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.
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