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.
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