Recombination may occur in the absence of transcription in the immunoglobulin heavy chain recombination centre

Developing B cells undergo V(D)J recombination to generate a vast repertoire of Ig molecules. V(D)J recombination is initiated by the RAG1/RAG2 complex in recombination centres (RCs), where gene segments become accessible to the complex. Whether transcription is the causal factor of accessibility or whether it is a side product of other processes that generate accessibility remains a controversial issue. At the IgHlocus, V(D)J recombination is controlled by E mu enhancer, which directs the transcriptional, epigenetic and recombinational events in the IgH RC. Deletion of ER enhancer affects both transcription and recombination, making it difficult to conclude if E mu controls the two processes through the same or different mechanisms. By using a mouse line carrying a CpG-rich sequence upstream of E mu enhancer and analyzing transcription and recombination at the single-cell level, we found that recombination could occur in the RC in the absence of detectable transcription, suggesting that E mu controls transcription and recombination through distinct mechanisms. Moreover, while the normally E mu-dependent transcription and demethylating activities were impaired, recruitment of chromatin remodeling complexes was unaffected. RAG1 was efficiently recruited, thus compensating for the defective transcription-associated recruitment of RAG2, and providing a mechanistic basis for RAG1/RAG2 assembly to initiate V(D)J recombination.