Danielle Feldmam

The immunoglobulin heavy chain (Igh) locus features at its 3’ end, a region of transcriptional enhancers known as the 3’ Regulatory Region (3’RR). The 3’RR is composed of enhancers HS3a, HS1,2, HS3b, and HS4 and has been shown to play a major role in immunoglobulin heavy chain class switch recombination (CSR). At present, no single enhancer deletion has been shown to substantially decrease CSR levels. However, a deletion of enhancer pair HS3b/HS4 resulted in markedly decreased levels of CSR and deletion of the entire 3’RR had an even more pronounced effect.  Preliminary data from our laboratory suggest that knockout of HS3b alone (and the nearly identical HS3a) has no effect on CSR.  Published studies from our laboratory demonstrate that HS4 deletion alone also does not mimic the effect of paired HS3b/HS4 deletion.  In the present study, we shift approach to ask not which 3’RR elements are necessary, but rather, which element(s) are sufficient to drive CSR within the IgH locus. CSR levels will be analyzed using a transgene in which HS3a and HS3b have been removed, and HS1,2 flanked by loxP sequences. Upon activation of Cre recombinase, the HS1,2 sequence will be lost, allowing comparison of CSR in the presence of both HS1,2 and HS4 or in the presence of HS4 alone. This transgene will be developed via homologous recombination of a Bacterial Artificial Chromosome (BAC) and a shuttle vector. The current stage of project development lies in the construction of the latter.