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

Fatmata Bah,John R Christin,Yi Yan,Zhongliang Ju,Shiniu Wei,Barbara K Birshtein,Joyce Pieretti,Laurel A Eckhardt

doi:10.1074/jbc.m111.230995

Abstract

Immunoglobulin heavy chain (IgH) genes are formed, tested, and modified to yield diverse, specific, and high affinity antibody responses to antigen. The processes involved must be regulated, however, to avoid unintended damage to chromosomes. The 3' regulatory region of the Igh locus plays a major role in regulating class-switch recombination (CSR), the process by which antibody effector functions are modified during an immune response. Loss of all known enhancer-like elements in this region dramatically impairs CSR, but individual element deletions have no effect on this process. In the present study, we explored the hypothesis that an underlying functional redundancy in the homologous elements hs3a and hs3b was masking the importance of either element to CSR. Several transgenic mouse lines were generated, each carrying a bacterial artificial chromosome transgene that mimicked Igh locus structure but in which hs3a was missing and hs3b was flanked by loxP sites. Matings to Cyclization Recombination Enzyme-expressing mice established "pairs" of lines that differed only in the presence or absence of hs3b. Remarkably, CSR remained robust in the absence of both hs3a and hs3b, suggesting that the remaining two elements of the 3' regulatory region, hs1.2 and hs4, although individually dispensable for CSR, are, together, sufficient to support CSR.

Highlights

Construction of Immunoglobulin heavy chain (IgH)⌬3alx3b bacterial artificial chromosome (BAC)—In a previous study, it was shown that class-switch recombination (CSR) was unimpaired in B cells from mice lacking the 3Ј regulatory region (3ЈRR) element hs3a [18]
A bacterial artificial chromosome containing much of the murine Igh locus (CHC BAC) [28] was first modified to replace the JH region with an assembled VH gene (Fig. 1A)
(397-bp ␤-globin cDNA segment) was inserted to uniquely tag this region of the BAC (Fig. 1A, TAG). hs3a deletion corresponded to the nucleotides shared by hs3a and hs3b, and, reciprocally, the hs3b region between loxP sites included all nucleotide sequences shared by these two 3ЈRR elements

Summary

Introduction

The modifications involved in deleting hs3a and flanking hs3b with loxP sites and tag DNA were confirmed by Southern blot analyses of the BACs (supplemental Fig. 1). Five of the lines (60, 69, 79, 81, and 83) were further analyzed to confirm intact BAC structure in regions mapping from the assembled VH gene through to the loxP-flanked hs3b

Results

Conclusion