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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: 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.

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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Related in: MedlinePlus

Southern blot analysis and quantitation of PCR amplified Vκ recombination products. (A) A representative blot for the amplification of the VκI family is shown. Aliquots were taken after 28, 31, and 34 cycles of amplification. The top panel shows samples from cells transfected with E47, EBF, or with RAG1 and RAG2 (RR) alone compared with mock-transfected BOSC23 cells. The bottom panel shows DNA from cells transfected with each transcription factor together with the RAG proteins. The bottom panel also shows amplification obtained from 10 fg of a plasmid control sample used for quantitation. The equality of genomic DNA used in each sample was confirmed by amplification of the same genomic DNA samples with primers for the ubiquitin-conjugating enzyme, shown under each lane of the blot, taken at 25, 28, and 31 cycles. (B) Levels of induced Vκ–Jκ rearrangements. Hybridization signals from PCR products in the linear amplification range were quantitated by PhosphorImager and normalized to the plasmid standards. Samples from cells that had only been transfected with the transcription factors alone did not result in measurable PCR products and were excluded from quantitation. Normalized values are expressed as the number of recombination events detected per 100 ng of transfected BOSC genomic DNA. The bar graph shows the average ±SEM of the normalized signals. The numbers under each family represent the number of recombining genes in the proximal half of the Vκ locus seen in large databases. The second and third row of numbers represents nonproductive (estimates of recombination frequency) and productive (expressed repertoire) sequences in vivo (references 28 and 30).
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Figure 1: Southern blot analysis and quantitation of PCR amplified Vκ recombination products. (A) A representative blot for the amplification of the VκI family is shown. Aliquots were taken after 28, 31, and 34 cycles of amplification. The top panel shows samples from cells transfected with E47, EBF, or with RAG1 and RAG2 (RR) alone compared with mock-transfected BOSC23 cells. The bottom panel shows DNA from cells transfected with each transcription factor together with the RAG proteins. The bottom panel also shows amplification obtained from 10 fg of a plasmid control sample used for quantitation. The equality of genomic DNA used in each sample was confirmed by amplification of the same genomic DNA samples with primers for the ubiquitin-conjugating enzyme, shown under each lane of the blot, taken at 25, 28, and 31 cycles. (B) Levels of induced Vκ–Jκ rearrangements. Hybridization signals from PCR products in the linear amplification range were quantitated by PhosphorImager and normalized to the plasmid standards. Samples from cells that had only been transfected with the transcription factors alone did not result in measurable PCR products and were excluded from quantitation. Normalized values are expressed as the number of recombination events detected per 100 ng of transfected BOSC genomic DNA. The bar graph shows the average ±SEM of the normalized signals. The numbers under each family represent the number of recombining genes in the proximal half of the Vκ locus seen in large databases. The second and third row of numbers represents nonproductive (estimates of recombination frequency) and productive (expressed repertoire) sequences in vivo (references 28 and 30).

Mentions: The human Igκ locus contains 76 Vκ and 5 Jκ gene segments located in a distal and a proximal region, carrying 36 and 40 genes respectively, with 800 kb of intervening sequence between the two halves of the locus 21. 4 Vκ families are used in the peripheral repertoire, VκI through VκIV. Importantly for our study, the VκI, II, and III genes are interspersed throughout the locus thus allowing us to investigate if accessibility is uniform throughout the locus. Therefore, we asked whether the various Vκ families would be induced to rearrange in a similar frequency upon ectopic expression of E2A or EBF. The embryonic kidney epithelial cell line, BOSC23, was transiently transfected with expression vectors encoding E12, E47, or EBF, either alone or in combination with expression vectors encoding the recombinase enzymes, RAG1 and RAG2. DNA from transfected cells was isolated 3 d later, and amplified with Vκ family-specific 5′ primers and a consensus Jκ 3′ primer. PCR products were blotted, probed with Vκ family-specific probes, and were quantitated using a PhosphorImager. To estimate the number of recombination events induced in each transfected sample, control PCR reactions were performed on known quantities of plasmids containing sequenced Vκ–Jκ fragments. To assure the linearity of amplification during the PCR, induced rearrangements for Vκ families as well as the control plasmids were analyzed between 27 and 37 cycles and only the data points in the linear range were used for quantitation. Fig. 1 A shows a representative blot for VκI–Jκ recombination. Cells transfected with expression constructs for either transcription factor in the absence of the RAG proteins did not show any recombination events. However, cells that had only been reconstituted with the RAG proteins alone in the absence of any transcription factor did result in very low levels of detectable VκI recombination in some transfections, while in other RAG-only transfectants, VκI rearrangements were completely absent (Fig. 1 A). The number of recombination events detected per 100 ng of BOSC genomic DNA was then calculated. Fig. 1 B represents the calculated recombination events determined for the members of the Vκ families. Fig. 1 B also indicates the number of rearranging genes in the proximal locus observed for each family. Since genes in the distal locus (located 1.4–1.9 Mb from the Jκ cluster) seldom rearrange, we did not include them in the count of rearranging genes 2830. The number of unique out-of-frame sequences identified in PBLs are indicated, representing the nonselected repertoire of individual Vκ families in vivo 2831.


