We want to understand the molecular mechanisms of Ig class switch DNA recombination (CSR). CSR entails generation of double-strand DNA breaks (DSBs) and DSB resolution, as mediated by AID, Ung and other DNA-binding proteins/repair enzymes. In this grant current funding period (years 06-10), we have made important discoveries, including the: (i) identification of HoxC4 as trans-activator of AID gene promoter and demonstration of HoxC4 promoter activation by estrogen; (ii) characterization of 5'-AGCT-3' as the major con- served repeats in all IgH switch (S) regions and specific targets of 14-3-3 proteins; (iii) demonstration that 14-3- 3 function as DNA/protein adaptors that bind S regions/AID and PKA for CSR; (iv) identification of a post- cleavage role of AID in CSR; and (v) demonstration of an important role of the Rev1 DNA polymerase in CSR. Here, we want to identify the role of 'open chromatin' in recruiting/stabilizing 14-3-3, AID, PKA, Rev1 and Ung to/on S regions, characterize the scaffolding functions of these factors in CSR and identify mechanisms of CSR inhibition. We hypothesize that 14-3-3, which have a high affinity for 5'-AGCT-3' repeats in all S regions, target only those that will undergo recombination due to the open chromatin state (reflected by germline transcription and combinatorial patterns of histone PTMs - our preliminary findings) of these regions, where they are stabilized by histone PTMs (Aim 1). Prompted by our preliminary findings that Rev1 recruits/stabilizes Ung to/on S regions, we contend that in addition to 14-3-3, which are bona fide 'scaffolding' adaptors, AID, PKA, Rev1 and Ung possess scaffolding functions that reciprocally contribute to their recruitment/stabilization to/on S regions, and disruption of such scaffolding functions by naturally occurring or synthetic molecules aborts CSR (Aim 2). We will test our hypotheses by:
(Aim 1. 1) assessing the open chromatin state of the recombining S regions, including germline transcription and combinatorial patterns of histone PTM H3S10ph, H3K9/K14ac H3K4me3, H2BK120ub and H2BS14ph;
(Aim 1. 2) analyzing the selective binding of 14-3-3, AID, PKA, Rev1 and Ung to the recombining S regions, and its dependency on histone PTMs;
(Aim 1. 3) addressing the role of H3K9acS10ph, H3S10phK14ac and/or H2BK120ub PTMs in recruitment/stabilization of 14-3-3, Rev1 and AID to/on the recombining S regions;
(Aim 2. 1) defining 14-3-3, AID, PKA, Rev1 and/or Ung molecular interactions and their scaffolding functions that are central to CSR;
(Aim 2. 2) further understanding 14-3-3, AID, PKA, Rev1 and/or Ung scaffolding functions and interfering with such scaffolding functions to abort CSR by using naturally occurring Vpr/short Vpr peptides;
(Aim 2. 3) identifying, by high-throughput screening (HTS), synthetic small molecules that interfere with 14-3-3, AID, PKA, Rev1 and/or Ung interactions to abort CSR. Our experiments are timely and based on convincing preliminary data. They use innovative approaches, such as BiFC, qChIP, ChIP-seq, Seq-ChIP, RNA-seq, FRET, Vpr peptides, HTS and new KO/mutant mice, and they will significantly advance the development of therapeutics that block unwanted CSR, e.g., to IgG autoantibodies or atopic IgE.
This proposal addresses the molecular mechanisms that underlie immunoglobulin (antibody) gene class switch DNA recombination (CSR). By changing the constant region of the antibody from IgM to IgG, IgA and/or IgE, CSR together with antibody somatic hypermutation play a central role in the generation of highly specific and protective antibodies against tumors and microbial pathogens, such as HIV, influenza virus and Salmonella. CSR impairment leads to hyper-IgM syndrome, which is characterized by low or undetectable serum IgG, IgA and IgE levels and profound susceptibility to infections. By exploring the regulation and new functions of the newly identified critical elements in CSR, our study will help understand how specific antibodies clear microbial pathogens and, therefore, how the immune system effectively fights infections, eventually leading to the development of better anti-infection therapies and effective vaccines. It is also highly relevant for the understanding of how autoantibodies (to self-antigens) can be pathogenic, as most of these antibodies are class-switched, mainly to IgG. Finally, by identifying molecular pathways of CSR, these experiments will have a significant impact on the development of therapeutics that block unwanted CSR, e.g., CSR to IgG autoantibodies or allergic IgE.
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