Limits...
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.

Show MeSH
(A) CBE at the Igκ locus in B cell development. Purified DNA from sorted subpopulations of B cells from wild-type Balb/c mice was subjected to LMPCR to detect CBE associated with Jκ1, Jκ2, and Jκ5 and most Vκ 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 CBE PCR products were  confirmed by DNA sequence analysis (data not shown). Labeled products were visualized with a PhosphorImager. 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.  (B) Diagram of possible mechanisms for secondary light chain gene rearrangements in immature (IgM+IgD−/low) B cells. (Left) replacement of the original  rearrangement (light hatched) by joining of an upstream Vκ to a downstream Jκ segment on the same chromosome (secondary rearrangement dark hatched).  (Right) direct rearrangement of a Vκ to a Jκ segment at the locus allelic to the original rearrangement.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196138&req=5

Figure 4: (A) CBE at the Igκ locus in B cell development. Purified DNA from sorted subpopulations of B cells from wild-type Balb/c mice was subjected to LMPCR to detect CBE associated with Jκ1, Jκ2, and Jκ5 and most Vκ 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 CBE PCR products were confirmed by DNA sequence analysis (data not shown). Labeled products were visualized with a PhosphorImager. 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. (B) Diagram of possible mechanisms for secondary light chain gene rearrangements in immature (IgM+IgD−/low) B cells. (Left) replacement of the original rearrangement (light hatched) by joining of an upstream Vκ to a downstream Jκ segment on the same chromosome (secondary rearrangement dark hatched). (Right) direct rearrangement of a Vκ to a Jκ segment at the locus allelic to the original rearrangement.

Mentions: DNA from equal numbers of cells was purified, treated with T4 DNA polymerase to blunt any overhanging CBE, and subjected to ligation with a double stranded linker capable of ligating in only one orientation. Linker-ligated DNA was then used for amplification by PCR using a linker-specific primer and a pair of nested locus-specific primers (Fig. 3 A). Control amplification of a non-rearranging locus showed that all samples contained similar amounts of amplifiable DNA. Because heavy chain gene rearrangements occur almost exclusively by deletion, using the RSS 5′ of DH genes for V-to-DJH joining (38), SBE upstream of DH genes represent intermediates in the VHto-DJH rearrangement step. We focused on SBE upstream of DFL16.1 because it is the most frequently used of the DH segments (40). At the κ locus we measured SBE upstream of the most frequently used Jκ segments, Jκ1 and Jκ2, as indicative of Vκ-to-Jκ rearrangement (Fig. 3 B). To distinguish between ongoing rearrangement and the persistence of unrepaired SBE in non-cycling cells at later stages of development, we also assayed for CBE at the Jκ1, Jκ2, and Jκ5 segments, as well as at Vκ gene segments (Fig. 4 A). In each instance, the data shown is representative of that obtained from at least three independent cell sorting experiments. The identities of the various SBE and CBE fragments have been confirmed by DNA sequence analysis (Schlissel, M.S., manuscript in preparation and 27).


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) CBE at the Igκ locus in B cell development. Purified DNA from sorted subpopulations of B cells from wild-type Balb/c mice was subjected to LMPCR to detect CBE associated with Jκ1, Jκ2, and Jκ5 and most Vκ 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 CBE PCR products were  confirmed by DNA sequence analysis (data not shown). Labeled products were visualized with a PhosphorImager. 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.  (B) Diagram of possible mechanisms for secondary light chain gene rearrangements in immature (IgM+IgD−/low) B cells. (Left) replacement of the original  rearrangement (light hatched) by joining of an upstream Vκ to a downstream Jκ segment on the same chromosome (secondary rearrangement dark hatched).  (Right) direct rearrangement of a Vκ to a Jκ segment at the locus allelic to the original rearrangement.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196138&req=5

Figure 4: (A) CBE at the Igκ locus in B cell development. Purified DNA from sorted subpopulations of B cells from wild-type Balb/c mice was subjected to LMPCR to detect CBE associated with Jκ1, Jκ2, and Jκ5 and most Vκ 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 CBE PCR products were confirmed by DNA sequence analysis (data not shown). Labeled products were visualized with a PhosphorImager. 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. (B) Diagram of possible mechanisms for secondary light chain gene rearrangements in immature (IgM+IgD−/low) B cells. (Left) replacement of the original rearrangement (light hatched) by joining of an upstream Vκ to a downstream Jκ segment on the same chromosome (secondary rearrangement dark hatched). (Right) direct rearrangement of a Vκ to a Jκ segment at the locus allelic to the original rearrangement.
Mentions: DNA from equal numbers of cells was purified, treated with T4 DNA polymerase to blunt any overhanging CBE, and subjected to ligation with a double stranded linker capable of ligating in only one orientation. Linker-ligated DNA was then used for amplification by PCR using a linker-specific primer and a pair of nested locus-specific primers (Fig. 3 A). Control amplification of a non-rearranging locus showed that all samples contained similar amounts of amplifiable DNA. Because heavy chain gene rearrangements occur almost exclusively by deletion, using the RSS 5′ of DH genes for V-to-DJH joining (38), SBE upstream of DH genes represent intermediates in the VHto-DJH rearrangement step. We focused on SBE upstream of DFL16.1 because it is the most frequently used of the DH segments (40). At the κ locus we measured SBE upstream of the most frequently used Jκ segments, Jκ1 and Jκ2, as indicative of Vκ-to-Jκ rearrangement (Fig. 3 B). To distinguish between ongoing rearrangement and the persistence of unrepaired SBE in non-cycling cells at later stages of development, we also assayed for CBE at the Jκ1, Jκ2, and Jκ5 segments, as well as at Vκ gene segments (Fig. 4 A). In each instance, the data shown is representative of that obtained from at least three independent cell sorting experiments. The identities of the various SBE and CBE fragments have been confirmed by DNA sequence analysis (Schlissel, M.S., manuscript in preparation and 27).

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