Spi-C and PU.1 counterregulate Rag1 and Igκ transcription to effect immunoglobulin kappa recombination in small pre-B cells

Abstract

B cell development requires the ordered rearrangement of Ig genes encoding H and L chain proteins that assemble into BCRs or Abs capable of recognizing specific Ags. Ig{kappa} rearrangement is promoted by chromatin accessibility and by relative abundance of RAG1/2 proteins. Expression of the E26-transformation-specific (ETS) transcription factor Spi-C is activated in response to double-stranded DNA breaks (DSBs) in small pre-B cells to negatively regulate pre-BCR signaling and Ig{kappa} rearrangement. However, it is not clear if Spi-C regulates Ig{kappa} rearrangement through transcription or by controlling RAG expression. In this study, we investigated the mechanism of Spi-C negative regulation of Ig{kappa} light chain rearrangement. Using an inducible expression system in a pre-B cell line, we found that Spi-C negatively regulated Ig{kappa} rearrangement, Ig{kappa} transcript levels, and Rag1 transcript levels. We found that Ig{kappa} and Rag1 transcript levels were increased in small pre-B cells from Spic-/- mice. In contrast, Ig{kappa} and Rag1 transcript levels were activated by PU.1 and were decreased in small pre-B cells from PU.1-deficient mice. Using chromatin immunoprecipitation analysis, we identified an interaction site for PU.1 and Spi-C located in the Rag1 promoter region. These results suggest that Spi-C and PU.1 counterregulate Ig{kappa} transcription and Rag1 transcription to effect Ig{kappa} recombination in small pre-B cells.