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)

Southern blot analysis and quantitation of PCR amplified Vκ recombination products. (A) A representative blot for the amplification of the VκI family is shown. Aliquots were taken after 28, 31, and 34 cycles of amplification. The top panel shows samples from cells transfected with E47, EBF, or with RAG1 and RAG2 (RR) alone compared with mock-transfected BOSC23 cells. The bottom panel shows DNA from cells transfected with each transcription factor together with the RAG proteins. The bottom panel also shows amplification obtained from 10 fg of a plasmid control sample used for quantitation. The equality of genomic DNA used in each sample was confirmed by amplification of the same genomic DNA samples with primers for the ubiquitin-conjugating enzyme, shown under each lane of the blot, taken at 25, 28, and 31 cycles. (B) Levels of induced Vκ–Jκ rearrangements. Hybridization signals from PCR products in the linear amplification range were quantitated by PhosphorImager and normalized to the plasmid standards. Samples from cells that had only been transfected with the transcription factors alone did not result in measurable PCR products and were excluded from quantitation. Normalized values are expressed as the number of recombination events detected per 100 ng of transfected BOSC genomic DNA. The bar graph shows the average ±SEM of the normalized signals. The numbers under each family represent the number of recombining genes in the proximal half of the Vκ locus seen in large databases. The second and third row of numbers represents nonproductive (estimates of recombination frequency) and productive (expressed repertoire) sequences in vivo (references 28 and 30).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195934&req=5

Figure 1: Southern blot analysis and quantitation of PCR amplified Vκ recombination products. (A) A representative blot for the amplification of the VκI family is shown. Aliquots were taken after 28, 31, and 34 cycles of amplification. The top panel shows samples from cells transfected with E47, EBF, or with RAG1 and RAG2 (RR) alone compared with mock-transfected BOSC23 cells. The bottom panel shows DNA from cells transfected with each transcription factor together with the RAG proteins. The bottom panel also shows amplification obtained from 10 fg of a plasmid control sample used for quantitation. The equality of genomic DNA used in each sample was confirmed by amplification of the same genomic DNA samples with primers for the ubiquitin-conjugating enzyme, shown under each lane of the blot, taken at 25, 28, and 31 cycles. (B) Levels of induced Vκ–Jκ rearrangements. Hybridization signals from PCR products in the linear amplification range were quantitated by PhosphorImager and normalized to the plasmid standards. Samples from cells that had only been transfected with the transcription factors alone did not result in measurable PCR products and were excluded from quantitation. Normalized values are expressed as the number of recombination events detected per 100 ng of transfected BOSC genomic DNA. The bar graph shows the average ±SEM of the normalized signals. The numbers under each family represent the number of recombining genes in the proximal half of the Vκ locus seen in large databases. The second and third row of numbers represents nonproductive (estimates of recombination frequency) and productive (expressed repertoire) sequences in vivo (references 28 and 30).
Mentions: The human Igκ locus contains 76 Vκ and 5 Jκ gene segments located in a distal and a proximal region, carrying 36 and 40 genes respectively, with 800 kb of intervening sequence between the two halves of the locus 21. 4 Vκ families are used in the peripheral repertoire, VκI through VκIV. Importantly for our study, the VκI, II, and III genes are interspersed throughout the locus thus allowing us to investigate if accessibility is uniform throughout the locus. Therefore, we asked whether the various Vκ families would be induced to rearrange in a similar frequency upon ectopic expression of E2A or EBF. The embryonic kidney epithelial cell line, BOSC23, was transiently transfected with expression vectors encoding E12, E47, or EBF, either alone or in combination with expression vectors encoding the recombinase enzymes, RAG1 and RAG2. DNA from transfected cells was isolated 3 d later, and amplified with Vκ family-specific 5′ primers and a consensus Jκ 3′ primer. PCR products were blotted, probed with Vκ family-specific probes, and were quantitated using a PhosphorImager. To estimate the number of recombination events induced in each transfected sample, control PCR reactions were performed on known quantities of plasmids containing sequenced Vκ–Jκ fragments. To assure the linearity of amplification during the PCR, induced rearrangements for Vκ families as well as the control plasmids were analyzed between 27 and 37 cycles and only the data points in the linear range were used for quantitation. Fig. 1 A shows a representative blot for VκI–Jκ recombination. Cells transfected with expression constructs for either transcription factor in the absence of the RAG proteins did not show any recombination events. However, cells that had only been reconstituted with the RAG proteins alone in the absence of any transcription factor did result in very low levels of detectable VκI recombination in some transfections, while in other RAG-only transfectants, VκI rearrangements were completely absent (Fig. 1 A). The number of recombination events detected per 100 ng of BOSC genomic DNA was then calculated. Fig. 1 B represents the calculated recombination events determined for the members of the Vκ families. Fig. 1 B also indicates the number of rearranging genes in the proximal locus observed for each family. Since genes in the distal locus (located 1.4–1.9 Mb from the Jκ cluster) seldom rearrange, we did not include them in the count of rearranging genes 2830. The number of unique out-of-frame sequences identified in PBLs are indicated, representing the nonselected repertoire of individual Vκ families in vivo 2831.

Bottom Line: 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.

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