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Initiation of V(D)J recombination by Dbeta-associated recombination signal sequences: a critical control point in TCRbeta gene assembly.

Franchini DM, Benoukraf T, Jaeger S, Ferrier P, Payet-Bornet D - PLoS ONE (2009)

Bottom Line: However the B12/23 restriction does not explain the order of TCRbeta assembly for which the regulation remains an unresolved issue.Herein, we probed RSS nicks at the TCRbeta locus and found that nicks were only detectable at Dbeta-associated RSSs.Altogether, our results provide the molecular explanation to the B12/23 constraint and also uncover a 'Dbeta1 23RSS-mediated' restriction operating beyond chromatin accessibility, which directs Dbeta1 ordered rearrangements.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie de Marseille-Luminy, Université Aix Marseille, Marseille, France.

ABSTRACT
T cell receptor (TCR) beta gene assembly by V(D)J recombination proceeds via successive Dbeta-to-Jbeta and Vbeta-to-DJbeta rearrangements. This two-step process is enforced by a constraint, termed beyond (B)12/23, which prohibits direct Vbeta-to-Jbeta rearrangements. However the B12/23 restriction does not explain the order of TCRbeta assembly for which the regulation remains an unresolved issue. The initiation of V(D)J recombination consists of the introduction of single-strand DNA nicks at recombination signal sequences (RSSs) containing a 12 base-pairs spacer. An RSS containing a 23 base-pairs spacer is then captured to form a 12/23 RSSs synapse leading to coupled DNA cleavage. Herein, we probed RSS nicks at the TCRbeta locus and found that nicks were only detectable at Dbeta-associated RSSs. This pattern implies that Dbeta 12RSS and, unexpectedly, Dbeta 23RSS initiate V(D)J recombination and capture their respective Vbeta or Jbeta RSS partner. Using both in vitro and in vivo assays, we further demonstrate that the Dbeta1 23RSS impedes cleavage at the adjacent Dbeta1 12RSS and consequently Vbeta-to-Dbeta1 rearrangement first requires the Dbeta1 23RSS excision. Altogether, our results provide the molecular explanation to the B12/23 constraint and also uncover a 'Dbeta1 23RSS-mediated' restriction operating beyond chromatin accessibility, which directs Dbeta1 ordered rearrangements.

