Somatic hypermutation and class switching in autoimmunity

描述(申请人提供)：我们想了解系统性自身免疫，特别是系统性红斑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，大多数致病自身抗体都是体细胞超突变和类别转换的。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(Auto)抗体突变体提供底物。CSR用下游的C3、C1或C5取代表达的免疫球蛋白恒定(CH)区，例如C5，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)由激活诱导的胞苷脱氨酶(AID)介导的DNA损伤的产生；(Ii)DNA损伤的修复，通过碱基旁路、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)及其解决(CSR)。我们认为在系统性红斑狼疮中，SHM和CSR是失调的，这是由于AID上调，从而增加了DNA损伤，并改变了DNA修复。我们认为，系统发育保守的同源结构域蛋白HoxC4，如我们所示，关键地调节SHM和CSR，通过直接结合我们在AID启动子中发现的保守的HoxC4/Oct结合的5‘-ATTTGAAT-3’位点来诱导AID的表达。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(ERE)的结合，极大地增强了CD154：CD40诱导的HoxC4的表达，从而进一步增强了AID的表达，并在狼疮SHM/CSR失调中起到了作用。我们还认为，跨病变DNA合成(TLS)聚合酶(Pol8)和pol7在自身抗体的SHM中起关键作用，在被泛素化的增殖细胞核抗原招募后修复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“聚合酶开关”。最后，我们假设HoxC4、AID和TLS聚合酶在狼疮易感小鼠中表达失调，切除HoxC4、Ung或TLS pol7会导致SHM/CSR降低，从而降低高亲和力和同型转换的自身抗体水平，并延迟免疫病理。为了验证我们的假设，我们将：(I)确定HoxC4介导的AID启动子激活和雌激素促进HoxC4从而增强AID表达的分子机制；(Ii)研究TLS pol8和pol7在SHM中的作用以及泛素化的PCNA对它们的募集；以及(Iii)分析SHM/CSR失调以及HoxC4、Ung org pol7缺失对狼疮易感小鼠产生高突变和类切换自身抗体的影响。这些实验将使用复杂的生化方法，以及我们新的KO HoxC4-/-，pol8-/-，双KO pol8-/-ung-/-和pol7-/-ung-/-和突变Aicdatm1(Pmut)和pol7mut小鼠，以及缺乏HoxC4，Ung或pol7的易患狼疮的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.