) or provide a special environment that allows efficient generation of A:T mutations not only in resolving AID-triggered U:G lesions but also during repair of endogenous DNA damage. Maeda, Y., Fujimura, L., O-wang, J., Hatano, M., Sakamoto, A., Arima, M., Ebara, M., Ino, H., Yamashita, T., Saisho, H., and Tokuhiasa,T. Role of Clast1 in development of cerebellar granule cells. Brain Res. 1104:18-26 (2006). Ukai, A., Maruyama, T., Mochizuki, S., Ouchida, R., Masuda, K., Kawamura, K., Tagawa, M., Kinoshita, K., Sakamoto, A., Tokuhisa, T. and O-Wang, J. Role of DNA polymerase θ in tolerance of endogenous and exogenous DNA damage in mouse B cells. Genes Cells 11: 111-121 (2006). Masuda, K., Ouchida, R., Takeuchi, A., Saito, T., Koseki, H., Kawamura, K., Tagawa, M., Tokuhisa, T., Azuma, T. and O-Wang, J. DNA polymerase θ contributes to the generation of C/G mutations during somatic hypermutation of Ig genes. Proc. Natl. Acad. Sci. USA 102: 13986-13991 (2005). 47
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