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

Abstract

V gene assembly, class switch recombination, and somatic hypermutation are gene-modifying processes essential to the development of an effective Ab response. If inappropriately applied, however, these processes can mediate genetic changes that lead to disease (e.g., lymphoma). A series of control elements within the Ig H chain (Igh) locus has been implicated in regulating these processes as well as in regulating IgH gene transcription. These include the intronic enhancer (Eμ) and several elements at the 3′ end of the locus (hs1,2, hs3a, hs3b, and hs4) known collectively as the 3′ regulatory region. Although it is clear that the Eμ plays a unique role in V gene assembly, it has not been established whether there are unique functions for each element within the 3′ regulatory region. In earlier studies in mice and in mouse cell lines, pairwise deletion of hs3b and hs4 had a dramatic effect on both class switch recombination and IgH gene transcription; deletion of an element almost identical with hs3b (hs3a), however, yielded no discernible phenotype. To test the resulting hypothesis that hs4 is uniquely required for these processes, we induced the deletion of hs4 within a bacterial artificial chromosome transgene designed to closely approximate the 3′ end of the natural Igh locus. When introduced into an Ig-secreting cell line, an Igα transcription unit within the bacterial artificial chromosome was expressed efficiently and the subsequent deletion of hs4 only moderately affected Igα expression. Thus, hs4 does not play a uniquely essential role in the transcription of a productively rearranged Ig VDJCα transcription unit.

Ig Synthesis and Class Switching Do Not Require the Presence of the hs4 Enhancer in the 3′ IgH Regulatory Region

“…Finally, the in vitro capacity to secrete various Ig isotypes was unaffected by the knockout. The fact that hs4 was not required for Ig secretion is in agreement with the recent study of Zhang et al (19), who reported, using various bacterial artificial chromosomes and the plasmocytoma cell line 9921, that transcription of a productively rearranged Ig VDJC␣ does not require the presence of hs4 in the 3ЈRR.…”

“…A predominant role for hs3b in this later study is difficult to support since deletion of the hs3a element (which shares 98% of homology with hs3a) has no effect (12). As previously suggested (12,19), this finding might reflect extensive functional redundancy among these elements (in cooperation or not with other elements in the locus). Deletion of only one of the four 3Ј IgH enhancers can be easily supplanted by the activity of the three remaining enhancers, with deletion of a pair of them becoming more problematic for the cell.…”

High-Affinity IgG Antibodies Develop Naturally in Ig-Knockout Rats Carrying Germline Human IgH/Igκ/Igλ Loci Bearing the Rat CH Region

“…However, a distinct attraction of using an IgH translocus in which the IgH C regions are of endogenous origin is that regions toward the 39 end of the IgH locus are known to play a major role in achieving efficient Ig class switch recombination as well, probably as optimal IgV gene somatic hypermutation (19,51,52). There are substantial differences between mouse/rat and human in the sequences in this enhancer region of the IgH locus, located some 30 kb downstream of Ca (53).…”

Homologous Elements hs3a and hs3b in the 3′ Regulatory Region of the Murine Immunoglobulin Heavy Chain (Igh) Locus Are Both Dispensable for Class-switch Recombination

“…Southern blot analyses were performed as described previously, using ϳ10 -30 g of mouse tail-tip DNA or liver DNA (30).…”