The genomic arrangement of T cell receptor variable genes is a determinant of the developmental rearrangement pattern

Abstract

Developmentally regulated V(D)J recombination profoundly influences immune repertoires, but the underlying mechanisms are poorly understood. In the endogenous T cell receptor Cgamma1 cluster, the 3' Vgamma3 gene (closest to Jgamma1) rearranges preferentially in the fetal period whereas rearrangement of the 5' Vgamma2 gene predominates in the adult. Reversing the positions of the Vgamma2 and Vgamma3 genes in a genomic transgene resulted in decreased rearrangement of the now 5' Vgamma3 gene in the fetal thymus and increased rearrangement of the now 3' Vgamma2 gene. The reversed rearrangement pattern was not accompanied by significant changes in chromatin accessibility of the relocated Vgamma genes. The results support a model in which the 3' location is the key determinant of rearrangement in the fetus, after which there is a promoter-dependent inactivation of Vgamma3 rearrangement in favor of Vgamma2 rearrangement.

Fig. 2.

Rearrangements in DECs of γB-Gn-Sw and γB transgenic mice. DECs from all three lines of γB-Gn-Sw transgenic mice were compared with γB (3) by semiquantitative PCR (9). Transgene copy number is indicated in brackets. Three-fold serial dilutions of DEC DNA samples were PCR amplified with the following primers (see Fig. 1): L2/J1 (for Vγ2), L3/J1 (for Vγ3), and 5′tubulin/3′tubulin (for Tub). PCR products were digested with NruI (for Vγ2) or EcoRI (for Vγ3) to distinguish endogenous (E) from transgene (Tg) rearrangements. Semiquantitative PCR of tubulin was used to normalize the DNA samples. Ratios of the levels of Vγ2 to Vγ3 rearrangements of the transgene are displayed below each data set (see Methods).

Fig. 1.

Comparison of γB, γB-Gn-Sw, and γB-Pr-Sw transgenes. The 5′ DNase hypersensitive site A (HSA) and the 3′ Cγ1 enhancer, 3′ E_Cγ1 (E) are indicated. All transgenes have the same backbone. Differences are depicted in the expanded segments. The boxes above the line are the coding (V and leader) sequences. Boxes below the line are segments subjected to exchange. Vγ2 sequences are in black and Vγ3 are hatched. Asterisks indicate restriction enzyme sites introduced into the transgene. Restriction enzymes are as follows: S, SpeI; H, HindIII; R1, EcoRI; and RV, EcoRV. Arrows indicate positions and orientation of primers: 1, L2; 2, PSVγ2; 3, VG2I; 4, V2-3′a; 5, L3; 6, PSVγ3; 7, V3-3′b; 8, V3-3′a; and 9, J1. The labeled line segments indicate probes used in Southern blots.

Fig. 3.

Semiquantitative PCR of Vγ2 and Vγ3 rearrangements in early fetal and adult thymocytes of transgenic mice. (A) Comparison of Vγ rearrangement in E14 thymus and E15 thymocytes. (B) Comparison of Vγ rearrangement in adult CD4^-CD8^- thymocytes. The Vγ2 standard is hybridoma DN2.3, which contains two copies of rearranged Vγ2. The Vγ3 standard is the WRD.34 cell line, which contains two copies of rearranged Vγ3. See Fig. 2 legend for other details. Similar data for other lines are summarized in Table 1.

Fig. 4.

RSS breaks and germ-line transcription of Vγ2 and Vγ3 genes in the transgenes. (A) Comparison of RSS breaks flanking Vγ2 and Vγ3 in the transgenes. Samples were serially diluted 5-fold (for E15 fetal samples). Semiquantitative PCR for tubulin was used as a loading control. (B) Vγ2 and Vγ3 germ-line transcripts emanating from transgenes in fetal and adult thymocytes. Shown is semiquantitative RT-PCR analysis of germ-line transcripts in total RNAs from adult CD4^-CD8^- thymocytes and E14 fetal thymus of γB and γB-Gn-Sw transgenic mice. Primers for reverse transcription (RT) included oligo(dT) (for the tubulin control), V2-3′a (for Vγ2), or V3-3′a (for Vγ3) (see Fig. 1). RT products were then 3-fold serially diluted and subjected to the semiquantitative PCR. Either 5′tubulin/3′tubulin primer set (for Tub) or L2/VG2I primer set (for Vγ2), or PSVγ3/V3-3′b primer set (for Vγ3) was used for PCR of corresponding RT products. Vγ2 and Vγ3 PCR products were digested with Nru1 (for Vγ2) or EcoRI (for Vγ3) before gel fractionation. RNA samples without RT were also directly subjected to the semiquantitative PCR to confirm the absence of DNA contamination in the samples. (C) Germ-line transcripts of γB and γB-Pr-Sw transgenes in adult CD4^-CD8^- thymocytes. The PSVγ2/VG2I primer set was used for Vγ2 PCR. All others were as in B.

Fig. 5.

Restriction enzyme accessibility of Vγ2 and Vγ3 genes in the transgenes. (A) Restriction enzyme sites in Vγ2 and Vγ3 genes used in the accessibility assays. The NruI and EcoRI sites indicated with asterisks are transgenespecific sites. Vertical arrows indicate the restriction sites probed in these analyses. Nuclei of E15 fetal thymocytes (B and C) or adult CD4^-CD8^- thymocytes (D and E) were treated with restriction enzymes to detect the accessibility of the Vγ2 (Asp-718) or Vγ3(EcoRI) genes. After DNA extraction, cleavage was assayed by radioactive LM-PCR. (B and D) The accessibility of the Asp-718 site in the Vγ2 gene. Samples were serially diluted 5-fold. Semiquantitative PCR for the tubulin gene was used as a loading control. A portion of each PCR product was digested with NruI before gel fractionation to confirm its origin from the transgene. (C and E) The accessibility of the EcoRI site in Vγ3 gene was determined. In this assay, the DNA was digested with a second restriction enzyme, ApaL1 (located 5′ of the EcoR1 site) after DNA extraction but before LM-PCR. Therefore, the ApaL1 PCR product corresponds to DNA that was uncleaved by EcoRI and serves as an internal reference. In C, two experiments are shown for comparison.