A15
Class switch recombination: consequence of the natural connection between transcription and recombination in mammalian cells?
Ponnari Gottipati1, Qiang Pan-Hammarstrm2, Thomas Helleday3
1Gray Institute for Radiation Oncology & Biology, University of Oxford, UK, 2Karolinska University Hospital Huddinge, Stockholm, Sweden, 3Arrhenius Laboratory, Stockholm University, Stockholm, Sweden
Transcription can enhance recombination; a ubiquitous phenomenon known as Transcription-Associated Recombination (TAR). Class switch recombination (CSR) in immunoglobulin (Ig) genes is a specialised mechanism that mediates isotype switching of Ig heavy chains during B cell development. It is developmentally coupled to transcription and seems to have evolutionarily taken advantage of this natural connection between transcription and recombination. CSR is initiated by a B cell specific protein activation-induced cytidine deaminase (AID). However the mechanisms of TAR and CSR are still elusive. The main objective of this study is to investigate the mechanisms of TAR in general and CSR in specific.
In order to investigate TAR, we used a specialised recombination construct with which recombination levels can be studied in the presence or absence of transcription. The TAR construct consists of a duplicated neomycin repeat, one copy of which is controlled by an inducible bi-directional promoter using the Tet-off system, which also controls the expression of luciferase gene on the other side, enabling the quantitative measurement of transcription. This construct was stably integrated into the SPD8 Chinese hamster cell line. The effect of the protein AID on TAR was studied by expressing AID in the fibroblasts carrying the TAR substrate. In fibroblasts, AID on its own showed no effect on levels of recombination or mutations.
We used an artificial CSR construct to further investigate CSR. The substrate contains S regions for and γ exons of Ig genes and a green fluorescent protein (GFP) tag inverted between the two S regions. When the S regions fuse during class switching, the GFP is expressed and this gives a measure of CSR. Using this construct and AID, we showed that phosphorylated AID is required and sufficient for class switching in fibroblasts, suggesting that all the downstream proteins in CSR are ubiquitously expressed.
