Role of MBD4 in double strand break formation during class switch recombination

描述(由申请人提供)：激活诱导脱氨酶(AID)是成熟B细胞中Ig体细胞超突变(SHM)和类切换重组(CSR)所必需的。援助使DC与DU脱钩。有四种尿嘧啶DNA糖基酶，UNG、SMUG1、TDG和甲基结合结构域4(MBD4)，能够识别和去除U：G错配中的Du。利用碱基切除修复(BER)途径，许多(但不是所有)AID诱导的Du碱基可以被尿嘧啶DNA糖基酶(UNG)切除，留下一个基本位点，然后容易出错的聚合酶复制该碱基，产生转换和颠换突变。遗传学研究表明，小鼠和人类的UNG缺乏导致类开关重组(CSR)的丢失和受损的体细胞超突变(SHM)。有证据表明，TDG不能替代UNG，SMUG1在这些过程中自然发挥的作用很小，因为它在活化的B细胞中表达很少。其他U：G错配可能是错配修复(MMR)MSH2/MSH6结合的底物，进而招募PMS2-MLH1。PMS2基因存在缺口，参与了S地区双链断裂的诱导。MBD4蛋白最初是通过与MLH1的相互作用而被发现的，MLH1是MMR蛋白复合体的一个组成部分，MMR深入参与了CSR和SHM。我们对斑马鱼中活跃的DNA去甲基化背景下MBD4和AID的功能关联很感兴趣，并试图探索参与CSR的成熟B细胞中这种潜在的相互作用。以前对Mbd4基因敲除小鼠的研究表明，没有CSR和SHM的表型。然而，我们注意到Mbd4mRNA的另一种剪接变体具有开放阅读框架，即使在外显子2-5缺失的情况下，它也可以使蛋白质的C末端表达。我们构建了另一种基因敲除，在正常情况下能够诱导CSR的CH12细胞中，Mbd4外显子6-8和3‘UTR被缺失。令人惊讶的是，我们发现在Mbd4缺陷的CH12细胞中，CSR显著受损，即使所有其他CSR标准保持不变。基于这些耐人寻味的新研究，我们建议更全面地研究MBD4的功能异构体，并构建一种新的小鼠，其中Mbd4外显子6-8和3‘UTR已通过靶向同源重组而缺失。后续研究将在CSR和SHM方面对这些小鼠进行特征描述。从长远来看，这只小鼠也将被用来研究Mbd4参与错配修复和基因组稳定性，这是理解肿瘤发生的关键。

DESCRIPTION (provided by applicant): Activation induced deaminase (AID) is essential for both Ig somatic hypermutation (SHM) and class switch recombination (CSR) in mature B cells. AID deaminates dC to dU. There are four uracil DNA glycosylases, UNG, SMUG1, TDG, and methyl binding domain 4 (MBD4), that are capable of recognizing and removing dU in U:G mismatches. Using the base excision repair (BER) pathway, many (but not all) AID induced dU bases can be excised by a uracil DNA glycosylase (UNG) leaving an abasic site that can then be replicated over by error prone polymerase to produce both transition and transversion mutations. Genetic studies show that UNG deficiency in mice and humans leads to loss of class switch recombination (CSR) and impaired somatic hypermutation (SHM). Evidence indicates that TDG cannot substitute for UNG and SMUG1 plays little natural role in these processes since it is poorly expressed in activated B cells. Other U:G mismatches could be substrates for mismatch repair (MMR) MSH2/MSH6 binding which in turn recruit PMS2-MLH1. PMS2 nicks the DNA and contributes to the induction of DSBs in S regions. MBD4 protein was originally discovered by virtue of its interaction with MLH1, a constituent of the MMR protein complex and MMR is deeply involved in CSR and SHM. We were intrigued by the functional association of MBD4 and AID in the context of active DNA demethylation in zebrafish and have sought to explore this potential interaction in mature B cells engaged in CSR. Previous studies focused on Mbd4 knockout mice indicated no phenotype with regard to CSR and SHM. However, we noticed that there are alternative splice variants of Mbd4 mRNA with open reading frames, which would enable expression of the C-terminal end of the protein even when exons 2-5 are deleted. We constructed another knockout in which Mbd4 exons 6-8 and the 3'UTR were deleted in CH12 cells that are normally capable of inducible CSR. Strikingly we found that in Mbd4 deficient CH12 cells, CSR was significantly impaired even while all other criteria for CSR remain intact. Based on these intriguing new studies we propose to more fully examine MBD4 for functional isoforms and to construct a new mouse in which Mbd4 exons 6-8 and the 3'UTR have been deleted by targeted homologous recombination. Follow-up studies will characterize these mice with respect to CSR and SHM. Long term, this mouse will also be used to investigate the involvement of Mbd4 in mismatch repair and genome stability, keys to understanding oncogenesis.