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Changes in locus-specific V(D)J recombinase activity induced by immunoglobulin gene products during B cell development.

Constantinescu A, Schlissel MS - J. Exp. Med. (1997)

Bottom Line: This switch in locus-specific recombinase activity results in allelic exclusion at the immunoglobulin heavy chain locus.We find that immature, but not mature, B cells that already express a functional light chain protein can undergo continued light chain gene rearrangement, by replacement of the original rearrangement on the same allele.Finally, we find that the developmentally regulated targeting of V(D)J recombination is unaffected by enforced rapid transit through the cell cycle induced by an E mu-myc transgene.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
The process of V(D)J recombination is crucial for regulating the development of B cells and for determining their eventual antigen specificity. Here we assess the developmental regulation of the V(D)J recombinase directly, by monitoring the double-stranded DNA breaks produced in the process of V(D)J recombination. This analysis provides a measure of recombinase activity at immunoglobulin heavy and light chain loci across defined developmental stages spanning the process of B cell development. We find that expression of a complete immunoglobulin heavy chain protein is accompanied by a drastic change in the targeting of V(D)J recombinase activity, from being predominantly active at the heavy chain locus in pro-B cells to being exclusively restricted to the light chain loci in pre-B cells. This switch in locus-specific recombinase activity results in allelic exclusion at the immunoglobulin heavy chain locus. Allelic exclusion is maintained by a different mechanism at the light chain locus. We find that immature, but not mature, B cells that already express a functional light chain protein can undergo continued light chain gene rearrangement, by replacement of the original rearrangement on the same allele. Finally, we find that the developmentally regulated targeting of V(D)J recombination is unaffected by enforced rapid transit through the cell cycle induced by an E mu-myc transgene.

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(A) Diagram of the LMPCR assay used to detect signal end and coding end double stranded DNA breaks at rearranging loci. A rearranging  locus is shown, with the RSS abutting the coding portion of a rearranging gene segment (J1, filled area). Cleavage by V(D)J recombinase at an RSS generates two kinds of ends: a signal end and a coding end. The signal end is blunt and 5′-phosphorylated and available for linker ligation. The coding end is  processed through a hairpin intermediate and the asterisk next to it signifies heterogeneity in the fine structure of the opened hairpin. Coding ends are  blunted with T4 DNA polymerase and then subjected to linker ligation. Amplification is subsequently carried out using a linker-specific primer (BW) and  a set of locus-specific primers (1, 2 or 1′, 2′). (B) SBE in developing B cells from wild-type and Eμ-myc mice. Purified DNA from sorted subpopulations  of B cells from wild-type Balb/c and Eμ-myc mice was subjected to LMPCR to detect SBE upstream of the DFL16.1, Jκ1, and Jκ2 segments. PCR products  were analyzed by electrophoresis on agarose gels, blot transfer to a nylon membrane, and hybridization with locus-specific oligonucleotide probes. The  identities of the indicated PCR products were confirmed by DNA sequence analysis (28). Labeled products were visualized with a PhosphorImager. Lanes  1–10, B cell development stages as defined in Table 1; lane 11, 6312, a pro-B cell line derived from a RAG-2-deficient mouse (3); lane 12, B220+ cells  from bone marrow, representing all stages of B cell development; lane 13, same as lane 12, with no ligase added at the linker ligation step. The bottom  strip shows an ethidium bromide-stained agarose gel of a control amplification of a non-rearranging locus (CD14), showing equivalent amounts of amplifiable DNA in all samples.
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Figure 3: (A) Diagram of the LMPCR assay used to detect signal end and coding end double stranded DNA breaks at rearranging loci. A rearranging locus is shown, with the RSS abutting the coding portion of a rearranging gene segment (J1, filled area). Cleavage by V(D)J recombinase at an RSS generates two kinds of ends: a signal end and a coding end. The signal end is blunt and 5′-phosphorylated and available for linker ligation. The coding end is processed through a hairpin intermediate and the asterisk next to it signifies heterogeneity in the fine structure of the opened hairpin. Coding ends are blunted with T4 DNA polymerase and then subjected to linker ligation. Amplification is subsequently carried out using a linker-specific primer (BW) and a set of locus-specific primers (1, 2 or 1′, 2′). (B) SBE in developing B cells from wild-type and Eμ-myc mice. Purified DNA from sorted subpopulations of B cells from wild-type Balb/c and Eμ-myc mice was subjected to LMPCR to detect SBE upstream of the DFL16.1, Jκ1, and Jκ2 segments. PCR products were analyzed by electrophoresis on agarose gels, blot transfer to a nylon membrane, and hybridization with locus-specific oligonucleotide probes. The identities of the indicated PCR products were confirmed by DNA sequence analysis (28). Labeled products were visualized with a PhosphorImager. Lanes 1–10, B cell development stages as defined in Table 1; lane 11, 6312, a pro-B cell line derived from a RAG-2-deficient mouse (3); lane 12, B220+ cells from bone marrow, representing all stages of B cell development; lane 13, same as lane 12, with no ligase added at the linker ligation step. The bottom strip shows an ethidium bromide-stained agarose gel of a control amplification of a non-rearranging locus (CD14), showing equivalent amounts of amplifiable DNA in all samples.