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RSS nicks at the TCRβ locus in mouse developing T cells.(A) Strategy to detect RSS nicks in vivo using oligo-capture as described by [9]. Vertical and horizontal arrows schematize, respectively, sites for restriction enzyme digestion and primers for PCR amplification. The bar schematizes the hybridization probe used for Southern blot analysis. (B) Schematic view of the TCRβ regions analyzed in this study (not drawn to scale); 12- and 23RSSs are figured by black and white triangles, respectively; the single strand nick positions are indicated by vertical arrows. The locations of the PCR primers and hybridization probes are shown; H (HindIII); G (BglII); Ss (SstI); S (SphI); RV (EcoRV); RI (EcoRI). (C, D) Autoradiographs of Southern blots of oligo-captured DNAs. Total genomic DNA from WT, RAG1−/− or Eβ−/− DN thymocytes was investigated for single strand nicks at TCRβ RSSs or Cβ2 sequences (used as a negative control). Nicks at Vβ4 and Vβ16 genes were analyzed together; Vβ8 and Vβ5 corresponded to RSSs from three (Vβ8.1, Vβ8.2 and Vβ8.3) and two (Vβ5.1 and Vβ5.2) genes, respectively; single-strand nicks at Dβ2 12- and 23RSSs were analyzed conjointly as these two RSSs possess identical heptamers. Jβ1 and Jβ2 corresponded to all functional Jβ1 and Jβ2 12RSSs, respectively. They were analyzed in one single round using a mixture of specific heptamers followed by PCR amplification of a genomic fragment located at the 3′ end of the Jβ1 (or Jβ2) cluster. PCR reactions were carried out using increasing amounts of template DNA from the bead release (0.5, 1 and 2% of captured DNA) or the flow through (10, 25 and 50 ng of non-captured DNA). Additional controls used 2% of captured (C) and 50 ng of non captured (NC) fractions from genomic DNA treated in parallel except that the biotinylated oligonucleotide was omitted (No 7mer). Estimation of the amount of captured DNA. Assuming that the amount of genomic DNA is 6 pg per cell, the cellular equivalent of 10 ng of genomic DNA is 1650 cells or 3300 alleles. For the less efficient Dβ RSS (5′Dβ1 12RSS) the intensity of the band (when 2% of captured DNA is analyzed) is tenfold lower than the band of non-captured DNA (10 ng of DNA analyzed). Hence, for 2% of captured 5′Dβ1, we estimated that 330 Dβ1 alleles were amplified and that the total amount of captured 5′Dβ1 DNA is around 16500 Dβ1 alleles. We observed that the signal is well detected when approximately 80 copies were analyzed (0.5% of 5′Dβ1 captured DNA). Conversely, nicked Vβ and Jβ RSSs were not detected (even with an input of 10% of captured DNA, not shown), suggesting that there is less than 80 copies of Vβ or Jβ DNA in the PCR tube. If we considered that the efficiency of the oligocapture assay is similar for all DNA targets and that only the amount of nicked DNA varies, we estimated that the amount of nicked Vβ or Jβ RSSs is at least twentyfold lower than the amount of nicked Dβ.
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pone-0004575-g002: RSS nicks at the TCRβ locus in mouse developing T cells.(A) Strategy to detect RSS nicks in vivo using oligo-capture as described by [9]. Vertical and horizontal arrows schematize, respectively, sites for restriction enzyme digestion and primers for PCR amplification. The bar schematizes the hybridization probe used for Southern blot analysis. (B) Schematic view of the TCRβ regions analyzed in this study (not drawn to scale); 12- and 23RSSs are figured by black and white triangles, respectively; the single strand nick positions are indicated by vertical arrows. The locations of the PCR primers and hybridization probes are shown; H (HindIII); G (BglII); Ss (SstI); S (SphI); RV (EcoRV); RI (EcoRI). (C, D) Autoradiographs of Southern blots of oligo-captured DNAs. Total genomic DNA from WT, RAG1−/− or Eβ−/− DN thymocytes was investigated for single strand nicks at TCRβ RSSs or Cβ2 sequences (used as a negative control). Nicks at Vβ4 and Vβ16 genes were analyzed together; Vβ8 and Vβ5 corresponded to RSSs from three (Vβ8.1, Vβ8.2 and Vβ8.3) and two (Vβ5.1 and Vβ5.2) genes, respectively; single-strand nicks at Dβ2 12- and 23RSSs were analyzed conjointly as these two RSSs possess identical heptamers. Jβ1 and Jβ2 corresponded to all functional Jβ1 and Jβ2 12RSSs, respectively. They were analyzed in one single round using a mixture of specific heptamers followed by PCR amplification of a genomic fragment located at the 3′ end of the Jβ1 (or Jβ2) cluster. PCR reactions were carried out using increasing amounts of template DNA from the bead release (0.5, 1 and 2% of captured DNA) or the flow through (10, 25 and 50 ng of non-captured DNA). Additional controls used 2% of captured (C) and 50 ng of non captured (NC) fractions from genomic DNA treated in parallel except that the biotinylated oligonucleotide was omitted (No 7mer). Estimation of the amount of captured DNA. Assuming that the amount of genomic DNA is 6 pg per cell, the cellular equivalent of 10 ng of genomic DNA is 1650 cells or 3300 alleles. For the less efficient Dβ RSS (5′Dβ1 12RSS) the intensity of the band (when 2% of captured DNA is analyzed) is tenfold lower than the band of non-captured DNA (10 ng of DNA analyzed). Hence, for 2% of captured 5′Dβ1, we estimated that 330 Dβ1 alleles were amplified and that the total amount of captured 5′Dβ1 DNA is around 16500 Dβ1 alleles. We observed that the signal is well detected when approximately 80 copies were analyzed (0.5% of 5′Dβ1 captured DNA). Conversely, nicked Vβ and Jβ RSSs were not detected (even with an input of 10% of captured DNA, not shown), suggesting that there is less than 80 copies of Vβ or Jβ DNA in the PCR tube. If we considered that the efficiency of the oligocapture assay is similar for all DNA targets and that only the amount of nicked DNA varies, we estimated that the amount of nicked Vβ or Jβ RSSs is at least twentyfold lower than the amount of nicked Dβ.

Mentions: The oligo-capture assay, initially described by Curry et al. [9] (Figure 2A), uncovers RAG1/2-mediated nicks generated at a given RSS site(s) in the genome. When applied to the analysis of nicking profiles within the Igκ, IgH and TCRα loci from RAG1/2-expressing cells, this methodology provided evidence that 12RSSs represent initial nicking targets, nucleating synaptic complex formation and the capture of a 23RSS partner [9].


