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ABSTRACT: TCRbeta, delta and gamma chain genes are assembled and expressed in double-negative thymocytes prior to alphabeta or gammadelta T cell lineage commitment. Thus, cells committed to the alphabeta T cell lineage can possess completely assembled TCRdelta and/or TCRgamma chain genes. However, these genes are not expressed. TCRgamma chain gene expression may be silenced through the activity of a cis-acting silencer element. In the TCRalpha/delta locus, the TCRdelta genes lie between the Valpha and Jalpha gene segments, which rearrange by deletion. Moreover, Valpha to Jalpha rearrangements occur on both alleles in essentially all developing alphabeta T cells. Consequently, both TCRdelta chain genes are excised from the chromosome and placed on extrachromosomal circles in mature alphabeta T cells. It has been proposed that this excision process is important for silencing TCRdelta gene expression and permitting alphabeta T cell lineage commitment. A gene-targeting Cre-loxP strategy was used to invert a 75-kb region of the TCRalpha/delta locus encompassing all the Jalpha gene segments, generating the TCRalpha/delta(I) allele. Initial Valpha to Jalpha rearrangements on the TCRalpha/delta(I) allele occur by inversion, resulting in chromosomal retention of TCRdelta chain genes. These TCRdelta chain genes can be productively rearranged and are expressed at levels similar to TCRdelta chain genes in gammadelta T cells. However, alphabeta T cell development appears unperturbed in TCRalpha/delta(I/I) mice. Thus, excision of TCRdelta genes from the chromosome per se is not required for commitment of developing lymphocytes to the alphabeta T cell lineage.
International Immunology 04/2005; 17(3):225-32. · 3.41 Impact Factor
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ABSTRACT: Ag receptor variable region gene assembly is initiated through the formation of a synaptic complex which minimally includes the recombination-activating gene (RAG) 1/2 proteins and a pair of recombination signals (RSs) flanking the recombining gene segments. RSs are composed of conserved heptamer and nonamer sequences flanking relatively nonconserved spacers of 12 or 23 bp. RSs regulate variable region gene assembly within the context of the 12/23 rule which mandates that recombination only occurs between RSs of dissimilar spacer length. RSs can exert additional constraints on variable region gene assembly beyond imposing spacer length requirements. At a minimum this restriction, termed B12/23, is imposed on the Vbeta to DJbeta rearrangement step by the 5' Dbeta RS and is enforced at or before the DNA cleavage step of the V(D)J recombination reaction. In this study, the components of the 5' Dbeta RS required for enforcing the B12/23 rule are assessed on chromosomal substrates in vivo in the context of normal murine thymocyte development and on extrachromosomal substrates induced to undergo recombination in nonlymphoid cell lines. These analyses reveal that the integrity of the nonamer sequence as well as the highly conserved spacer nucleotides of the 5' Dbeta1 RS are critical for enforcing the B12/23 restriction. These findings have important implications for understanding the B12/23 restriction and the manner in which RS synaptic complexes are assembled in vivo.
The Journal of Immunology 01/2004; 171(12):6604-10. · 5.79 Impact Factor
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ABSTRACT: The third complementarity-determining region (CDR) of the TCR alpha and beta chains forms loops that engage amino acid residues of peptides complexed with MHC. This interaction is central to the specific discrimination of antigenic-peptide-MHC complexes by the TCR. The TCRbeta chain CDR3 loop is encoded by the Dbeta gene segment and flanking portions of the Vbeta and Jbeta gene segments. The joining of these gene segments is imprecise, leading to significant variability in the TCRbeta chain CDR3 loop length and amino acid composition. In marked contrast to other pairing antigen-receptor chains, the TCR beta and alpha chain CDR3 loop size distributions are relatively narrow and closely matched. Thus, pairing of TCR alpha and beta chains with relatively similar CDR3 loop sizes may be important for generating a functional repertoire of alpha beta TCR. Here we show that the TCRbeta chain CDR3 loop size distribution is minimally impacted by TCRbeta chain or alpha beta TCR selection during thymocyte development. Rather, this distribution is determined primarily at the level of variable-region gene assembly, and is critically dependent on unique features of the V(D)J recombination reaction that ensure Dbeta gene segment utilization.
