We propose to study IgH class switch recombination (CSR) and Ig variable region (VDJ) exon somatic hypermutation (SHM) with a focus on elucidating mechanisms that direct Activation Induced Cytidine Deaminase (AID), the initiator of CSR and SHM, to specific targets. Based on various lines of evidence, we hypothesize that particular sequence motifs and unique transcription features target AID to particular genomic sites. Based on supporting preliminary data, we propose to test this hypothesis with powerful new approaches. We propose to elucidate mechanisms of DSB generation and directional joining during CSR. IgH switch (S) regions are targets of AID-initiated DSBs that are joined between two different S regions to achieve CSR and are composed of repetitive sequences with unusual characteristics. We developed a powerful new V (D) J passenger allele assay system that allowed us to show that S regions undergo both SHMs and DSBs when inserted in place of a V (D) J exon in CSR-activated B cells and in GC B cells and to perform, for the first time, in depth analysis of SHM patterns of core S regions. We propose to use the passenger allele system to perform a systematic analysis of mouse and human S region motifs, as well as those from other species to identify AID targeting motifs and to elucidate how orientation-specific joining of S region DSBs is enforced to promote productive, deletional CSR. V (D) J exons are targeted by AID activity during SHM in germinal center (GC) B cells through different mechanisms than S regions in B cells activated for CSR. Within V(D)J exons, complementarity determining region (CDRs) are prime AID targets even though they are not enriched for known AID hotspot motifs. We propose to use the passenger allele system to dissect potential AID targeting elements in CDRs and test the hypothesis that high levels of SHMs, deletions and insertions in anti-HIV broadly neutralizing antibody variable regions are promoted by acquisition of AID hotspots motifs during the antibody affinity maturation process. We also will test the hypothesis that convergent transcription of VH(D)JH exons in GC B cells targets AID for SHM and that that elements of the IgH 3'regulatory region regulate such transcription. Many human B cell lymphomas are of GC origin and harbor suspected AID-initiated oncogenic translocations. We now propose to identify AID-dependent DSB/translocation off-targets in mouse GC B cells by our newly developed high throughput translocation sequencing method and to characterize their transcriptional signatures by Gro-Seq, another high throughput approach. We also will test activity of previously defined and newly identified AID non-Ig gene targets (""""""""off-targets"""""""") in our passenger allele system to elucidate targeting motifs and roles of transcription in making them AID targets. Finally, we will extend our passenger allele analyses to test translocated human B cell oncogenes to test whether they are, indeed, preferential AID targets and, if so, to elucidate potential target motifs within them and roles for transcription.
Our studies propose to elucidate how target sequences and transcription differentially and specifically direct AID to appropriate substrates in the context o CSR and SHM. Answers to these questions will enhance understanding of how specific immune responses are generated and may contribute to designing approaches to optimize immune responses such as those that underlie the origin of anti-HIV broadly neutralizing antibodies. Our proposed experiments should also enhance understanding of how AID collateral damage occurs and, thereby, contribute to elucidating mechanisms that underlie the most common forms of human B cell lymphomas.
|Kumar, Vipul; Alt, Frederick W; Frock, Richard L (2016) PAXX and XLF DNA repair factors are functionally redundant in joining DNA breaks in a G1-arrested progenitor B-cell line. Proc Natl Acad Sci U S A 113:10619-24|
|Hu, Jiazhi; Meyers, Robin M; Dong, Junchao et al. (2016) Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing. Nat Protoc 11:853-71|
|Tian, Ming; Cheng, Cheng; Chen, Xuejun et al. (2016) Induction of HIV Neutralizing Antibody Lineages in Mice with Diverse Precursor Repertoires. Cell 166:1471-1484.e18|
|Hwang, Joyce K; Alt, Frederick W; Yeap, Leng-Siew (2015) Related Mechanisms of Antibody Somatic Hypermutation and Class Switch Recombination. Microbiol Spectr 3:MDNA3-0037-2014|
|Yeap, Leng-Siew; Hwang, Joyce K; Du, Zhou et al. (2015) Sequence-Intrinsic Mechanisms that Target AID Mutational Outcomes on Antibody Genes. Cell 163:1124-37|
|Dong, Junchao; Panchakshari, Rohit A; Zhang, Tingting et al. (2015) Orientation-specific joining of AID-initiated DNA breaks promotes antibody class switching. Nature 525:134-9|
|Meng, Fei-Long; Du, Zhou; Federation, Alexander et al. (2014) Convergent transcription at intragenic super-enhancers targets AID-initiated genomic instability. Cell 159:1538-48|
|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|
|Boboila, Cristian; Alt, Frederick W; Schwer, Bjoern (2012) Classical and alternative end-joining pathways for repair of lymphocyte-specific and general DNA double-strand breaks. Adv Immunol 116:1-49|
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