Initiation of V(D)J recombination by Dbeta-associated recombination signal sequences: a critical control point in TCRbeta gene assembly.

Franchini DM, Benoukraf T, Jaeger S, Ferrier P, Payet-Bornet D - PLoS ONE (2009)

RSS nicks at the TCRβ locus in mouse developing T cells.(A) Strategy to detect RSS nicks in vivo using oligo-capture as described by [9]. Vertical and horizontal arrows schematize, respectively, sites for restriction enzyme digestion and primers for PCR amplification. The bar schematizes the hybridization probe used for Southern blot analysis. (B) Schematic view of the TCRβ regions analyzed in this study (not drawn to scale); 12- and 23RSSs are figured by black and white triangles, respectively; the single strand nick positions are indicated by vertical arrows. The locations of the PCR primers and hybridization probes are shown; H (HindIII); G (BglII); Ss (SstI); S (SphI); RV (EcoRV); RI (EcoRI). (C, D) Autoradiographs of Southern blots of oligo-captured DNAs. Total genomic DNA from WT, RAG1−/− or Eβ−/− DN thymocytes was investigated for single strand nicks at TCRβ RSSs or Cβ2 sequences (used as a negative control). Nicks at Vβ4 and Vβ16 genes were analyzed together; Vβ8 and Vβ5 corresponded to RSSs from three (Vβ8.1, Vβ8.2 and Vβ8.3) and two (Vβ5.1 and Vβ5.2) genes, respectively; single-strand nicks at Dβ2 12- and 23RSSs were analyzed conjointly as these two RSSs possess identical heptamers. Jβ1 and Jβ2 corresponded to all functional Jβ1 and Jβ2 12RSSs, respectively. They were analyzed in one single round using a mixture of specific heptamers followed by PCR amplification of a genomic fragment located at the 3′ end of the Jβ1 (or Jβ2) cluster. PCR reactions were carried out using increasing amounts of template DNA from the bead release (0.5, 1 and 2% of captured DNA) or the flow through (10, 25 and 50 ng of non-captured DNA). Additional controls used 2% of captured (C) and 50 ng of non captured (NC) fractions from genomic DNA treated in parallel except that the biotinylated oligonucleotide was omitted (No 7mer). Estimation of the amount of captured DNA. Assuming that the amount of genomic DNA is 6 pg per cell, the cellular equivalent of 10 ng of genomic DNA is 1650 cells or 3300 alleles. For the less efficient Dβ RSS (5′Dβ1 12RSS) the intensity of the band (when 2% of captured DNA is analyzed) is tenfold lower than the band of non-captured DNA (10 ng of DNA analyzed). Hence, for 2% of captured 5′Dβ1, we estimated that 330 Dβ1 alleles were amplified and that the total amount of captured 5′Dβ1 DNA is around 16500 Dβ1 alleles. We observed that the signal is well detected when approximately 80 copies were analyzed (0.5% of 5′Dβ1 captured DNA). Conversely, nicked Vβ and Jβ RSSs were not detected (even with an input of 10% of captured DNA, not shown), suggesting that there is less than 80 copies of Vβ or Jβ DNA in the PCR tube. If we considered that the efficiency of the oligocapture assay is similar for all DNA targets and that only the amount of nicked DNA varies, we estimated that the amount of nicked Vβ or Jβ RSSs is at least twentyfold lower than the amount of nicked Dβ.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2642999&req=5

