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

MBD4 在类别转换重组过程中双链断裂形成中的作用

基本信息

  • 批准号:
    8702378
  • 负责人:
  • 金额:
    $ 23.97万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2014
  • 资助国家:
    美国
  • 起止时间:
    2014-08-01 至 2016-07-31
  • 项目状态:
    已结题

项目摘要

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.
描述(由申请方提供):活化诱导的脱氨酶(AID)对于成熟B细胞中的IG体细胞超突变(SHM)和类别转换重组(CSR)都是必需的。AID将dC脱氨为dU。有四种尿嘧啶DNA糖基化酶,UNG,SMUG 1,TDG和甲基结合结构域4(MBD 4),能够识别和去除U:G错配中的dU。使用碱基切除修复(BER)途径,许多(但不是全部)AID诱导的dU碱基可以被尿嘧啶DNA糖基化酶(UNG)切除,留下脱碱基位点,然后该脱碱基位点可以被易错聚合酶复制以产生转换和颠换突变。遗传学研究表明,小鼠和人类的UNG缺乏导致类别转换重组(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,SMUG 1在这些过程中几乎没有发挥天然作用,因为它在活化的B细胞中表达不足。其他U:G错配可能是错配修复(MMR)MSH 2/MSH 6结合的底物,其进而募集PMS 2-MLH 1。PMS 2使DNA产生切口,并有助于在S区诱导DSB。MBD 4蛋白最初是由于其与MMR蛋白复合物的组分MLH 1的相互作用而被发现的,并且MMR深深地参与CSR和SHM。我们对MBD 4和AID在斑马鱼主动DNA去甲基化背景下的功能关联感兴趣,并试图探索参与CSR的成熟B细胞中的这种潜在相互作用。以前的研究集中在Mbd 4基因敲除小鼠表明没有关于CSR和SHM的表型。然而,我们注意到Mbd 4 mRNA存在具有开放阅读框的可变剪接变体,即使外显子2-5缺失,这也能够表达蛋白质的C末端。我们构建了另一种敲除,其中Mbd 4外显子6-8和3 'UTR在CH 12细胞中缺失,所述CH 12细胞通常能够诱导CSR。令人惊讶的是,我们发现,在Mbd 4缺陷的CH 12细胞中,即使在CSR的所有其他标准保持完整的情况下,CSR也显著受损。基于这些有趣的新研究,我们建议更全面地检查MBD 4的功能亚型,并构建一个新的小鼠,其中Mbd 4外显子6-8和3 'UTR已被删除的靶向同源重组。后续研究将在CSR和SHM方面表征这些小鼠。从长远来看,这种小鼠也将用于研究Mbd 4参与错配修复和基因组稳定性,这是理解肿瘤发生的关键。

项目成果

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Amy L Kenter其他文献

Amy L Kenter的其他文献

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{{ truncateString('Amy L Kenter', 18)}}的其他基金

Impact of novel enhancers on Igh repertoire diversity
新型增强子对 Igh 库多样性的影响
  • 批准号:
    10716628
  • 财政年份:
    2023
  • 资助金额:
    $ 23.97万
  • 项目类别:
Igh locus function in immunosenescent mice
免疫衰老小鼠中的 Igh 基因座功能
  • 批准号:
    10303603
  • 财政年份:
    2021
  • 资助金额:
    $ 23.97万
  • 项目类别:
Igh locus function in immunosenescent mice
免疫衰老小鼠中的 Igh 基因座功能
  • 批准号:
    10427437
  • 财政年份:
    2021
  • 资助金额:
    $ 23.97万
  • 项目类别:
Identification of a CSR specific checkpoint
识别 CSR 特定检查点
  • 批准号:
    10198743
  • 财政年份:
    2020
  • 资助金额:
    $ 23.97万
  • 项目类别:
Identification of a CSR specific checkpoint
识别 CSR 特定检查点
  • 批准号:
    10063761
  • 财政年份:
    2020
  • 资助金额:
    $ 23.97万
  • 项目类别:
Characterization of chromatin loops responsible for Igh locus contraction
负责 Igh 基因座收缩的染色质环的表征
  • 批准号:
    8873312
  • 财政年份:
    2015
  • 资助金额:
    $ 23.97万
  • 项目类别:
Class switch recombination during early B cell development
早期 B 细胞发育过程中的类别转换重组
  • 批准号:
    8594576
  • 财政年份:
    2013
  • 资助金额:
    $ 23.97万
  • 项目类别:
Class switch recombination during early B cell development
早期 B 细胞发育过程中的类别转换重组
  • 批准号:
    8664344
  • 财政年份:
    2013
  • 资助金额:
    $ 23.97万
  • 项目类别:
Lymphocytes/Immune System:Cellular/Interactive Mechanism
淋巴细胞/免疫系统:细胞/相互作用机制
  • 批准号:
    7000871
  • 财政年份:
    2005
  • 资助金额:
    $ 23.97万
  • 项目类别:
Factors and DNA Motifs Involved in Ig Class Switch
参与 Ig 类别转换的因素和 DNA 基序
  • 批准号:
    6629967
  • 财政年份:
    2003
  • 资助金额:
    $ 23.97万
  • 项目类别:

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