This application proposes studies of the mechanisms of immunoglobulin heavy chain (IgH) class switch recombination (CSR) and Somatic Hypermutation (SHM). We have shown that Activation Induced Cytidine Deaminase (AID), the initiator of CSR, is a single strand DNA (ssDNA) specific cytidine deaminase and we employed a series of novel biochemical and genetic approaches to elucidate mechanisms by which AID gains access to transcribed double strand (ds)DNA sequences in the context transcription-generated ssDNA structures and/or certain AID modifications or co-factors. We also showed that CSR may employ general processes for synapsis of AID-initiated DNA double strand breaks (DSBs), that general DNA repair factors function in CSR, and that two distinct end-joining pathways fuse S region breaks to complete CSR. Our current proposal builds on these observations in the context of three specific Aims.
Our first aim proposes use of biochemical and genetic approaches to elucidate basic mechanisms of AID function and regulation. In this regard, we developed methods to purify AID from normal B cells, in vitro assays for transcription-dependent AID deamination of dsDNA DNA, and genetic approaches to evaluate in vivo AID functions elucidated biochemically.
Our second aim addresses mechanisms by which DNA sequences influence AID activity and its outcome. For these studies, we developed targeted mutation assays to replace endogenous IgH class switch (S) regions and exons encoding IgH variable regions with test sequences that will allow us to determine how substrate sequences influence activities of AID and other relevant factors in CSR and SHM. Together, the complementary biochemical and genetic assays of Aims 1 and 2 offer a powerful approach for elucidating factors and mechanisms involved in initiation and regulation of IgH CSR and SHM. A third proposed aim is to elucidate processes involved in the repair of AID induced DSBs to complete CSR. For these studies, we again have developed a large array of reagents and novel approaches, including cytogenetic methods to follow CSR related breaks in chromosomes, novel genetic approaches to study factors involved in long range synapsis of DSBs, and genetic models to elucidate DSB repair pathways that complete IgH CSR. Our proposed studies should provide novel insights into the mechanism of antibody production via IgH CSR and, therefore, be relevant to understanding immunodeficiencies, vaccine immunology, and autoimmune diseases. As CSR is required for IgE production, the work will also be relevant to understanding pathogenesis of allergic diseases and asthma. Finally, the work is relevant to B cell malignancies as they often involve chromosomal translocations that link translocated oncogenes to IgH S regions via aberrant CSR.
Our proposed studies will continue to provide novel insights into the mechanism by which different types of antibodies are produced through the gene rearrangement process termed immunoglobulin heavy chain class switch recombination (CSR). Elucidation of the CSR mechanism has great relevance for understanding immunodeficiency and autoimmune diseases. Elucidation of the CSR mechanism also has importance for fully understanding allergic diseases and asthma, as increased production of a particular class of antibodies is an important component of the pathogenesis of these diseases. Finally, the work will help elucidate factors that underlie certain cancers of the immune system, such as lymphomas, which activate cancer causing genes through aberrant CSR.
|Gostissa, Monica; Schwer, Bjoern; Chang, Amelia et al. (2014) IgH class switching exploits a general property of two DNA breaks to be joined in cis over long chromosomal distances. Proc Natl Acad Sci U S A 111:2644-9|
|Shi, Wei; Bain, Amanda L; Schwer, Bjoern et al. (2013) Essential developmental, genomic stability, and tumour suppressor functions of the mouse orthologue of hSSB1/NABP2. PLoS Genet 9:e1003298|
|Wesemann, Duane R; Magee, Jennifer M; Boboila, Cristian et al. (2011) Immature B cells preferentially switch to IgE with increased direct S? to S? recombination. J Exp Med 208:2733-46|
|Basu, Uttiya; Meng, Fei-Long; Keim, Celia et al. (2011) The RNA exosome targets the AID cytidine deaminase to both strands of transcribed duplex DNA substrates. Cell 144:353-63|
|Eccleston, Jennifer; Yan, Catherine; Yuan, Karen et al. (2011) Mismatch repair proteins MSH2, MLH1, and EXO1 are important for class-switch recombination events occurring in B cells that lack nonhomologous end joining. J Immunol 186:2336-43|
|Zhang, Tingting; Franklin, Andrew; Boboila, Cristian et al. (2010) Downstream class switching leads to IgE antibody production by B lymphocytes lacking IgM switch regions. Proc Natl Acad Sci U S A 107:3040-5|
|Boboila, Cristian; Jankovic, Mila; Yan, Catherine T et al. (2010) Alternative end-joining catalyzes robust IgH locus deletions and translocations in the combined absence of ligase 4 and Ku70. Proc Natl Acad Sci U S A 107:3034-9|
|Boboila, Cristian; Yan, Catherine; Wesemann, Duane R et al. (2010) Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase 4. J Exp Med 207:417-27|
|Wang, Jing H; Gostissa, Monica; Yan, Catherine T et al. (2009) Mechanisms promoting translocations in editing and switching peripheral B cells. Nature 460:231-6|
|Basu, Uttiya; Franklin, Andrew; Schwer, Bjoern et al. (2009) Regulation of activation-induced cytidine deaminase DNA deamination activity in B-cells by Ser38 phosphorylation. Biochem Soc Trans 37:561-8|
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