Abstract
The immunoglobulin heavy chain (IGH) gene loci are subject to specific recombination events during B-cell differentiation including somatic hypermutation and class switch recombination which mark the end of immunoglobulin gene maturation in germinal centers of secondary lymph nodes. These two events rely on the activity of activation-induced cytidine deaminase (AID) which requires DNA double strand breaks be created, a potential danger to the cell. Applying 3D-fluorescence in situ hybridization coupled with immunofluorescence staining to a previously described experimental system recapitulating normal B-cell differentiation ex vivo, we have kinetically analyzed the radial positioning of the two IGH gene loci as well as their proximity with the nucleolus, heterochromatin and γH2AX foci. Our observations are consistent with the proposal that these IGH gene rearrangements take place in a specific perinucleolar “recombination compartment” where AID could be sequestered thus limiting the extent of its potentially deleterious off-target effects.
Highlights
For a functional immunoglobulin to be synthesized in a mature B lymphocyte, one of its two immunoglobulin heavy chain (IGH) genes must first be rearranged during the so-called V(D)J recombination process which takes place in bone-marrow B-cells
At each of these three steps of IGH gene rearrangement, DNA double strand breaks (DSBs) are created through enzymatic activities www.impactjournals.com/oncotarget performed by the RAG1/2 complex in bone marrow, and the result of activation-induced cytidine deaminase (AID) in lymph nodes [3]
The radial distribution of the human IGH gene loci was studied at the various steps of B-cell differentiation recapitulated in vitro in the Fest system. 3D-fluorescence in situ hybridization (3D-FISH) images were produced and computer analyzed as previously described [24]
Summary
For a functional immunoglobulin to be synthesized in a mature B lymphocyte, one of its two immunoglobulin heavy chain (IGH) genes must first be rearranged during the so-called V(D)J recombination process which takes place in bone-marrow B-cells. Fine tuning to the immunogen relies on somatic hypermutation (SHM) events occurring within the variable part of the IGH gene (reviewed in [1]) This is followed by a last intragenic recombination called class-switch recombination (CSR) which determines the final isotype of the antibody eventually produced in the mature plasma cell [2]. At each of these three steps of IGH gene rearrangement, DNA double strand breaks (DSBs) are created through enzymatic activities www.impactjournals.com/oncotarget performed by the RAG1/2 complex in bone marrow, and the result of activation-induced cytidine deaminase (AID) in lymph nodes [3]. Changes in gene activity are expected to occur concomitantly with a relocalization to specific nuclear compartments
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