Mentions: As shown in Fig. 3 A, cleavage by V(D)J recombinase at an RSS results in two broken-ended species, a signal end and a coding end. Signal broken ends (SBE) are the more abundant, as they remain in the blunt-ended conformation until they are joined. This broken-ended species can persist in a resting cell for an extended period of time. However, cell cycling places a limit on the persistence of these SBE, because they are joined before the onset of DNA synthesis (27, 29). Thus, in a population of cycling cells, SBE can be a reliable measure of locus-specific V(D)J recombinase activity. This is in contrast to analysis of completed joints, which can be affected by pre-existing rearrangements or by selection at the cellular level occurring after rearrangement. Coding broken ends (CBE) are a more specific indicator of V(D)J recombinase activity, because they are rapidly processed to coding joints regardless of the cycling status of the cell (29). However, they are initially sealed in a hairpin structure and become available to linker ligation only in the interval between hairpin opening and coding joint formation (Schlissel, M.S., manuscript in preparation and 26, 29). The short half-life and the intermediate processing steps of coding ends make them much less abundant than the corresponding signal ends and more difficult to detect reliably.


Changes in locus-specific V(D)J recombinase activity induced by immunoglobulin gene products during B cell development.

Constantinescu A, Schlissel MS - J. Exp. Med. (1997)

