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Localized gene-specific induction of accessibility to V(D)J recombination induced by E2A and early B cell factor in nonlymphoid cells.

Goebel P, Janney N, Valenzuela JR, Romanow WJ, Murre C, Feeney AJ - J. Exp. Med. (2001)

Bottom Line: However, here we show that Ig loci are not opened globally but that recombination is localized.The induced repertoire seen at 24 hours does not change significantly over time indicating the absence of many secondary rearrangements and also suggesting a direct targeting mechanism.We propose that accessibility occurs in a local manner, and that binding sites for factors facilitating accessibility are therefore likely to be associated with individual gene segments.

View Article: PubMed Central - PubMed

Affiliation: The Scripps Research Institute, Department of Immunology IMM-22, La Jolla, CA 92037, USA.

ABSTRACT
Accessibility of immunoglobulin (Ig) gene segments to V(D)J recombination is highly regulated and is normally only achieved in B cell precursors. We previously showed that ectopic expression of E2A or early B cell factor (EBF) with recombination activating gene (RAG) induces rearrangement of IgH and IgL genes in nonlymphoid cells. VkappaI genes throughout the locus were induced to rearrange after transfection with E2A, suggesting that the entire Vkappa locus was accessible. However, here we show that Ig loci are not opened globally but that recombination is localized. Gene families are interspersed in the D(H), Vkappa, and Vlambda loci, and we show that certain families and individual genes undergo high levels of recombination after ectopic expression of E2A or EBF, while other families within the same locus are not induced to rearrange. Furthermore, in some families, induction of germline transcription correlates with the level of induced recombination, while in others there is no correlation, suggesting that recombination is not simply initiated by induction of germline transcription. The induced repertoire seen at 24 hours does not change significantly over time indicating the absence of many secondary rearrangements and also suggesting a direct targeting mechanism. We propose that accessibility occurs in a local manner, and that binding sites for factors facilitating accessibility are therefore likely to be associated with individual gene segments.

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Levels of induced Vλ–Jλ and DH–JH recombination events after transient transfection Induced recombination events for the members of several Vλ and DH gene families were analyzed as outlined in the legend to Fig. 1. Hybridization signals were quantitated, normalized to plasmid standards, and are represented as recombination events per 100 ng of transfected BOSC genomic DNA. (A) Levels of induced Vλ–Jλ rearrangements. The number of rearranging genes within each family are indicated. As an estimate of the contribution of each family to the peripheral repertoire, the number of nonproductive sequences identified in PBL 34 and their contribution within a large cDNA library (reference 35) are shown. (B) Levels of induced DH3 and DH4 rearrangements. The number of rearranging genes within each family and the contribution of each family to complete VDJ rearrangements isolated from peripheral blood (reference 23) are shown.
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Figure 3: Levels of induced Vλ–Jλ and DH–JH recombination events after transient transfection Induced recombination events for the members of several Vλ and DH gene families were analyzed as outlined in the legend to Fig. 1. Hybridization signals were quantitated, normalized to plasmid standards, and are represented as recombination events per 100 ng of transfected BOSC genomic DNA. (A) Levels of induced Vλ–Jλ rearrangements. The number of rearranging genes within each family are indicated. As an estimate of the contribution of each family to the peripheral repertoire, the number of nonproductive sequences identified in PBL 34 and their contribution within a large cDNA library (reference 35) are shown. (B) Levels of induced DH3 and DH4 rearrangements. The number of rearranging genes within each family and the contribution of each family to complete VDJ rearrangements isolated from peripheral blood (reference 23) are shown.

Mentions: The Igλ locus contains ∼70 Vλ genes which are organized in three clusters all upstream of seven Jλ-Cλ pairs 22. The Jλ-proximal A-cluster carries interspersed members of the VλII and VλIII family, while genes belonging to the VλI family are found further upstream in the B cluster. In the peripheral repertoire, VλII and VλI are the predominantly used V genes 3435. Our previous study had only analyzed rearrangements using VλIII and Jλ1 segments. In this study we extended our analysis to include the VλI, VλII, and VλIII gene families, which together account for >90% of all Vλ genes expressed in the peripheral repertoire. We also used a primer detecting Jλ2 and Jλ3 since they are more frequently used in the peripheral repertoire than Jλ1. Recombination of VλIII was strongest with EBF, but low levels of rearrangement were also induced by E2A (Fig. 3 A). Induction of VλII recombination by EBF was eightfold lower compared with VλIII. The low levels of rearrangement detected for VλIII after E2A expression were confirmed by extending the PCR reactions to 40 cycles of amplification (data not shown). Although there are about twice as many VλIII genes than there are VλII genes in the locus, the VλII family contributes at a twofold higher level to the expressed peripheral repertoire (Fig. 3 A). Induced recombination in BOSC cells however is clearly dominated by the VλIII family. In addition, we analyzed VλI–Jλ rearrangements. The VλI genes contribute almost to the same extent to the peripheral repertoire as the VλII family. However, only negligible VλI rearrangement was detected in EBF transfected BOSC cells while no recombination was seen in E2A transfectants even at 40 cycles.


