Molecular Mechanisms of Class Switch Recombination

在正常的体液免疫反应中，除IgM和IGD外，所有类型的Ig的表达都需要免疫球蛋白重链开关重组(CSR)。此外，免疫球蛋白重链类别转换的调节或机制方面的异常可能是许多疾病的基础，从过敏到某些具有S区域/癌基因易位的癌症。此外，CSR过程很可能采用导致基因组不稳定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，结合胚系突变或RAG-2缺陷的囊胚互补来产生突变的B细胞，并对其进行分析，以研究CSR的基本调控和机制。第一个目标是分别和联合产生假定的3‘IgH调节区中各种已知增强子的替换/缺失突变，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，将对其他3‘序列进行类似的分析。第二个目的是通过在CH基因座的不同位置插入PGK-neor盒或仅PGK启动子(或各种I区启动子)来测试启动子竞争机制调节CSR的假设。这一目的还将通过将CGamma3的5‘端染色体区域倒置到Ca的3’端染色体区域来检验启动子相对于3‘IgH基因座的作用。第三个目标将测试3‘IgH调节区是否能够补偿内含子iEmu增强子元件的功能，方法是在含有iEmu缺失突变的染色体背景上分析各种3’IgH位点插入/缺失突变的影响，并通过删除CDelta和Gamma3基因之间的染色体区域来确定Emu和3‘IgH影响的染色体区域之间的潜在边界元素。最后，我们将通过评估颠倒SGamma2b或SMU区域与颠倒整个生殖系IGamma2b/SGamma2b/CGamma2b或IMU/SMU/CMU转录单位对CSR的影响，以及通过删除S区域并用更早的接受序列取代它们，来阐明生殖系转录和/或转录本在CSR中的作用。

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.