Immunoglobulin class switch DNA recombination

描述（由申请人提供）：我们想了解Ig类开关DNA重组（CSR）的分子机制。CSR涉及双链DNA断裂（DSB）的产生和DSB的分解，由AID、Ung和其他DNA结合蛋白/修复酶介导。在本基金当前资助期内（06-10年），我们取得了重要发现，包括：(i)鉴定HoxC4为AID基因启动子的反式激活子，并证明了HoxC4启动子可被雌激素激活；（ii）鉴定5′- agct -3′是所有IgH开关(S)区域的主要保守重复序列和14-3-3蛋白的特异性靶点；（iii）证明14-3- 3作为DNA/蛋白质接头，结合S区/AID和PKA用于CSR；（iv）确定AID在CSR中的裂解后作用；(v)证明Rev1 DNA聚合酶在CSR中的重要作用。在此，我们希望确定“开放染色质”在招募/稳定14-3-3、AID、PKA、Rev1和Ung to/on S区域中的作用，表征这些因子在CSR中的支架功能，并确定CSR抑制的机制。我们假设14-3-3对所有S区域的5‘- agct -3’重复序列具有高亲和力，仅针对那些由于这些区域的开放染色质状态（通过种系转录和组蛋白PTMs的组合模式反映-我们的初步发现）而进行重组的区域，在这些区域中，它们被组蛋白PTMs稳定（Aim 1）。根据我们的初步研究结果，Rev1招募/稳定Ung到S区域，我们认为除了14-3-3是真正的“脚手架”适配器，AID， PKA， Rev1和Ung具有脚手架功能，相互促进其招募/稳定到S区域，并且自然发生或合成分子破坏这种脚手架功能会导致CSR（目的2）。我们将通过（Aim 1.1）评估重组S区域的开放染色质状态，包括组蛋白PTM H3S10ph、H3K9/K14ac H3K4me3、H2BK120ub和H2BS14ph的种系转录和组合模式来验证我们的假设；（Aim 1.2）分析14-3-3、AID、PKA、Rev1和Ung对重组S区的选择性结合及其对组蛋白ptm的依赖性；（Aim 1.3）研究H3K9acS10ph、H3S10phK14ac和/或H2BK120ub ptm在重组S区14-3-3、Rev1和AID的募集/稳定中的作用；（Aim 2.1）定义14-3-3、AID、PKA、Rev1和/或Ung分子相互作用及其对CSR至关重要的脚手架功能；（Aim 2.2）进一步了解14-3-3，AID， PKA， Rev1和/或Ung支架功能，并通过使用天然存在的Vpr/短Vpr肽干扰这些支架功能以终止CSR；（Aim 2.3）通过高通量筛选（HTS）鉴定干扰14-3-3、AID、PKA、Rev1和/或Ung相互作用以终止CSR的合成小分子。我们的实验是及时的，基于令人信服的初步数据。他们使用创新的方法，如BiFC、qChIP、ChIP-seq、Seq-ChIP、RNA-seq、FRET、Vpr肽、HTS和新的KO/突变小鼠，他们将显著推进阻断不需要的CSR的治疗方法的发展，例如IgG自身抗体或特应性IgE。

DESCRIPTION (provided by applicant): 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.