The 3' end of the heavy chain constant region locus enhances germline transcription and switch recombination of the four gamma genes

Abstract

The switch in immunoglobulin (Ig) heavy chain class is preceded by germline transcription and then mediated by a DNA recombination event. To study germline transcription and class switch recombination we used transgenic mice with a 230-kilobase bacterial artificial chromosome that included a rearranged VDJ gene and the entire heavy chain constant region locus. In addition to several lines with intact transgenes, we identified two lines in which the heavy chain locus transgene lacked Calpha and everything 3' of it, including the regulatory elements HS3a, HS1-2, HS3b, and HS4. B cells from both lines with the truncated transgenes make abundant transgenic (Tg) VDJCmu transcripts and IgM protein. Deletion of the 3' end of the locus results in dramatically reduced expression of both germline transcripts and switched VDJCH transcripts of the gamma3, gamma2b, gamma2a, and epsilon genes. In addition, the transgenes lacking the 3' end of the locus express reduced amounts of gamma1 germline transcripts and 2-3% of the amount of Tg IgG1 in tissue culture compared with intact transgenes. Finally, switch recombination to gamma1 is undetectable in the transgenes lacking the 3' elements, as measured by digestion circularization-polymerase chain reaction or by the expression of VDJCgamma1 transcripts.

Figure 1.

Structure of the transgenes of the heavy chain constant region locus. (A) The DNA segments included are indicated for Tg mice with a wild-type γ1 gene (black lines) or the mutated Stat6 binding site in the γ1 promoter region (gray lines). The number designation and transgene copy number of each Tg line are indicated below each type of transgene. (B) Southern hybridization (JH3-4 and the various S segments, indicated below each set of lanes) or PCR results (Cɛ) define the presence and copy number of the transgene. In the “Sα” panel, the faint band in each sample above the Tg “Sα” fragment is the cross-hybridizing Sμ fragment.

Figure 2.

Transgenic heavy chain expression as determined by RT-PCR. (A) Transgenic VDJCμ expression. Two amounts of cDNA, differing by fivefold, were tested. Line 858 has three copies of the Tg μ gene; the other four lines have two copies. (B) Transgenic γ3 expression. Lines 822, 580, 551, and 847 have one copy of the transgene; the other lines have two or three copies. The expression of Tg γ3 germline transcripts by B cells from lines 847, 551, and 580 is documented in Fig. S3 (available at http://www.jem.org/cgi/content/full/jem.20041988/DC1). (C) Transgenic γ2b expression. The HPRT expression for some of these LPS samples is presented in Fig. 3 C. (D) Transgenic ɛ expression. For γ3, γ2b, and ɛ, the same amount of cDNA was used for IμCH and for Tg VDJCH amplifications.

Figure 3.

Transgenic γ2a expression. (A) RT-PCR for germline transcripts. The size difference between germline transcripts of the End gene and the transgene in lines 556, 578, and 1001 without the four-bp insertion in Iγ2a is noted by the lower Tg arrow. The size difference between germline transcripts of the End gene and the transgene with the four-bp insertion in Iγ2a in lines 858 and 822 with the four-bp insertion is noted by the upper Tg arrow. (B and D) RT-PCR for Tg VDJCγ2a and IμCγ2a transcripts. Lines 556, 578, and 858 have two or three copies of the ARS/Igh transgene. Lines 1001 and 822 have two Tg VH copies, but one Tg γ2a copy. Lines 847, 551, and 580 have one intact copy of the ARS/Igh transgene, but line 551 has two copies of Tg γ2a. (C) RT-PCR for HPRT transcripts. Although we normalized the LPS + IFNγ samples using IμCγ2a expression, we present HPRT transcripts to allow comparison of RNA levels between LPS and LPS + IFNγ samples. HPRT transcripts were amplified beginning with two cDNA concentrations, one fivefold greater than the other. The amount of cDNA that was used for each founder line was identical for germline, Tg VDJCγ2a, and IμCγ2a transcripts. The more abundant HPRT transcripts were amplified from smaller amounts of cDNA, but the relative quantities of the cDNA samples were consistent among the HPRT amplifications and the other three reactions.

Figure 4.

Transgenic IgG1 expression. (A) Means of quadruplicate samples, with SD error bars, for a single set of cultures. Supernatant fluids were tested for IgG1 with the Tg allotype by ELISA. The signals for Tg IgG1 in lines 1001 and 822, for all four culture conditions, are near the limit of detection, and therefore, can fluctuate between 0 and 20 ng/ml. In particular, the relative signals for line 822 B cells that were cultured in LPS compared with LPS + IL-4 are not representative of the average twofold (not statistically significant) reduction in LPS + IL-4 compared with LPS alone. Transgenic γ1 copy number for each line is given directly below the line number. (B) The portion of Tg IgM was calculated by dividing the amount of AD8+ (Tg idiotype) IgM by total IgM for LPS cultures. Means, with SEM bars, are presented for two to four independent cultures. The portion of Tg IgG1 was calculated by dividing the amount of IgG1^a by the amount of total IgG1 for LPS + IL-4 or CD40L + IL-4 cultures. Means, with SEM bars, are presented for 5 to 11 cultures. As a negative control, the mean IgG1^a/total IgG1 value from LPS + IL-4 and CD40L + IL-4 cultures of NTg B cells is presented as an open box. (C) Endogenous and Tg germline transcripts were identified by TaqI-digestion of RT-PCR products of RNA from B cells induced with LPS + IL-4 (23). The line 578 sample was derived from an independent experiment. B cells from line 847 express smaller amounts of γ1 germline transcripts as a result of the mutation of the Stat6 site in the γ1 promoter (26). (D) Transgenic VDJCγ1 and IμCγ1 transcripts were determined by RT-PCR, using the same amount of cDNA for the two reactions. RT-PCR for HPRT used less cDNA, but the relative amounts for each sample were held constant. (E) DC-PCR for Tg γ1 switch recombination. Transgenic and End recombination products were distinguished by MboI digestion. MboI sites are indicated by arrows at the bottom of the figure. Sequences flanking the 5′ portion of Sμ are indicated by a gray bar and those flanking the 3′ portion of Sγ1 are indicated by a black bar. These DNA segments are joined at the junction of the gray and black bars by ligation at EcoRI ends to form a circle. “Percent Tg” was calculated by dividing the cpm in the Tg band by the sum of the cpm in the Tg and the two End bands.