Biallelic germline transcription at the kappa immunoglobulin locus

Abstract

Rearrangement of antigen receptor genes generates a vast array of antigen receptors on lymphocytes. The establishment of allelic exclusion in immunoglobulin genes requires differential treatment of the two sequence identical alleles. In the case of the kappa immunoglobulin locus, changes in chromatin structure, methylation, and replication timing of the two alleles are all potentially involved in regulating rearrangement. Additionally, germline transcription of the kappa locus which precedes rearrangement has been proposed to reflect an opening of the chromatin structure rendering it available for rearrangement. As the initial restriction of rearrangement to one allele is critical to the establishment of allelic exclusion, a key question is whether or not germline transcription at the kappa locus is monoallelic or biallelic. We have used a sensitive reverse transcription-polymerase chain reaction (RT-PCR) assay and an RNA-fluorescence in situ hybridization (FISH) to show that germline transcription of the kappa locus is biallelic in wild-type immature B cells and in recombination activating gene (RAG)-/-, mu+ B cells. Therefore, germline transcription is unlikely to dictate which allele will be rearranged first and rather reflects a general opening on both alleles that must be accompanied by a mechanism allowing one of the two alleles to be rearranged first.

Figure 1.

(A) The κ germline transcripts initiate from two promoters yielding a 8.4 and a 4.7 kb products which are further spliced to 1.1 and a 0.8 kb transcripts, respectively. The arrowheads indicate PCR primers used to amplify the 0.8 or the 1.1 kb transcript. Restriction site polymorphism (Hga I) between the Mus musculus and Mus spretus allele is indicated by the asterisk. (B) The 1.1 kb or the 0.8 kb transcript was subjected to RT-PCR with Mus musculus RNA, Mus spretus RNA or a mix of RNA from both Mus musculus and Mus spretus. The PCR products were then digested with the restriction endonuclease Hga I.

Figure 1.

(A) The κ germline transcripts initiate from two promoters yielding a 8.4 and a 4.7 kb products which are further spliced to 1.1 and a 0.8 kb transcripts, respectively. The arrowheads indicate PCR primers used to amplify the 0.8 or the 1.1 kb transcript. Restriction site polymorphism (Hga I) between the Mus musculus and Mus spretus allele is indicated by the asterisk. (B) The 1.1 kb or the 0.8 kb transcript was subjected to RT-PCR with Mus musculus RNA, Mus spretus RNA or a mix of RNA from both Mus musculus and Mus spretus. The PCR products were then digested with the restriction endonuclease Hga I.

Figure 2.

(A) Single-cell RT-PCR analysis of 1.1 kb κ germline transcript in BM cells from Rag^−/− μ⁺ mice. RT-PCR products were digested with Hga1 restriction endonuclease which recognizes only the Mus musculus allele. M is the Mus musculus allele, S is the Mus spretus allele, and B indicates the presence of both the Mus musculus and Mus spretus allele. The number of PCR products obtained ranged from one to eight out of eight. Cell # 1 and #2 are examples with four PCR products. Cell # 3 is an example with 8 PCR products. (B) Single-cell RT-PCR analysis of 0.8 kb κ germline transcript in BM cells from Rag^−/− μ^+/+ mice. RT-PCR products were digested with HgaI restriction endonuclease which recognizes only the Mus musculus allele. M is the Mus musculus allele and S is the Mus spretus allele. The number of PCR products obtained ranged from one to eight out of eight. Cell # 1 is an example with three PCR products. Cell #2 is an example with two PCR products.

Figure 3.

(A) RT-PCR analysis of 0.8 kb κ germline transcript in BCC' fraction of BM preB cells from WT mice. 50 cells were FACS^® sorted into a single tube and subjected to a RT reaction. This RT reaction was divided to eight PCR tubes. The RT-PCR products were digested with Hga1 restriction endonuclease which recognizes only the Mus musculus allele. M is the Mus musculus allele, S is the Mus spretus allele, and B indicates the presence of both the Mus musculus and Mus spretus allele. (B) Single-cell RT-PCR analysis of 0.8 kb κ germline transcript in BCC' fraction of BM preB cells from WT mice. RT-PCR products were digested with Hga1 restriction endonuclease which recognizes only the Mus musculus allele. M is the Mus musculus allele, S is the Mus spretus allele, and B indicates the presence of both the Mus musculus and Mus spretus allele. The number of PCR products obtained ranged from one to eight out of eight. Cell # 1 and #2 are examples with four PCR products.

Figure 4.

Graph of κ germline transcription in pre-B cells. Left panel: the percentage of cells appearing to be monoallelic is plotted as a function of the number of positive PCR reactions observed per cell. 16 cells revealed one PCR product/cell, 17 cells revealed two, 8 cells revealed three, 7 cells revealed four, 2 cells revealed five, six and seven PCR product/cell, and 3 cells revealed eight PCR products/cell. Note that grouping either the cells with 2 or 3 products per cell or the cells with 4 or more products per cell gives a fraction of the cells appearing monoallelic which is slightly below and statistically indistinguishable from the theoretical prediction for a biallelically expressed gene. Right panel: the theoretical expectation for an analysis of a monoallelically expressed gene. The formula 2/2ⁿ (where n is the number of PCR products observed) approximates the fractions for low values of n. The percentages presented leave out a minor correction in the formula for rare instances in which a given signal actually represents more than one cDNA product. The line across at the 100% level indicates what would be theoretically expected if monoallelic expression were absolute.

Figure 5.

RNA-FISH confirms biallelic κ germline transcription. A Cy3 labeled probe (pSPIg8) for the κ germline transcript revealed biallelic transcription in 91% of the RAG-1^−/− Abelson-transformed pre-B cells in which any κ germline transcript was detected. DAPI allows visualization of DNA in nuclei. Two of the four cells in the field presented are expressing the κ germline transcript and both of them have two RNA-FISH signals.