Coordination of Immunoglobulin DJ_H Transcription and D-to-J_H Rearrangement by Promoter-Enhancer Approximation

Abstract

The genes that encode the variable regions of immunoglobulin (Ig) heavy chains are encoded by three DNA segments: V_H, D, and J_H. During B-cell development these segments are brought together by a pair of sitespecific DNA rearrangements. The first of these joins a D segment to a J_H segment; the second brings a V_H segment in apposition to a DJ_H unit. B-cell precursors that have undergone D-to-J_H joining express transcripts that initiate at the 5′ flanks of rearranged D segments (DJ_H transcription). In this study we have examined the coordination of D-to-J_H rearrangement and DJ_H transcription. The B-lymphoid progenitor cell line HAFTL-1 undergoes D-to-J_H rearrangement when propagated in culture. In progeny of a single HAFTL-1 cell clone, joining of distal D segments (D_SP2 and D_FL16) to J_H is accompanied by an increase in the steady-state level of transcripts initiating 5′ of the D coding region. Steady-state transcription of a D_SP2 gene segment was undetectable prior to rearrangement and was observed to increase at least 20-fold upon joining to J_H. In contrast, transcription from the 5′ flank of D_Q52, which lies within 700 bp of the J_H cluster, was detected prior to rearrangement and did not increase significantly after rearrangement. The 5′ flank of a D_SP2 segment was found to support expression of a heterologous gene upon transfection into B progenitor cell lines. Expression from this D_SP2 promoter was at least 30-fold higher in the presence of the Ig heavy-chain enhancer, in either orientation, than in its absence. A DNA fragment spanning the interval from -165 to +19 bp relative to the major D_SP2 transcriptional start site retained enhancer-dependent promoter activity. These observations imply that activation of D_SP2J_H and D_FL16J_H transcription is coordinated with D-to-J_H rearrangement by approximation of enhancer-dependent D promoter elements to the Ig heavy-chain enhancer. This interpretation is consistent with our observation that the D_Q52 segment, which is closely linked to the J_H cluster, is transcribed both before and after rearrangement.