Epigenetic modifications of the VH region after DJH recombination in Pro-B cells

Abstract

The variable region of murine immunoglobulin heavy chain (Igh) is assembled by sequential D_H -J_H and V_H -DJ_H recombination. The accessibility of the Igh locus determines the order of rearrangement. Because of the large number of V_H genes and the lack of a suitable model, the epigenetic modifications of V_H genes after DJ_H recombination have not previously been characterized. Here, we employed two v-Abl pro-B cell lines, in which the Igh locus is in germline and DJ_H -recombined configurations, respectively. The DJ_H junction displays the characteristics of a recombination centre, such as high levels of activation-associated histone modifications and recombination-activating gene protein (RAG) binding in DJ_H -rearranged pro-B cells, which extend the recombination centre model proposed for the germline Igh locus. The different domains of the V_H region have distinct epigenetic characteristics after DJ_H recombination. Distal V_H genes have higher levels of active histone modifications, germline transcription and Pax5 binding, and good quality recombination signal sequences. Proximal V_H genes are relatively close to the DJ_H recombination centre, which partially compensates for the low levels of the above active epigenetic modifications. DJ_H recombination centre might serve as a cis-acting element to regulate the accessibility of the V_H region. Furthermore, we demonstrate that RAG weakly binds to functional V_H genes, which is the first detailed assessment of RAG dynamic binding to V_H genes. We provide a way for V_H -DJ_H recombination in which the V_H gene is brought into close proximity with the DJ_H recombination centre for RAG binding by a Pax5-dependent chromosomal compaction event, and held in this position for subsequent cleavage and V_H -DJ_H joining.

Keywords: Pax5; germline transcription; histone modification; recombination activating gene protein; recombination centre.