Antigen receptor gene assembly is regulated during lymphocyte development by modulating both the levels of V(D)J recombinase activity and the accessibility of V7 D, and J gene segments to recombinase. Previously, we have shown that recombination and expression of gene segments in a TCR minilocus construct critically depends upon the presence of an active enhancer element. To examine the role of transcriptional activation in targeting V(D)J recombination, we identified the promoter element responsible for expression of germline D/3 and J/3 gene segments in this minilocus. Si nuclease protection assays of B cells transfected with a TCR/3-EK minilocus demonstrated a major transcription initiation site located 3' to the D/31.2 gene segment. Analysis of the sequences that neighbor D/31.2 revealed a consensus TATA box within the 5' recombination signal sequence and multiple consensus bindings sites for known transcription factors (including SP1, C/EBP and PU.l). Heterologous luciferase constructs containing the SV40 enhancer were used to locate the minimal promoter element (PD/3) within a 450 bp fragment that spans the D/31.2 element. Maximal PD/3 activity required the presence of both the SPl motif and the TATA box. Current experiments are directed towards defining the developmental/tissue specificity of this promoter and its activity in developing thymocytes.
|State||Published - Dec 1 1996|