Position-dependent inhibition of class-switch recombination by PGK-neor cassettes inserted into the immunoglobulin heavy chain constant region locus

Abstract

The Ig heavy chain (IgH) constant region (CH) genes are organized from 5' to 3' in the order Cmicro, Cdelta, Cgamma3, Cgamma1, Cgamma2b, Cgamma2a, Cepsilon, and Calpha. Expression of CH genes downstream of Cdelta involves class-switch recombination (CSR), a process that is targeted by germ-line transcription (GT) of the corresponding CH gene. Previously, we demonstrated that insertion of a PGK-neor cassette at two sites downstream of Calpha inhibits, in cultured B cells, GT of and CSR to a subset of CH genes (including Cgamma3, Cgamma2a, Cgamma2b, and Cepsilon) that lie as far as 120 kb upstream. Here we show that insertion of the PGK-neor cassette in place of sequences in the Igamma2b locus inhibits GT of and CSR to the upstream Cgamma3 gene, but has no major effect on the downstream Cgamma2a and Cepsilon genes. Moreover, replacement of the Cepsilon exons with a PGK-neor cassette in the opposite transcriptional orientation also inhibits, in culture, GT of and CSR to the upstream Cgamma3, Cgamma2b, and Cgamma2a genes. As with the PGK-neor insertions 3' of Calpha studied previously, the Cgamma1 and Calpha genes were less affected by these mutations both in culture and in mice, whereas the Cgamma2b gene appeared less affected in vivo. Our findings support the existence of a long-range 3' IgH regulatory region required for GT of and CSR to multiple CH genes and suggest that PGK-neor cassette insertion into the locus short circuits the ability of this region to facilitate GT of dependent CH genes upstream of the insertion.

Figure 1

Insertion of the PGK-neo^r gene in the IgH locus disrupts class switching upstream of the insertion. Summary of the effects on CSR in gene-targeting experiments within the IgH locus using the PGK-neo^r gene as a selectable marker. Iγ2b and IgE C_H1–4 summarize experiments presented here, and HS1,2 and HS3a summarize previously published studies (24, 25). The effect on CSR is noted as inhibition (downward-pointing arrow), partial inhibition (+/−), or CSR levels comparable to wt controls (+). Transcripts and transcriptional orientation are represented by horizontal arrows. ND, not determined. I, I exon; S, switch region; C, constant region genes for corresponding isotype.

Figure 2

Class-switching defects in Iγ2b N/N and IgE N/N mice. (Upper) Concentrations of specific Ig isotypes in sera from Iγ2b−/− (□), Iγ2bN/N (⋄), Iγ2b +/N (○), wt (TC1/WT) (▵), and IgE N/N (♦) mice as measured by ELISA. Serum from some neo^r-inserted mice contained undetectable levels of IgG3 (symbols on axis). (Lower) ELISA analyses of culture supernatants from splenic B cells from Iγ2b−/− (squares), Iγ2bN/N (diamonds), Iγ2b +/N (circles), wt (TC1/WT) (triangles), or IgE N/N (◃) mice stimulated in vitro for 4.5 days in the presence of LPS (open symbols) (IgM, IgG3, IgG2b), LPS plus interferon γ (closed symbols for IgG2a), LPS plus IL-4 (closed symbols for IgG1 and IgE), or LPS plus transforming growth factor β (closed symbols for IgA). For Iγ2bN/N cultures, IgG3 was reduced 40- to 200-fold and IgG1 was partially inhibited (reduced 10-fold) compared with controls. For IgEN/N cultures, IgG3 was down 40- to 200-fold, IgG2b was down 20- to 50-fold, IgG2a was down 5- to 20-fold, IgG1 was reduced slightly (down 10-fold), and IgE was reduced severely.

Figure 3

Analysis of C_H germ-line transcripts in Iγ2bN/N- and IgEN/N-activated B cells. (A) Northern analysis of RNA from cells cultured with LPS or LPS + IL-4 for 2 or 5 days as indicated. Probes used were (in this order) Cγ2b C_H3 region, Cγ3, mb-1, Cɛ, and Cμ. Transcript sizes for productive (p) and germ-line (g) Cμ are 2 kb; for Cγ3 transcript, sizes are 3.3 membrane (m) and 1.9 secreted (s), where p and g transcripts are a similar size. P and g Cγ2b transcripts are 3.6 (m) and 1.7 (s) (53). P and g γ3 transcripts are 3.3 (m) and 1.9 (s) (54), the ɛ g transcript is 1.7, and the p is 2.3 (14). 129, Wild type. One representative experiment of three is shown. (B) RT-PCR for γ3 (LPS-stimulated) and ɛ (LPS + IL-4-stimulated) germ-line transcripts. cDNA derived from cells stimulated for 2 days in culture was diluted 1:1, 1:5, and 1:25 for all samples. Mb-1 RT-PCR was used as a control for amount of cDNA per reaction. Wild-type controls are indicated as WT. One representative experiment of three is shown.