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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI077595-09
Application #
9228317
Study Section
Cellular and Molecular Immunology - B Study Section (CMIB)
Program Officer
Nasseri, M Faraz
Project Start
2008-12-15
Project End
2019-02-28
Budget Start
2017-03-01
Budget End
2018-02-28
Support Year
9
Fiscal Year
2017
Total Cost
$442,500
Indirect Cost
$192,500
Name
Boston Children's Hospital
Department
Type
Independent Hospitals
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115
Nicolas, Laura; Cols, Montserrat; Choi, Jee Eun et al. (2018) Generating and repairing genetically programmed DNA breaks during immunoglobulin class switch recombination. F1000Res 7:458
Panchakshari, Rohit A; Zhang, Xuefei; Kumar, Vipul et al. (2018) DNA double-strand break response factors influence end-joining features of IgH class switch and general translocation junctions. Proc Natl Acad Sci U S A 115:762-767
Crowe, Jennifer L; Shao, Zhengping; Wang, Xiaobin S et al. (2018) Kinase-dependent structural role of DNA-PKcs during immunoglobulin class switch recombination. Proc Natl Acad Sci U S A 115:8615-8620
Qiao, Qi; Wang, Li; Meng, Fei-Long et al. (2017) AID Recognizes Structured DNA for Class Switch Recombination. Mol Cell 67:361-373.e4
Nguyen, Hai Vu; Dong, Junchao; Panchakshari, Rohit A et al. (2017) Histone methyltransferase MMSET promotes AID-mediated DNA breaks at the donor switch region during class switch recombination. Proc Natl Acad Sci U S A 114:E10560-E10567
Compagno, Mara; Wang, Qi; Pighi, Chiara et al. (2017) Phosphatidylinositol 3-kinase ? blockade increases genomic instability in B cells. Nature 542:489-493
Verkoczy, Laurent; Alt, Frederick W; Tian, Ming (2017) Human Ig knockin mice to study the development and regulation of HIV-1 broadly neutralizing antibodies. Immunol Rev 275:89-107
Hwang, Joyce K; Wang, Chong; Du, Zhou et al. (2017) Sequence intrinsic somatic mutation mechanisms contribute to affinity maturation of VRC01-class HIV-1 broadly neutralizing antibodies. Proc Natl Acad Sci U S A 114:8614-8619
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
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

Showing the most recent 10 out of 30 publications