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ABSTRACT: Non-productive antigen receptor genes with frame shifts generated during the assembly of these genes are found in many mature lymphocytes. Transcripts from these genes have premature termination codons (PTCs) and could encode truncated proteins if they are not either inactivated or destroyed by nonsense-mediated decay (NMD). In mammalian cells, NMD can be activated by pathways that rely on the presence of an intron downstream of the PTC; however, NMD can also be activated by pathways that do not rely on these downstream introns, and pathways independent of NMD can inactivate PTC-containing transcripts. Here, through the generation and analysis of mice with gene-targeted modifications of the endogenous T cell receptor beta (Tcrb) locus, we demonstrate that in T cells in vivo, optimal clearance of PTC-containing Tcrb transcripts depends on the presence of an intron downstream of the PTC.
PLoS ONE 01/2011; 6(7):e21627. · 4.09 Impact Factor
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Elsa Callén,
Sam Bunting,
Ching-Yu Huang,
Michael J Difilippantonio,
Nancy Wong, Bernard Khor,
Grace Mahowald,
Michael J Kruhlak,
Thomas Ried,
Barry P Sleckman,
André Nussenzweig
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ABSTRACT: Translocations involving the T cell receptor alpha/delta (TCRalpha/delta) chain locus, which bring oncogenes in the proximity of the TCRalpha enhancer, are one of the hallmark features of human T cell malignancies from ataxia telangiectasia (AT) and non-AT patients. These lesions are frequently generated by the fusion of DNA breaks at the TCRalpha/delta locus to a disperse region centromeric of the immunoglobulin heavy chain (IgH) locus. Aberrant VDJ joining accounts for TCRalpha/delta associated DNA cleavage, but the molecular mechanism that leads to generation of the "oncogene partner" DNA break is unclear. Here we show that in ATM deficient primary mouse T cells, IgH/TCRalpha/delta fusions arise at a remarkably similar frequency as in human AT lymphocytes. Recombinase-activating gene (RAG) is responsible for both TCRalpha/delta as well as IgH associated breaks on chromosome 12 (Chr12), which are subject to varying degrees of chromosomal degradation. We suggest a new model for how oncogenic translocations can arise from two non-concerted physiological DSBs.
Cell cycle (Georgetown, Tex.) 09/2009; 8(15):2408-12. · 5.36 Impact Factor
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ABSTRACT: The second exon of lymphocyte antigen receptor genes is assembled in developing lymphocytes from component V, J and, in some cases, D gene segments through the process of V(D)J recombination. This process is initiated by an endonuclease comprised of the Rag-1 and Rag-2 proteins, collectively referred to as Rag. Rag binds to recombination signals (RSs) and catalyzes the pair-wise introduction of DNA double strand breaks (DSBs) at recombining gene segments. DNA cleavage by Rag is restricted both by intrinsic features of RSs, as well as the activity of other cis-acting elements, such as promoters and enhancers that regulate the accessibility of gene segments to Rag. In the TCRbeta locus, accessibility of the Dbeta1-Jbeta1 gene segment cluster relies on the function of an enhancer, Ebeta, and a promoter, PDbeta1. Here we demonstrate that deletion of a small genomic region containing five of the six Jbeta1 gene segments, but no known transcriptional regulatory elements, leads to a marked decrease in transcription and rearrangements involving the Dbeta1 and Jbeta1.1 gene segments. Surprisingly, point mutations in the RS of the Jbeta1.1 gene segment not only impact Rag cleavage, but also lead to diminished transcription through the Dbeta1-Jbeta1 gene segment cluster. Our findings demonstrate that cis-acting elements that regulate transcription and accessibility of the TCRbeta locus may functionally overlap with RS sequences, which are known primarily to direct Rag-mediated cleavage.