European Journal of Immunology 07/2003; 33(6):1568-75. · 5.10 Impact Factor
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ABSTRACT: Assembly of TCRbeta variable region genes is ordered during thymocyte development with Dbeta to Jbeta rearrangement preceding Vbeta to DJbeta rearrangement. The 5'Dbeta 12-RSS is required to precisely and efficiently target Vbeta rearrangement beyond simply enforcing the 12/23 rule. By prohibiting direct Vbeta to Jbeta rearrangement, this restriction ensures Dbeta gene segment use in the assembly of essentially all TCRbeta variable region genes. In this study, we show that rearrangement of Vbeta 23-RSSs is significantly biased to the Dbeta 12-RSS over Jbeta 12-RSSs on extrachromosomal recombination substrates in nonlymphoid cells that express the recombinase-activating gene-1/2 proteins. These findings demonstrate that targeting of Vbeta to Dbeta rearrangement can be enforced by the V(D)J recombinase in the absence of lymphoid-specific factors other than the recombinase-activating gene-1/2 proteins.
The Journal of Immunology 02/2003; 170(1):5-9. · 5.79 Impact Factor
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ABSTRACT: Antigen receptor loci are composed of numerous variable (V), diversity (D), and joining (J) gene segments, each flanked by recombination signal sequences (RSSs). The V(D)J recombination reaction proceeds through RSS recognition and DNA cleavage steps making it possible for multiple DNA double strand breaks (DSBs) to be introduced at a single locus. Here we use ligation-mediated PCR to analyze DNA cleavage intermediates in thymocytes from mice with targeted RSS mutations at the endogenous TCRbeta locus. We show that DNA cleavage does not occur at individual RSSs but rather must be coordinated between RSS pairs flanking gene segments that ultimately form coding joins. Coordination of the DNA cleavage step occurs over great distances in the chromosome and favors intra- over interchromosomal recombination. Furthermore, through several restrictions imposed on the generation of both nonpaired and paired DNA DSBs, this requirement promotes antigen receptor gene integrity and genomic stability in developing lymphocytes undergoing V(D)J recombination.
Journal of Experimental Medicine 03/2002; 195(3):309-16. · 13.85 Impact Factor
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ABSTRACT: TCRβ, δ and γ chain genes are assembled and expressed in double-negative thymocytes prior to αβ or γδ T cell lineage commitment. Thus, cells committed to the αβ T cell lineage can possess completely assembled TCRδ and/or TCRγ chain genes. However, these genes are not expressed. TCRγ chain gene expression may be silenced through the activity of a cis -acting silencer element. In the TCRα/δ locus, the TCRδ genes lie between the Vα and Jα gene segments, which rearrange by deletion. Moreover, Vα to Jα rearrangements occur on both alleles in essentially all developing αβ T cells. Consequently, both TCRδ chain genes are excised from the chromosome and placed on extrachromosomal circles in mature αβ T cells. It has been proposed that this excision process is important for silencing TCRδ gene expression and permitting αβ T cell lineage commitment. A gene-targeting Cre– loxP strategy was used to invert a 75-kb region of the TCRα/δ locus encompassing all the Jα gene segments, generating the TCRα/δI allele. Initial Vα to Jα rearrangements on the TCRα/δI allele occur by inversion, resulting in chromosomal retention of TCRδ chain genes. These TCRδ chain genes can be productively rearranged and are expressed at levels similar to TCRδ chain genes in γδ T cells. However, αβ T cell development appears unperturbed in TCRα/δI/I mice. Thus, excision of TCRδ genes from the chromosome per se is not required for commitment of developing lymphocytes to the αβ T cell lineage.
