Immunoglobulin gene transcription ceases upon deletion of a distant enhancer.

Abstract

The tissue‐specific E mu enhancer within the immunoglobulin heavy chain (IgH) locus has recently been shown to be essential for efficient V region gene assembly in early B lineage cells. However, we and others have shown that late stage, Ig‐secreting cells can produce IgH in the absence of E mu. In the present study we have explored the notion that another enhancer found in the far 3′ region of the IgH locus (3′ alpha E) takes on an important regulatory role in cells that have reached this terminal stage in B cell development. The technique of homologous recombination was used to disrupt the 3′ alpha E region in an E mu‐deficient, Ig gamma 2a‐secreting cell line. Loss of 3′ alpha E completely abolished Ig heavy chain gene expression, demonstrating that transcription of this gene was dependent upon sequences that reside over 70 kb downstream. The ability of these sequences to function efficiently in the absence of E mu may also provide an explanation for deregulated c‐myc expression in many Ig‐secreting tumors.

Transcription of a Productively Rearranged Ig VDJCα Does Not Require the Presence of HS4 in the Igh 3′ Regulatory Region

“…a cell line that already lacks E , enhancer-replacement within the 3ЈRR by a selectable marker gene completely arrested IgH gene transcription, supporting the hypothesis that IgH transcription in an E -deficient locus is 3ЈRR dependent (10). In other studies, c-myc was juxtaposed with the 3ЈRR both in transgenes and by genereplacement within the natural IgH and c-myc loci (19,36,37).…”

“…This argues against there being any compensatory effect of the neo r gene upon hs4 deletion in the BACs of the present studies. Certainly, when a neo r gene is inserted directly into the 3ЈRR it can affect locus activity (10), but the present studies do not involve insertion of the neo r gene into the BAC. Rather, in this study the neo r gene remains on a plasmid that has no homology to the BAC and, at best, will integrate at the 5Ј or 3Ј end of the BAC.…”

“…These two findings led tocould be supplanted by the 3Ј IgH enhancers. In one of the Edeficient cell lines (9921), a technical knockout of the 3Ј IgH enhancer region downstream of the active IgH locus (by replacing hs1,2 with a neo r gene) abolished IgH transcription, confirming that this region was indeed required for locus expression (10).…”

“…The first of these, E , is found in an intron separating the V region and C region coding sequences and, although initially discovered because of its ability to enhance transcription from the promoter of an assembled IgH gene, it has since been shown to be essential for efficient V-D-J recombination (3)(4)(5)(6)(7)(8)(9). The other four enhancers lie at the far 3Ј end of the IgH locus and have been shown to contribute substantially to IgH gene transcription in Ig-secreting cells as well as play an important role in CSR (10,11).…”

“…9921 is a plasmacytoma that secretes IgG2a. The single, functional Ig␥2a gene lacks E (as the result of an aberrant CSR event) and so its expression is entirely dependent upon 3Ј IgH regulatory sequences (10,15), making it a particularly appropriate cell line for studying 3ЈRR function. This cell line also carries three copies of a translocated chromosome that juxtaposes the oncogene c-myc with C␥2a and all Igh locus sequences downstream (including the 3ЈRR) (25).…”