Molecular Immunology 01/2009; 46(3):321-6. · 2.90 Impact Factor
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Bernard Khor,
Andrea L Bredemeyer,
Ching-Yu Huang,
Isaiah R Turnbull,
Ryan Evans,
Leonard B Maggi,
J Michael White,
Laura M Walker,
Kay Carnes,
Rex A Hess,
Barry P Sleckman
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ABSTRACT: The PA200 proteasome activator is a broadly expressed nuclear protein. Although how PA200 normally functions is not fully understood, it has been suggested to be involved in the repair of DNA double-strand breaks (DSBs). The PA200 gene (Psme4) is composed of 45 coding exons spanning 108 kb on mouse chromosome 11. We generated a PA200 null allele (PA200(Delta)) through Cre-loxP-mediated interchromosomal recombination after targeting loxP sites at either end of the locus. PA200(Delta/Delta) mice are viable and have no obvious developmental abnormalities. Both lymphocyte development and immunoglobulin class switching, which rely on the generation and repair of DNA DSBs, are unperturbed in PA200(Delta/Delta) mice. Additionally, PA200(Delta/Delta) embryonic stem cells do not exhibit increased sensitivity to either ionizing radiation or bleomycin. Thus, PA200 is not essential for the repair of DNA DSBs generated in these settings. Notably, loss of PA200 led to a marked reduction in male, but not female, fertility. This was due to defects in spermatogenesis observed in meiotic spermatocytes and during the maturation of postmeiotic haploid spermatids. Thus, PA200 serves an important nonredundant function during spermatogenesis, suggesting that the efficient generation of male gametes has distinct protein metabolic requirements.
Molecular and Cellular Biology 05/2006; 26(8):2999-3007. · 5.53 Impact Factor
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ABSTRACT: Allelic exclusion at the TCRbeta locus mandates that gene assembly be regulated in a manner that permits feedback inhibition of further complete TCRbeta rearrangements upon pre-TCR expression. Here we show that assembly of TCRbeta chain genes from Vbeta, Dbeta and Jbeta gene segments is intra-allelically ordered, proceeding primarily through DJbeta, and not VDbeta, intermediates. This ensures that Vbeta to DJbeta rearrangement, which can be feedback inhibited, is the final step in the assembly process. A newly assembled VDJbeta rearrangement must be tested to determine if it is in-frame before Vbeta to DJbeta rearrangement is permitted on the alternate allele. This inter-allelic ordering may occur through a general inefficiency of Vbeta to DJbeta rearrangement and/or through static differences in accessibility of the two TCRbeta alleles. However, we find that within the regulatory context of allelic exclusion, Vbeta to DJbeta rearrangement proceeds to completion on both alleles. Furthermore, all possible VDJbeta rearrangements are not completed on one allele before Vbeta to DJbeta rearrangement is initiated on the alternate allele. Together, these data support a dynamic model of inter-allelic accessibility that permits the ordered and efficient assembly of complete variable region genes on both TCRbeta alleles during T cell development.
European Journal of Immunology 04/2005; 35(3):964-70. · 5.10 Impact Factor
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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: Allelic exclusion of V(beta)-to-DJ(beta) recombination depends on asynchronous rearrangement of alleles of the gene encoding T cell receptor beta in double-negative thymocytes and feedback inhibition that is maintained in double-positive thymocytes. Feedback is thought to be enforced through downregulation of V(beta) accessibility. In an attempt to override this negative regulation, we introduced the enhancer of the gene encoding T cell receptor alpha into the V(beta) gene cluster downstream of V(beta)12. In double-negative thymocytes, the introduced enhancer had no measurable effect on accessibility, but V(beta)12 rearrangement was stimulated and V(beta)12 allelic exclusion was partially subverted. In contrast, double-positive thymocytes showed increased V(beta) transcription and accessibility, but feedback inhibition of V(beta)-to-DJ(beta) recombination remained intact. Our results indicate additional regulatory constraints on V(beta)-to-DJ(beta) recombination that operate beyond the accessibility barrier.
Nature Immunology 03/2005; 6(2):189-97. · 26.01 Impact Factor
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ABSTRACT: Assembly of antigen receptor genes is regulated in several important contexts during lymphocyte development. This regulation occurs through modulation of gene segment accessibility to the V(D)J recombinase and/or at the level of the recombination reaction due, in part, to constraints imposed by recombination signal (RS) sequences. RSs are composed of conserved heptamer and nonamer sequences that flank relatively non-conserved spacer sequences of either 12 or 23 base pairs. Recombination occurs only between RSs of dissimilar spacer lengths, a restriction known as the 12/23 rule. Recently, we have shown that RSs can impose significant constraints on antigen receptor gene assembly beyond enforcing the 12/23 rule. This restriction, termed B12/23, was revealed by analysis of T-cell receptor beta (TCRbeta) locus rearrangements, where Dbeta 12RSs and not Jbeta 12RSs are capable of efficiently targeting Vbeta 23RSs' rearrangement. The B12/23 restriction occurs at or prior to the DNA-cleavage step of the V(D)J recombination reaction, relies on features of the Dbeta 12RSs and Vbeta 23RSs, and is not absolutely dependent on lymphoid-specific factors other than the recombinase-activating gene-1 (RAG-1) and RAG-2 proteins. By preserving Dbeta gene segment utilization, the B12/23 restriction is required, at a minimum, for the generation of a diverse repertoire of TCRbeta chains.
