Somatic hypermutation and class switching in autoimmunity

描述（由申请人提供）：我们希望了解系统性自身免疫，特别是系统性狼疮中抗体体细胞超突变（SHM）和类开关DNA重组（CSR）的分子机制。像针对微生物病原体的抗体一样，大多数致病性自身抗体在体细胞上发生超突变和类别转换。SHM主要在免疫球蛋白（Ig）重链和轻链V(D)J区引入点突变，从而为（自身）抗原选择高亲和力（自身）抗体突变体提供底物。CSR用下游的C3、C1或C5取代表达的IgH常数（CH）区域，从而赋予（自身）抗体新的生物效应功能。SHM和CSR都需要两个连续的阶段：(i) DNA损伤的产生，由激活诱导的胞苷脱氨酶（AID）介导；（ii） DNA损伤修复通过基本位点旁路、碱基切除修复（BER）或错配修复（MMR）机制，导致错配（SHM）或双链DNA断裂（dsb）的出现及其解决（CSR）。我们认为，在系统性狼疮中，SHM和CSR是失调的，这是AID上调的结果，因此，DNA损伤增加，DNA修复改变。我们认为，系统发育上保守的同源结构域蛋白HoxC4通过直接结合我们在AID启动子中发现的保守的HoxC4/ oct结合5‘-ATTTGAAT-3’位点诱导AID表达，正如我们已经证明的那样，HoxC4对SHM和CSR具有关键调控作用。我们还认为，雌激素通过触发雌激素受体（er）与我们最近在HoxC4启动子中发现的两个保守的雌激素反应元件（EREs）的结合，从而大大增强CD154: cd40诱导的HoxC4表达，从而进一步增强AID表达，并导致狼疮患者的SHM/CSR失调。我们还认为翻译DNA合成（TLS）聚合酶（pol） 8和pol7在（自体）抗体的SHM中至关重要，它们在被泛素化的PCNA募集后修复DNA损伤，介导从高保真复制DNA聚合酶到容易出错的TLS聚合酶的“聚合酶转换”。最后，我们假设在狼疮易感小鼠中，HoxC4、AID和TLS聚合酶的表达失调，HoxC4、Ung或TLS pol7的消融导致SHM/CSR降低，因此，高亲和力和同型转换自身抗体水平降低，免疫病理延迟。为了验证我们的假设，我们将：(i)定义HoxC4介导的AID启动子激活的分子机制，并通过雌激素增强HoxC4，从而增强AID表达；（ii）研究tlspol8和pol7在SHM中的作用及其在泛素化PCNA中的募集；最后，（iii）分析SHM/CSR失调以及HoxC4、Ung org pol7缺乏对狼疮易感小鼠产生超突变和类别转换自身抗体的影响。这些实验将使用复杂的生化方法和我们的新KO HoxC4-/-， pol8-/-，双KO pol8-/-Ung-/-和pol7-/- /-和突变Aicdatm1（Pmut）和pol7mut小鼠，以及缺乏HoxC4， Ung或pol7的狼疮易感MRL/Faslpr/lpr小鼠。

DESCRIPTION (provided by applicant): We want to understand the molecular mechanisms that underlie antibody somatic hypermutation (SHM) and class switch DNA recombination (CSR) in systemic autoimmunity, particularly systemic lupus. Like antibodies to microbial pathogens, most pathogenic autoantibodies are somatically hypermutated and class-switched. SHM introduces mainly point-mutations in immunoglobulin (Ig) heavy and light chain V(D)J regions, thereby providing the substrate for selection by (self) antigen of higher affinity (auto)antibody mutants. CSR replaces the expressed IgH constant (CH) region, e.g., C5, with downstream C3, C1 or C5, thereby endowing (auto)antibodies with new biological effector functions. Both SHM and CSR entail two sequential stages: (i) generation of DNA lesions, as mediated by activation-induced cytidine-deaminase (AID), and (ii) DNA lesion repair by the basic site bypass, base-excision repair (BER) or mismatch repair (MMR) machineries, leading to the emergence of mismatches (SHM) or double-strand DNA breaks (DSBs) and their resolution (CSR). We argue here that in systemic lupus, SHM and CSR are dysregulated, as a result of upregulated AID and, therefore, increased DNA lesions, and altered DNA repair. We contend that the phylogenetically conserved homeodomain protein HoxC4, which, as we have shown, critically regulates SHM and CSR, induces AID expression by directly binding to a conserved HoxC4/Oct-binding 5'-ATTTGAAT-3' site we have identified in the AID promoter. We also contend that estrogen greatly enhances CD154:CD40-induced HoxC4 expression by triggering the binding of estrogen receptors (ERs) to two conserved estrogen responsive elements (EREs) we have recently identified in the HoxC4 promoter, thereby further enhancing AID expression and contributing to SHM/CSR dysregulation in lupus. We also argue that the translesion DNA synthesis (TLS) polymerase (pol) 8 and pol7 are critical in SHM of (auto)antibodies, by repairing DNA lesions after being recruited by ubiquitinated PCNA, which mediates the "polymerase switch" from high-fidelity replicative DNA polymerases to error-prone TLS polymerases. Finally, we hypothesize that expression of HoxC4, AID and TLS polymerases is dysregulated in lupus-prone mice and ablation of HoxC4, Ung or TLS pol7 leads to decreased SHM/CSR and, therefore, decreased level of high-affinity and isotype-switched autoantibodies and delayed immunopathology. To test our hypotheses, we will: (i) define the molecular mechanisms of HoxC4-mediated AID promoter activation and the enhancement of HoxC4 and, therefore, AID expression by estrogen; (ii) address the role of TLS pol8 and pol7 and their recruitment by ubiquitinated PCNA in SHM; and, finally, (iii) analyze the SHM/CSR dysregulation and the impact of deficiency of HoxC4, Ung org pol7 on the generation of hypermutated and class-switched autoantibodies in lupus-prone mice. These experiments will use sophisticated biochemical methods and our new KO HoxC4-/-, pol8-/-, double KO pol8-/-Ung-/- and pol7-/-Ung-/- and mutant Aicdatm1(Pmut) and pol7mut mice, and lupus-prone MRL/Faslpr/lpr mice deficient in HoxC4, Ung or pol7. PUBLIC HEALTH RELEVANCE: This proposal addresses the molecular mechanisms of the generation of pathogenic autoantibodies in systemic lupus erythematosus (SLE): immunoglobulin (antibody) somatic hypermutation (SHM) and class switch DNA recombination (CSR). By increasing the affinity of antibodies for antigens and by changing the constant region of the antibody from IgM to IgG, IgA and/or IgE, SHM and CSR play a central role in the generation of highly specific and advantageous antibody responses to microbial pathogens and tumors. However, by increasing the affinity and changing the class, mainly to IgG, of antibodies to components of the body, SHM and CSR can also lead to the generation of autoantibodies that can damage tissues and organs (pathogenic autoantibodies). Thus, dysregulation of SHM and CSR can lead to the emergence of autoimmunity, including SLE. By addressing the mechanisms underlying SHM and CSR and their dysregulation, these studies will help understand how specific pathogenic autoantibodies, as those occurring in patients with SLE, are generated in the human body, eventually leading to the development of better therapeutic approaches.