(A) Diagram of the LMPCR assay used to detect signal end and coding end double stranded DNA breaks at rearranging loci. A rearranging  locus is shown, with the RSS abutting the coding portion of a rearranging gene segment (J1, filled area). Cleavage by V(D)J recombinase at an RSS generates two kinds of ends: a signal end and a coding end. The signal end is blunt and 5′-phosphorylated and available for linker ligation. The coding end is  processed through a hairpin intermediate and the asterisk next to it signifies heterogeneity in the fine structure of the opened hairpin. Coding ends are  blunted with T4 DNA polymerase and then subjected to linker ligation. Amplification is subsequently carried out using a linker-specific primer (BW) and  a set of locus-specific primers (1, 2 or 1′, 2′). (B) SBE in developing B cells from wild-type and Eμ-myc mice. Purified DNA from sorted subpopulations  of B cells from wild-type Balb/c and Eμ-myc mice was subjected to LMPCR to detect SBE upstream of the DFL16.1, Jκ1, and Jκ2 segments. PCR products  were analyzed by electrophoresis on agarose gels, blot transfer to a nylon membrane, and hybridization with locus-specific oligonucleotide probes. The  identities of the indicated PCR products were confirmed by DNA sequence analysis (28). Labeled products were visualized with a PhosphorImager. Lanes  1–10, B cell development stages as defined in Table 1; lane 11, 6312, a pro-B cell line derived from a RAG-2-deficient mouse (3); lane 12, B220+ cells  from bone marrow, representing all stages of B cell development; lane 13, same as lane 12, with no ligase added at the linker ligation step. The bottom  strip shows an ethidium bromide-stained agarose gel of a control amplification of a non-rearranging locus (CD14), showing equivalent amounts of amplifiable DNA in all samples.
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Figure 3: (A) Diagram of the LMPCR assay used to detect signal end and coding end double stranded DNA breaks at rearranging loci. A rearranging locus is shown, with the RSS abutting the coding portion of a rearranging gene segment (J1, filled area). Cleavage by V(D)J recombinase at an RSS generates two kinds of ends: a signal end and a coding end. The signal end is blunt and 5′-phosphorylated and available for linker ligation. The coding end is processed through a hairpin intermediate and the asterisk next to it signifies heterogeneity in the fine structure of the opened hairpin. Coding ends are blunted with T4 DNA polymerase and then subjected to linker ligation. Amplification is subsequently carried out using a linker-specific primer (BW) and a set of locus-specific primers (1, 2 or 1′, 2′). (B) SBE in developing B cells from wild-type and Eμ-myc mice. Purified DNA from sorted subpopulations of B cells from wild-type Balb/c and Eμ-myc mice was subjected to LMPCR to detect SBE upstream of the DFL16.1, Jκ1, and Jκ2 segments. PCR products were analyzed by electrophoresis on agarose gels, blot transfer to a nylon membrane, and hybridization with locus-specific oligonucleotide probes. The identities of the indicated PCR products were confirmed by DNA sequence analysis (28). Labeled products were visualized with a PhosphorImager. Lanes 1–10, B cell development stages as defined in Table 1; lane 11, 6312, a pro-B cell line derived from a RAG-2-deficient mouse (3); lane 12, B220+ cells from bone marrow, representing all stages of B cell development; lane 13, same as lane 12, with no ligase added at the linker ligation step. The bottom strip shows an ethidium bromide-stained agarose gel of a control amplification of a non-rearranging locus (CD14), showing equivalent amounts of amplifiable DNA in all samples.
Mentions: As shown in Fig. 3 A, cleavage by V(D)J recombinase at an RSS results in two broken-ended species, a signal end and a coding end. Signal broken ends (SBE) are the more abundant, as they remain in the blunt-ended conformation until they are joined. This broken-ended species can persist in a resting cell for an extended period of time. However, cell cycling places a limit on the persistence of these SBE, because they are joined before the onset of DNA synthesis (27, 29). Thus, in a population of cycling cells, SBE can be a reliable measure of locus-specific V(D)J recombinase activity. This is in contrast to analysis of completed joints, which can be affected by pre-existing rearrangements or by selection at the cellular level occurring after rearrangement. Coding broken ends (CBE) are a more specific indicator of V(D)J recombinase activity, because they are rapidly processed to coding joints regardless of the cycling status of the cell (29). However, they are initially sealed in a hairpin structure and become available to linker ligation only in the interval between hairpin opening and coding joint formation (Schlissel, M.S., manuscript in preparation and 26, 29). The short half-life and the intermediate processing steps of coding ends make them much less abundant than the corresponding signal ends and more difficult to detect reliably.

Bottom Line: This switch in locus-specific recombinase activity results in allelic exclusion at the immunoglobulin heavy chain locus.We find that immature, but not mature, B cells that already express a functional light chain protein can undergo continued light chain gene rearrangement, by replacement of the original rearrangement on the same allele.Finally, we find that the developmentally regulated targeting of V(D)J recombination is unaffected by enforced rapid transit through the cell cycle induced by an E mu-myc transgene.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
The process of V(D)J recombination is crucial for regulating the development of B cells and for determining their eventual antigen specificity. Here we assess the developmental regulation of the V(D)J recombinase directly, by monitoring the double-stranded DNA breaks produced in the process of V(D)J recombination. This analysis provides a measure of recombinase activity at immunoglobulin heavy and light chain loci across defined developmental stages spanning the process of B cell development. We find that expression of a complete immunoglobulin heavy chain protein is accompanied by a drastic change in the targeting of V(D)J recombinase activity, from being predominantly active at the heavy chain locus in pro-B cells to being exclusively restricted to the light chain loci in pre-B cells. This switch in locus-specific recombinase activity results in allelic exclusion at the immunoglobulin heavy chain locus. Allelic exclusion is maintained by a different mechanism at the light chain locus. We find that immature, but not mature, B cells that already express a functional light chain protein can undergo continued light chain gene rearrangement, by replacement of the original rearrangement on the same allele. Finally, we find that the developmentally regulated targeting of V(D)J recombination is unaffected by enforced rapid transit through the cell cycle induced by an E mu-myc transgene.

Show MeSH