Immunological Reviews 09/2004; 200:36-43. · 11.15 Impact Factor
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ABSTRACT: Glucocorticoids, acting through the glucocorticoid receptor, potently modulate immune function and are a mainstay of therapy for treatment of inflammatory conditions, autoimmune diseases, leukemias and lymphomas. Moreover, removal of systemic glucocorticoids, by adrenalectomy in animal models or adrenal insufficiency in humans, has shown that endogenous glucocorticoid production is required for regulation of physiologic immune responses. These effects have been attributed to suppression of cytokines, although the crucial cellular and molecular targets remain unknown. In addition, considerable controversy remains as to whether glucocorticoids are required for thymocyte development. To assess the role of the glucocorticoid receptor in immune system development and function, we generated T-cell-specific glucocorticoid receptor knockout mice. Here we show that the T-cell is a critical cellular target of glucocorticoid receptor signaling, as immune activation in these mice resulted in significant mortality. This lethal activation is rescued by cyclooxygenase-2 (COX-2) inhibition but not steroid administration or cytokine neutralization. These studies indicate that glucocorticoid receptor suppression of COX-2 is crucial for curtailing lethal immune activation, and suggest new therapeutic approaches for regulation of T-cell-mediated inflammatory diseases.
Nature Medicine 11/2003; 9(10):1318-22. · 22.46 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: Assembly of TCRbeta chain variable-region genes is regulated in the context of allelic exclusion. Differential epigenetic modifications of the two TCRbeta alleles established early in embryonic development may be important for permitting allelic exclusion by ordering rearrangement of the two alleles in double-negative thymocytes. Expression of a TCRbeta chain, as part of the pre-TCR complex, activates signaling pathways that enforce allelic exclusion in double-positive thymocytes. These signaling pathways, which utilize p56(lck) and SLP-76, may be distinct from those used to promote other processes initiated by pre-TCR expression. In double-positive thymocytes allelic exclusion is enforced, in part, by changes in Vbeta gene segment accessibility promoted by cis-acting elements that may be distinct from those regulating accessibility of D/Jbeta gene segments.
Current Opinion in Immunology 05/2002; 14(2):230-4. · 9.52 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.
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[show abstract]
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ABSTRACT: The second exon of lymphocyte antigen receptor genes is assembled in developing lymphocytes from component V, J and, in some cases, D gene segments through the process of V(D)J recombination. This process is initiated by an endonuclease comprised of the Rag-1 and Rag-2 proteins, collectively referred to as Rag. Rag binds to recombination signals (RSs) and catalyzes the pair-wise introduction of DNA double strand breaks (DSBs) at recombining gene segments. DNA cleavage by Rag is restricted both by intrinsic features of RSs, as well as the activity of other cis-acting elements, such as promoters and enhancers that regulate the accessibility of gene segments to Rag. In the TCRβ locus, accessibility of the Dβ1–Jβ1 gene segment cluster relies on the function of an enhancer, Eβ, and a promoter, PDβ1. Here we demonstrate that deletion of a small genomic region containing five of the six Jβ1 gene segments, but no known transcriptional regulatory elements, leads to a marked decrease in transcription and rearrangements involving the Dβ1 and Jβ1.1 gene segments. Surprisingly, point mutations in the RS of the Jβ1.1 gene segment not only impact Rag cleavage, but also lead to diminished transcription through the Dβ1–Jβ1 gene segment cluster. Our findings demonstrate that cis-acting elements that regulate transcription and accessibility of the TCRβ locus may functionally overlap with RS sequences, which are known primarily to direct Rag-mediated cleavage.
Molecular Immunology.
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[show abstract]
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ABSTRACT: Assembly of TCRβ chain variable-region genes is regulated in the context of allelic exclusion. Differential epigenetic modifications of the two TCRβ alleles established early in embryonic development may be important for permitting allelic exclusion by ordering rearrangement of the two alleles in double-negative thymocytes. Expression of a TCRβ chain, as part of the pre-TCR complex, activates signaling pathways that enforce allelic exclusion in double-positive thymocytes. These signaling pathways, which utilize p56lck and SLP-76, may be distinct from those used to promote other processes initiated by pre-TCR expression. In double-positive thymocytes allelic exclusion is enforced, in part, by changes in Vβ gene segment accessibility promoted by cis-acting elements that may be distinct from those regulating accessibility of D/Jβ gene segments.
Current Opinion in Immunology.
