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
- 来源:
- 关键词:3&apos Untranslated RegionsAPEXL2 GeneAlternative SplicingAntigensB-Cell ActivationB-LymphocytesBase Excision RepairsBindingBiological AssayC-terminalCell physiologyCellsDNADNA DamageDNA RepairDNA Repair PathwayDNA lesionDistalDistantExonsGene RearrangementGenesGeneticGenetic RecombinationGenomeGenome StabilityHealthHomologous GeneHumanIgEIgG1IgG3Immune systemImmunoglobulin AImmunoglobulin Class SwitchingImmunoglobulin MImmunoglobulin Somatic HypermutationImmunoglobulin Switch RecombinationKnock-outKnockout MiceLaboratoriesLarge Intestine CarcinomaLeftLinkMLH1 geneMSH2 geneMSH6 geneMalignant NeoplasmsMature B-LymphocyteMessenger RNAMismatch RepairMusMutateOpen Reading FramesPMS2 genePathway interactionsPhenotypePhysiologicalPlayPolymerasePost-Translational Protein ProcessingProcessProductionProtein Binding DomainProtein IsoformsProteinsReagentRecruitment ActivityRoleSeriesSiteSystemTimeLineTranscriptUntranslated RegionsUracilVariantZebrafishactivation-induced cytidine deaminasebasedemethylationdesignendonucleasefollow-uphomologous recombinationhuman APEX1 proteinprotein complexresponsetransition mutationtransversion mutationtumorigenesisuracil-DNA glycosylase
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(由申请人提供):激活诱导脱氨酶(AID)对于成熟 B 细胞中 Ig 体细胞超突变(SHM)和类别转换重组(CSR)都是必需的。 AID 将 dC 脱氨基为 dU。有四种尿嘧啶 DNA 糖基化酶 UNG、SMUG1、TDG 和甲基结合域 4 (MBD4),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,留下无碱基位点,然后可以通过容易出错的聚合酶复制该位点,以产生转变和颠换突变。遗传学研究表明,小鼠和人类的 UNG 缺乏会导致类别转换重组 (CSR) 丢失和体细胞超突变 (SHM) 受损。有证据表明 TDG 不能替代 UNG,并且 SMUG1 在这些过程中几乎没有发挥天然作用,因为它在活化的 B 细胞中表达较差。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 在 DNA 上产生切口并有助于在 S 区诱导 DSB。 MBD4 蛋白最初是通过其与 MLH1 的相互作用而被发现的,MLH1 是 MMR 蛋白复合物的一个组成部分,并且 MMR 深入参与 CSR 和 SHM。我们对斑马鱼 DNA 主动去甲基化背景下 MBD4 和 AID 的功能关联很感兴趣,并试图探索参与 CSR 的成熟 B 细胞中的这种潜在相互作用。先前针对 Mbd4 敲除小鼠的研究表明,CSR 和 SHM 没有表型。然而,我们注意到 Mbd4 mRNA 存在具有开放阅读框的替代剪接变体,即使外显子 2-5 被删除,也可以使蛋白质的 C 末端表达。我们构建了另一种敲除方法,其中 Mbd4 外显子 6-8 和 3'UTR 在通常能够诱导 CSR 的 CH12 细胞中被删除。令人惊讶的是,我们发现在 Mbd4 缺陷的 CH12 细胞中,即使 CSR 的所有其他标准保持不变,CSR 也显着受损。基于这些有趣的新研究,我们建议更全面地检查 MBD4 的功能亚型,并构建一种新小鼠,其中 Mbd4 外显子 6-8 和 3'UTR 已通过靶向同源重组删除。后续研究将在 CSR 和 SHM 方面描述这些小鼠的特征。从长远来看,该小鼠还将用于研究 Mbd4 在错配修复和基因组稳定性中的参与,这是了解肿瘤发生的关键。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
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Amy L Kenter其他文献
Amy L Kenter的其他文献
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