pone-0004575-g002: RSS nicks at the TCRβ locus in mouse developing T cells.(A) Strategy to detect RSS nicks in vivo using oligo-capture as described by [9]. Vertical and horizontal arrows schematize, respectively, sites for restriction enzyme digestion and primers for PCR amplification. The bar schematizes the hybridization probe used for Southern blot analysis. (B) Schematic view of the TCRβ regions analyzed in this study (not drawn to scale); 12- and 23RSSs are figured by black and white triangles, respectively; the single strand nick positions are indicated by vertical arrows. The locations of the PCR primers and hybridization probes are shown; H (HindIII); G (BglII); Ss (SstI); S (SphI); RV (EcoRV); RI (EcoRI). (C, D) Autoradiographs of Southern blots of oligo-captured DNAs. Total genomic DNA from WT, RAG1−/− or Eβ−/− DN thymocytes was investigated for single strand nicks at TCRβ RSSs or Cβ2 sequences (used as a negative control). Nicks at Vβ4 and Vβ16 genes were analyzed together; Vβ8 and Vβ5 corresponded to RSSs from three (Vβ8.1, Vβ8.2 and Vβ8.3) and two (Vβ5.1 and Vβ5.2) genes, respectively; single-strand nicks at Dβ2 12- and 23RSSs were analyzed conjointly as these two RSSs possess identical heptamers. Jβ1 and Jβ2 corresponded to all functional Jβ1 and Jβ2 12RSSs, respectively. They were analyzed in one single round using a mixture of specific heptamers followed by PCR amplification of a genomic fragment located at the 3′ end of the Jβ1 (or Jβ2) cluster. PCR reactions were carried out using increasing amounts of template DNA from the bead release (0.5, 1 and 2% of captured DNA) or the flow through (10, 25 and 50 ng of non-captured DNA). Additional controls used 2% of captured (C) and 50 ng of non captured (NC) fractions from genomic DNA treated in parallel except that the biotinylated oligonucleotide was omitted (No 7mer). Estimation of the amount of captured DNA. Assuming that the amount of genomic DNA is 6 pg per cell, the cellular equivalent of 10 ng of genomic DNA is 1650 cells or 3300 alleles. For the less efficient Dβ RSS (5′Dβ1 12RSS) the intensity of the band (when 2% of captured DNA is analyzed) is tenfold lower than the band of non-captured DNA (10 ng of DNA analyzed). Hence, for 2% of captured 5′Dβ1, we estimated that 330 Dβ1 alleles were amplified and that the total amount of captured 5′Dβ1 DNA is around 16500 Dβ1 alleles. We observed that the signal is well detected when approximately 80 copies were analyzed (0.5% of 5′Dβ1 captured DNA). Conversely, nicked Vβ and Jβ RSSs were not detected (even with an input of 10% of captured DNA, not shown), suggesting that there is less than 80 copies of Vβ or Jβ DNA in the PCR tube. If we considered that the efficiency of the oligocapture assay is similar for all DNA targets and that only the amount of nicked DNA varies, we estimated that the amount of nicked Vβ or Jβ RSSs is at least twentyfold lower than the amount of nicked Dβ.
Mentions: The oligo-capture assay, initially described by Curry et al. [9] (Figure 2A), uncovers RAG1/2-mediated nicks generated at a given RSS site(s) in the genome. When applied to the analysis of nicking profiles within the Igκ, IgH and TCRα loci from RAG1/2-expressing cells, this methodology provided evidence that 12RSSs represent initial nicking targets, nucleating synaptic complex formation and the capture of a 23RSS partner [9].

Bottom Line: However the B12/23 restriction does not explain the order of TCRbeta assembly for which the regulation remains an unresolved issue.Herein, we probed RSS nicks at the TCRbeta locus and found that nicks were only detectable at Dbeta-associated RSSs.Altogether, our results provide the molecular explanation to the B12/23 constraint and also uncover a 'Dbeta1 23RSS-mediated' restriction operating beyond chromatin accessibility, which directs Dbeta1 ordered rearrangements.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie de Marseille-Luminy, Université Aix Marseille, Marseille, France.

ABSTRACT
T cell receptor (TCR) beta gene assembly by V(D)J recombination proceeds via successive Dbeta-to-Jbeta and Vbeta-to-DJbeta rearrangements. This two-step process is enforced by a constraint, termed beyond (B)12/23, which prohibits direct Vbeta-to-Jbeta rearrangements. However the B12/23 restriction does not explain the order of TCRbeta assembly for which the regulation remains an unresolved issue. The initiation of V(D)J recombination consists of the introduction of single-strand DNA nicks at recombination signal sequences (RSSs) containing a 12 base-pairs spacer. An RSS containing a 23 base-pairs spacer is then captured to form a 12/23 RSSs synapse leading to coupled DNA cleavage. Herein, we probed RSS nicks at the TCRbeta locus and found that nicks were only detectable at Dbeta-associated RSSs. This pattern implies that Dbeta 12RSS and, unexpectedly, Dbeta 23RSS initiate V(D)J recombination and capture their respective Vbeta or Jbeta RSS partner. Using both in vitro and in vivo assays, we further demonstrate that the Dbeta1 23RSS impedes cleavage at the adjacent Dbeta1 12RSS and consequently Vbeta-to-Dbeta1 rearrangement first requires the Dbeta1 23RSS excision. Altogether, our results provide the molecular explanation to the B12/23 constraint and also uncover a 'Dbeta1 23RSS-mediated' restriction operating beyond chromatin accessibility, which directs Dbeta1 ordered rearrangements.

Show MeSH