Chromosome choice for initiation of V-(D)-J recombination is not governed by genomic imprinting

Abstract

V-(D)-J recombination generates the antigen receptor diversity necessary for immune cell function, while allelic exclusion ensures that each cell expresses a single antigen receptor. V-(D)-J recombination of the Ig, Tcrb, Tcrg and Tcrd antigen receptor genes is ordered and sequential so that only one allele generates a productive rearrangement. The mechanism controlling sequential rearrangement of antigen receptor genes, in particular how only one allele is selected to initiate recombination while at least temporarily leaving the other intact, remains unresolved. Genomic imprinting, a widespread phenomenon wherein maternal or paternal allele inheritance determines allele activity, could represent a regulatory mechanism for controlling sequential V-(D)-J rearrangement. We used strain-specific single-nucleotide polymorphisms within antigen receptor genes to determine if maternal vs paternal inheritance could underlie chromosomal choice for the initiation of recombination. We found no parental chromosomal bias in the initiation of V-(D)-J recombination in T or B cells, eliminating genomic imprinting as a potential regulator for this tightly regulated process.

Figure 1. Rationale for experimental approach

Models of V-(D)-J recombination where the choice of initial substrate is biased toward the maternal (dark gray) chromosome, biased toward the paternal (light gray) chromosome, or not influenced by chromosome parental origin. Maternal and paternal alleles are distinguished by SNPs within the region that is always deleted by V-DJ (or V-J) recombination. By assaying the relative proportions of maternal and paternal alleles in mature antigen-receptor cell populations we can distinguish these three mutually exclusive models. Alleles are distinguished and then quantitated by restriction enzyme digestion and also by pyrosequencing. Analyzing mice generated by reciprocal crosses eliminates the possibility that allelic bias will mask parent-of-origin bias in the selection of the initial chromosome that will undergo recombination.

Figure 2. Analysis of parent-of-origin bias by restriction enzyme digestion

(A) Tcrb locus: genomic DNA was prepared from tail biopsy and from purified TCRγδ cells (γδ TCR+), purified TCRαβ CD4+ cells (TCRβ+, γδ TCR−, CD4+, CD8−), purified TCRαβ CD8+ cells (TCRβ+, γδ TCR−, CD8+, CD4−), and purified B cells (TCRβ−, γδ TCR−, CD19+, B220+) from adult mice generated by intercrosses of B6 × FVB (lane 1) or FVB × B6 (lane 2). To determine allelic frequency, gDNAs were amplified across known SNPs, digested with informative restriction enzymes, and electrophoresed. Four total analyses were done and representative data are shown. To identify a parent-of-origin bias in the choice of chromosome to initiate V-DJ recombination, we looked for distinct patterns in cells that have undergone recombination (marked by grey boxes) relative to cells that did not undergo recombination. Recombination of the β locus will have occurred in CD4+ and CD8+ cells but not in tail or in TCRγδ or B cells. However, the relative frequencies of B6 and FVB are not different in those cell types. The use of reciprocal crosses ensures that allelic biases (in either recombination or in detection) did not obscure parent-of-origin biases. N = 4. (B) Tcrg locus was analyzed as in (A) except for the heterozygotes being B6 × DBA (lane 1) and DBA × B6 (lane 2). (C) Igh locus was exactly analyzed as described in panel A.

Figure 3. Analysis of parent-of-origin bias by pyrosequencing

Genomic DNAs were prepared essentially as described for Figure 2 except that for Igh, we included samples from B6 × FVB, FVB × B6, B6 × DBA, and DBA × B6 F1 heterozgyotes since both crosses included informative SNPs. After amplification across known SNPs, allelic frequency was analyzed by pyrosequencing. The relative frequency of the maternally inherited allele is depicted as mean ± standard error of the mean. For Tcrb, n=4 (2 B6 × FVB and 2 FVB × B6). For Tcrg, n=4 (2 B6 × DBA and 2 DBA × B6). For Igh, n = 8 (2 B6 × FVB, 2 FVB × B6, 2 B6 × DBA, and 2 DBA × B6). For each locus, the dark grey bars depict the cell population that will have undergone V-DJ recombination. Non-recombined cells are shown in light grey. The absence of detectable differences between these two types of cells and maternal allele frequencies of 50% together indicate that there was no parent-of-origin bias in the choice of chromosome to initiate recombination.