We want to understand the mechanisms that underlie the maturation ofthe antibody response to antigen. Central to such maturation are somatic hypermutation (SHM)and class switch DNArecombination (CSR). In SHM, point-mutations accumulate in immunoglobulin(Ig) heavy (H) and light chain V(D)J regions, thereby providing the substrate for selection of higher affinity mutants by antigen. In CSR,the constant (C) i chain gene is replaced by CY,Ca or CE,resulting in new biological effector functions, such as extravascular clearance of pathogens (IgG)or mucosal protection (IgA,IgE). In this proposal, we will address the mechanisms of CSR. We argue that the conserved S region AGCT motif recruits specifictrans-factors, particularly 14-3-3 adaptor proteins, to mediate CSR,which proceeds through generation ofactivation-induced cytidine deaminase (AID)- processed double-strand DNAbreak (DSBs). We also argue that such DSBs are resolved through a process entailing intervention of DNAmismatch repair (MMR) proteins, includingMlhs, the last complement of the mammalian set ofMMR proteins, and error-prone translesion synthesis (TLS) DNApolymerases, such as polymerase (pol) ? and the recently characterizedpol 6, eventuallyleading to S-S DNAjunctionsinvolving nucleotide """"""""microhomologies""""""""or """"""""insertions"""""""". Finally,we hypothesizethat germline IH-CH transcription and CSR are upregulated bythe IgH 3'enhancer (s'Ea) elements as induced by HoxC4, a highly conserved homeodomain protein that, as we showed, binds to and induces the human s'Ea hsi,2 enhancer. To test our hypotheses, we propose to: (i) address the role of S region AGCT motifs in recruiting specifictrans- factors, namely14-3-3 adaptors, and outline the role ofthese proteins in CSR;(ii) define the role ofthe MMR proteins Mlhs, Pmsi and other MutL homologs,and TLS DNApol 6 and pol ?, (Revs catalytic subunit) inCSR; and (iii) address the role ofthe phylogenetically conserved HoxC4 homeodomain protein in the regulation of CSR through induction ofthe IgH s'Ea enhancers and, possibly, modulation ofAID expression using our newly derived hoxCf/'mice. These experimentswill be performedusing novel mice and reagents, including14-3-3? /-, mlh3-/~, prnsr/',pol d'/', conditional reu^4/4, hypomorphic rettf^0/-, and hoxCf/- knockout mice,and our human monoclonal Bcell lines that undergo CSRat a high rate. Relevance to public health: ByunveilingmechanismsunderlyingCSR, these studies will help understand how antibodies to bacteria and viruses or pathogenicautoantibodies, such as those occurring in patients with autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis and type I diabetes, are generated bythe human body, eventually leading to the development ofbetter vaccines and therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI045011-10
Application #
7994852
Study Section
Cellular and Molecular Immunology - A Study Section (CMIA)
Program Officer
Peyman, John A
Project Start
2000-05-01
Project End
2012-07-31
Budget Start
2010-12-01
Budget End
2012-07-31
Support Year
10
Fiscal Year
2011
Total Cost
$478,772
Indirect Cost
Name
University of California Irvine
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
Lam, Tonika; Kulp, Dennis V; Wang, Rui et al. (2016) Small Molecule Inhibition of Rab7 Impairs B Cell Class Switching and Plasma Cell Survival To Dampen the Autoantibody Response in Murine Lupus. J Immunol 197:3792-3805
Pone, Egest J; Lam, Tonika; Lou, Zheng et al. (2015) B cell Rab7 mediates induction of activation-induced cytidine deaminase expression and class-switching in T-dependent and T-independent antibody responses. J Immunol 194:3065-78
Li, Guideng; Zan, Hong; Xu, Zhenming et al. (2013) Epigenetics of the antibody response. Trends Immunol 34:460-70
Mai, Thach; Pone, Egest J; Li, Guideng et al. (2013) Induction of activation-induced cytidine deaminase-targeting adaptor 14-3-3γ is mediated by NF-κB-dependent recruitment of CFP1 to the 5'-CpG-3'-rich 14-3-3γ promoter and is sustained by E2A. J Immunol 191:1895-906
Zan, Hong; Casali, Paolo (2013) Regulation of Aicda expression and AID activity. Autoimmunity 46:83-101
Pone, Egest J; Zhang, Jinsong; Mai, Thach et al. (2012) BCR-signalling synergizes with TLR-signalling for induction of AID and immunoglobulin class-switching through the non-canonical NF-κB pathway. Nat Commun 3:767
Pone, Egest J; Xu, Zhenming; White, Clayton A et al. (2012) B cell TLRs and induction of immunoglobulin class-switch DNA recombination. Front Biosci (Landmark Ed) 17:2594-615
Li, Guideng; Pone, Egest J; Tran, Daniel C et al. (2012) Iron inhibits activation-induced cytidine deaminase enzymatic activity and modulates immunoglobulin class switch DNA recombination. J Biol Chem 287:21520-9
Xu, Zhenming; Zan, Hong; Pone, Egest J et al. (2012) Immunoglobulin class-switch DNA recombination: induction, targeting and beyond. Nat Rev Immunol 12:517-31
Zan, Hong; White, Clayton A; Thomas, Lisa M et al. (2012) Rev1 recruits ung to switch regions and enhances du glycosylation for immunoglobulin class switch DNA recombination. Cell Rep 2:1220-32

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