Localized gene-specific induction of accessibility to V(D)J recombination induced by E2A and early B cell factor in nonlymphoid cells.

Goebel P, Janney N, Valenzuela JR, Romanow WJ, Murre C, Feeney AJ - J. Exp. Med. (2001)

Levels of induced Vλ–Jλ and DH–JH recombination events after transient transfection Induced recombination events for the members of several Vλ and DH gene families were analyzed as outlined in the legend to Fig. 1. Hybridization signals were quantitated, normalized to plasmid standards, and are represented as recombination events per 100 ng of transfected BOSC genomic DNA. (A) Levels of induced Vλ–Jλ rearrangements. The number of rearranging genes within each family are indicated. As an estimate of the contribution of each family to the peripheral repertoire, the number of nonproductive sequences identified in PBL 34 and their contribution within a large cDNA library (reference 35) are shown. (B) Levels of induced DH3 and DH4 rearrangements. The number of rearranging genes within each family and the contribution of each family to complete VDJ rearrangements isolated from peripheral blood (reference 23) are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195934&req=5

Figure 3: Levels of induced Vλ–Jλ and DH–JH recombination events after transient transfection Induced recombination events for the members of several Vλ and DH gene families were analyzed as outlined in the legend to Fig. 1. Hybridization signals were quantitated, normalized to plasmid standards, and are represented as recombination events per 100 ng of transfected BOSC genomic DNA. (A) Levels of induced Vλ–Jλ rearrangements. The number of rearranging genes within each family are indicated. As an estimate of the contribution of each family to the peripheral repertoire, the number of nonproductive sequences identified in PBL 34 and their contribution within a large cDNA library (reference 35) are shown. (B) Levels of induced DH3 and DH4 rearrangements. The number of rearranging genes within each family and the contribution of each family to complete VDJ rearrangements isolated from peripheral blood (reference 23) are shown.
Mentions: The Igλ locus contains ∼70 Vλ genes which are organized in three clusters all upstream of seven Jλ-Cλ pairs 22. The Jλ-proximal A-cluster carries interspersed members of the VλII and VλIII family, while genes belonging to the VλI family are found further upstream in the B cluster. In the peripheral repertoire, VλII and VλI are the predominantly used V genes 3435. Our previous study had only analyzed rearrangements using VλIII and Jλ1 segments. In this study we extended our analysis to include the VλI, VλII, and VλIII gene families, which together account for >90% of all Vλ genes expressed in the peripheral repertoire. We also used a primer detecting Jλ2 and Jλ3 since they are more frequently used in the peripheral repertoire than Jλ1. Recombination of VλIII was strongest with EBF, but low levels of rearrangement were also induced by E2A (Fig. 3 A). Induction of VλII recombination by EBF was eightfold lower compared with VλIII. The low levels of rearrangement detected for VλIII after E2A expression were confirmed by extending the PCR reactions to 40 cycles of amplification (data not shown). Although there are about twice as many VλIII genes than there are VλII genes in the locus, the VλII family contributes at a twofold higher level to the expressed peripheral repertoire (Fig. 3 A). Induced recombination in BOSC cells however is clearly dominated by the VλIII family. In addition, we analyzed VλI–Jλ rearrangements. The VλI genes contribute almost to the same extent to the peripheral repertoire as the VλII family. However, only negligible VλI rearrangement was detected in EBF transfected BOSC cells while no recombination was seen in E2A transfectants even at 40 cycles.

Bottom Line: However, here we show that Ig loci are not opened globally but that recombination is localized.The induced repertoire seen at 24 hours does not change significantly over time indicating the absence of many secondary rearrangements and also suggesting a direct targeting mechanism.We propose that accessibility occurs in a local manner, and that binding sites for factors facilitating accessibility are therefore likely to be associated with individual gene segments.

View Article: PubMed Central - PubMed

Affiliation: The Scripps Research Institute, Department of Immunology IMM-22, La Jolla, CA 92037, USA.

ABSTRACT
Accessibility of immunoglobulin (Ig) gene segments to V(D)J recombination is highly regulated and is normally only achieved in B cell precursors. We previously showed that ectopic expression of E2A or early B cell factor (EBF) with recombination activating gene (RAG) induces rearrangement of IgH and IgL genes in nonlymphoid cells. VkappaI genes throughout the locus were induced to rearrange after transfection with E2A, suggesting that the entire Vkappa locus was accessible. However, here we show that Ig loci are not opened globally but that recombination is localized. Gene families are interspersed in the D(H), Vkappa, and Vlambda loci, and we show that certain families and individual genes undergo high levels of recombination after ectopic expression of E2A or EBF, while other families within the same locus are not induced to rearrange. Furthermore, in some families, induction of germline transcription correlates with the level of induced recombination, while in others there is no correlation, suggesting that recombination is not simply initiated by induction of germline transcription. The induced repertoire seen at 24 hours does not change significantly over time indicating the absence of many secondary rearrangements and also suggesting a direct targeting mechanism. We propose that accessibility occurs in a local manner, and that binding sites for factors facilitating accessibility are therefore likely to be associated with individual gene segments.

Show MeSH
Related in: MedlinePlus