Molecular Mechanisms of Class Switch Recombination

免疫球蛋白重链类别转换重组（CSR）是在正常体液免疫应答期间除了IgM和IgD之外的所有类别的IG的表达所必需的。 此外，IG重链类别转换的调节或机制方面的异常可能是从过敏到具有S区/癌基因易位的某些癌症的许多疾病的基础。 此外，CSR过程可能充分利用了导致基因组不稳定性的其他过程中涉及的基本机制。 我们建议在ES细胞中进行基因靶向突变实验，结合种系突变或RAG-2缺陷的胚泡互补，以产生突变的B细胞，将对其进行分析，以研究CSR的基本调控和机制方面。 第一个目的是在推定的3 ′ IgH调节区中产生各种已知增强子的替换/缺失突变，单独地和组合地，以确定该区域在CH基因种系转录和CSR调节中的作用。 根据结果，将类似地分析另外的3'序列。 第二个目的是通过在CH基因座的不同位点插入pgk-neor盒或仅插入pgk启动子（或各种I区启动子）来测试用于调节CSR的启动子竞争机制的假设。 该目的还将通过将染色体区域从C γ 3的5'反转到Ca的3'来测试启动子相对于3 'IgH基因座的距离的功能。第三个目的是测试3 ′ IgH调节区是否可以补偿内含子iEmu增强子元件的功能，方法是当在含有iEmu缺失突变的染色体的背景上进行测定时，测定各种3 ′ IgH基因座插入/缺失突变的作用，还通过缺失Cdelta和γ 3基因之间的染色体区域来鉴定受影响的鸸鹋和3 ′ IgH之间的潜在边界元件染色体区域 最后，我们将阐明生殖系转录和/或转录本在CSR中的作用，通过评估反转Sgamma 2b或Smu区域与反转整个生殖系Igamma 2b/Sgamma 2b/Cgamma 2b或Imu/Smu/Cmu转录单位对CSR的影响，以及删除S区域并用旧的受体序列取代它们。

Immunoglobulin heavy chain class switch recombination (CSR) is required for expression of all classes of Ig except for IgM and IgD during a normal humoral immune response. Moreover, abnormalities in the regulation or mechanistic aspects of Ig heavy chain class switching likely underlie a number of diseases ranging from allergies to certain cancers which have S region/oncogene translocations. Furthermore, the CSR process may well employ basic mechanisms involved in other processes that lead to genome instability. We propose gene targeted mutation experiments in ES cells coupled with germline mutation or RAG-2 deficient blastocyst complementation to generate mutant B cells which will be analyzed to study fundamental regulatory and mechanistic aspects of CSR. The first aim will be to generate replacement/deletion mutations of the various know enhancers in the putative 3'IgH regulatory region, both separately and in combination, to determine the role of this region in regulation of CH gene germline transcription and CSR. Depending on the results, additional 3' sequences will be similarly analyzed. A second aim will test postulates of the promoter competition mechanism for regulation of CSR by inserting the pgk-neor cassette or just the pgk promoter (or various I region promoters) at different points in the CH locus. This aim also will test the function of promoter distance relative to the 3'IgH locus by inverting the chromosomal region from 5' of Cgamma3 to 3' of Ca. A third aim will test whether the 3'IgH regulatory region can compensate for functions of the intronic iEmu enhancer element by assaying the effects of various 3'IgH locus insertion/deletion mutations when assayed on the background of a chromosome that harbors an iEmu deletion mutation, and also by deleting the chromosomal region between Cdelta and gamma3 genes to identify potential boundary elements between the Emu and 3'IgH influenced chromosomal regions. Finally, we will elucidate the role of germline transcription and/or transcripts in CSR by assessing the effects on CSR of inverting the Sgamma2b or Smu regions versus inverting the entire germline Igamma2b/Sgamma2b/Cgamma2b or Imu/Smu/Cmu transcription units, and also by deleting S regions and replacing them with older receptive sequences.