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

• 批准号: 8702378
• 负责人: Amy L Kenter
• 金额: $ 23.97万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 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: https://reporter.nih.gov/project-details/8702378
• 关键词: 3' Untranslated Regions; APEXL2 Gene; Alternative Splicing; Antigens; B-Cell Activation; B-Lymphocytes; Base Excision Repairs; Binding; Biological Assay; C-terminal; Cell physiology; Cells; DNA; DNA Damage; DNA Repair; DNA Repair Pathway; DNA lesion; Distal; Distant; Exons; Gene Rearrangement; Genes; Genetic; Genetic Recombination; Genome; Genome Stability; Health; Homologous Gene; Human; IgE; IgG1; IgG3; Immune system; Immunoglobulin A; Immunoglobulin Class Switching; Immunoglobulin M; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Knock-out; Knockout Mice; Laboratories; Large Intestine Carcinoma; Left; Link; MLH1 gene; MSH2 gene; MSH6 gene; Malignant Neoplasms; Mature B-Lymphocyte; Messenger RNA; Mismatch Repair; Mus; Mutate; Open Reading Frames; PMS2 gene; Pathway interactions; Phenotype; Physiological; Play; Polymerase; Post-Translational Protein Processing; Process; Production; Protein Binding Domain; Protein Isoforms; Proteins; Reagent; Recruitment Activity; Role; Series; Site; System; TimeLine; Transcript; Untranslated Regions; Uracil; Variant; Zebrafish; activation-induced cytidine deaminase; base; demethylation; design; endonuclease; follow-up; homologous recombination; human APEX1 protein; protein complex; response; transition mutation; transversion mutation; tumorigenesis; uracil-DNA glycosylase

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）：成熟B细胞中激活诱导的脱氨酶（AID）对于Ig体细胞超痕（SHM）和类开关重组（CSR）都是必不可少的。帮助将DC脱离到DU。有四种尿嘧啶DNA糖基酶，UNG，SMUG1，TDG和甲基结合结构域4（MBD4），能够识别和删除U：G不匹配中的DU。使用碱基切除修复（BER）途径，可以通过尿嘧啶DNA糖基化酶（UNG）切除许多（但不是全部）辅助诱导的DU碱基，然后将无碱性位点通过，然后可以通过误差的ProNE聚合酶来复制，以产生过渡和转移突变。遗传研究表明，小鼠和人类的UNG缺乏会导致类转换重组（CSR）的丧失和损害的体细胞超偏（SHM）。证据表明，TDG无法代替UNG，并且自鸣得意在这些过程中扮演的自然作用很少，因为它在活化的B细胞中表达不佳。其他U：g不匹配可能是不匹配修复（MMR）MSH2/MSH6结合的底物，进而募集PMS2-MLH1。 PMS2刻有DNA，并有助于S区域的DSB诱导。 MBD4蛋白最初是通过与MLH1的相互作用（MMR蛋白质复合物的组成部分）发现的，MMR与CSR和SHM深度参与。 MBD4的功能关联使我们感兴趣，并在斑马鱼中主动DNA脱甲基化的背景下有助​​于探索参与CSR的成熟B细胞中的这种潜在相互作用。以前针对MBD4基因敲除小鼠的研究表明与CSR和SHM有关。但是，我们注意到MBD4 mRNA的替代剪接变体带有开放式读取框架，即使删除了外显子2-5，这也能够表达蛋白质的C末端端。我们构建了另一个敲除，其中MBD4外显子6-8和3'UTR在通常能够诱导的CSR的CH12细胞中删除。令人惊讶的是，我们发现在MBD4缺陷的CH12细胞中，即使CSR的所有其他标准仍然完好无损，CSR也会受到显着损害。基于这些有趣的新研究，我们建议更全面地检查MBD4的功能同工型，并构建一种新的小鼠，其中MBD4外显子6-8和3'UTR已被靶向的同源重组删除。随访研究将为CSR和SHM而言这些小鼠的特征。长期，该小鼠还将用于研究MBD4在不匹配修复和基因组稳定性中的参与，这是理解肿瘤发生的关键。