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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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The Dβ1 23RSS impairs RAG1/2-mediated cleavages at the adjacent Dβ1 12RSS in vitro.(A) RAG1/2-mediated nicking assays using substrates comprised of the Dβ1 gene segment flanked by various combinations of 5′ and/or 3′ RSSs. The various Dβ-containing fragments were radio-labeled at the 5′ ends and incubated for 5 min without (−) or with (+) the RAG1/2 extract. (B) RAG1/2-mediated coupled cleavage assays of the substrates illustrated on the left. Depending on the substrate, Southern blot analysis used probes A or B, as indicated. (A and B) 12- and 23RSSs are depicted as black and white triangles respectively. Dβ1 RSSs are highlighted by a dot within the triangle. All results shown are representative of at least three separate experiments.
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pone-0004575-g005: The Dβ1 23RSS impairs RAG1/2-mediated cleavages at the adjacent Dβ1 12RSS in vitro.(A) RAG1/2-mediated nicking assays using substrates comprised of the Dβ1 gene segment flanked by various combinations of 5′ and/or 3′ RSSs. The various Dβ-containing fragments were radio-labeled at the 5′ ends and incubated for 5 min without (−) or with (+) the RAG1/2 extract. (B) RAG1/2-mediated coupled cleavage assays of the substrates illustrated on the left. Depending on the substrate, Southern blot analysis used probes A or B, as indicated. (A and B) 12- and 23RSSs are depicted as black and white triangles respectively. Dβ1 RSSs are highlighted by a dot within the triangle. All results shown are representative of at least three separate experiments.

Mentions: In vivo, in the context of an intact chromatin structure, we showed that nicking of the Dβ1 12RSS (and thus initiation of Vβ-to-Dβ1 rearrangement) requires the previous elimination of the Dβ1 23RSS. To test if the inhibition of RAG1/2 cleavage activity on the Dβ1 12RSS is mediated by the neighboring Dβ1 23RSS and not by the chromatin structure, we performed in vitro cleavage assays. We first carried out in vitro nicking assays using Dβ-based substrates. As previously shown, nicking at a single RSS can occur in presence of Mg2+ ions in the buffer [33]. Substrates containing Dβ1 coding sequence flanked by either the Dβ1 12- or 23RSS (5′Dβ1 and 3′Dβ1, respectively) were cleaved in the presence of the RAG1/2 extract to produce the corresponding nicking product (gels 1 and 2, Figure 5A). However, a substrate containing the Dβ1 coding sequence flanked by both RSS mostly produced the 23RSS-derived fragment (gel 3) indicating preferential nicking at the Dβ1 23RSS. We observed no such bias towards the 23RSS when using a modified substrate (D1V14), in which the Dβ1 23RSS is replaced by the Vβ14 23RSS (gel 4). On the contrary, preferential cleavage fell on the 12RSS. These data therefore suggest a regulatory function unique to the Dβ1 23RSS which, in the germline situation, might anchor RAG1/2 catalytic activity at the expense of the neighboring Dβ1 12RSS.


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)

The Dβ1 23RSS impairs RAG1/2-mediated cleavages at the adjacent Dβ1 12RSS in vitro.(A) RAG1/2-mediated nicking assays using substrates comprised of the Dβ1 gene segment flanked by various combinations of 5′ and/or 3′ RSSs. The various Dβ-containing fragments were radio-labeled at the 5′ ends and incubated for 5 min without (−) or with (+) the RAG1/2 extract. (B) RAG1/2-mediated coupled cleavage assays of the substrates illustrated on the left. Depending on the substrate, Southern blot analysis used probes A or B, as indicated. (A and B) 12- and 23RSSs are depicted as black and white triangles respectively. Dβ1 RSSs are highlighted by a dot within the triangle. All results shown are representative of at least three separate experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004575-g005: The Dβ1 23RSS impairs RAG1/2-mediated cleavages at the adjacent Dβ1 12RSS in vitro.(A) RAG1/2-mediated nicking assays using substrates comprised of the Dβ1 gene segment flanked by various combinations of 5′ and/or 3′ RSSs. The various Dβ-containing fragments were radio-labeled at the 5′ ends and incubated for 5 min without (−) or with (+) the RAG1/2 extract. (B) RAG1/2-mediated coupled cleavage assays of the substrates illustrated on the left. Depending on the substrate, Southern blot analysis used probes A or B, as indicated. (A and B) 12- and 23RSSs are depicted as black and white triangles respectively. Dβ1 RSSs are highlighted by a dot within the triangle. All results shown are representative of at least three separate experiments.
Mentions: In vivo, in the context of an intact chromatin structure, we showed that nicking of the Dβ1 12RSS (and thus initiation of Vβ-to-Dβ1 rearrangement) requires the previous elimination of the Dβ1 23RSS. To test if the inhibition of RAG1/2 cleavage activity on the Dβ1 12RSS is mediated by the neighboring Dβ1 23RSS and not by the chromatin structure, we performed in vitro cleavage assays. We first carried out in vitro nicking assays using Dβ-based substrates. As previously shown, nicking at a single RSS can occur in presence of Mg2+ ions in the buffer [33]. Substrates containing Dβ1 coding sequence flanked by either the Dβ1 12- or 23RSS (5′Dβ1 and 3′Dβ1, respectively) were cleaved in the presence of the RAG1/2 extract to produce the corresponding nicking product (gels 1 and 2, Figure 5A). However, a substrate containing the Dβ1 coding sequence flanked by both RSS mostly produced the 23RSS-derived fragment (gel 3) indicating preferential nicking at the Dβ1 23RSS. We observed no such bias towards the 23RSS when using a modified substrate (D1V14), in which the Dβ1 23RSS is replaced by the Vβ14 23RSS (gel 4). On the contrary, preferential cleavage fell on the 12RSS. These data therefore suggest a regulatory function unique to the Dβ1 23RSS which, in the germline situation, might anchor RAG1/2 catalytic activity at the expense of the neighboring Dβ1 12RSS.

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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