Regulation of TCR Allelic Exclusion by Gene Segment Proximity and Accessibility (original) (raw)
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Nature Immunology, 2008
Earlier studies of antigen-receptor loci implicated directed monoallelic association with pericentromeric heterochromatin in the initiation or maintenance of allelic exclusion. Here we provide evidence for a fundamentally different basis for Tcrb allelic exclusion. Using 3D Immuno-FISH we found that germline Tcrb alleles associated stochastically and at high frequency with the nuclear lamina or with peri-centromeric heterochromatin in developing thymocytes, and that such interactions inhibited V β -to-D β J β recombination prior to β-selection. Introduction of an ectopic enhancer into Tcrb reduced these interactions and impaired allelic exclusion. We propose that initial V β -to-D β J β recombination events are generally monoallelic in developing thymocytes due to frequent stochastic, rather than directed, interactions of Tcrb alleles with repressive nuclear compartments. Such interactions may be essential for Tcrb allelic exclusion.
Regulation of Tcra/Tcrd Locus Conformation during Thymocyte Development
2012
The chromatin architecture of antigen receptor loci has been hypothesized to facilitate the assembly of variable (V), diversity (D), and joining (J) gene segments during lymphocyte development. The 1.6 megabase Tcra/Tcrd locus is unique since it undergoes highly divergent Tcrd and Tcra recombination programs in CD4 − CD8 − double negative (DN) thymocytes and CD4 + CD8 + double positive (DP) thymocytes, respectively. In this dissertation, we asked whether these divergent recombination programs are supported by distinct conformational states of the Tcra/Tcrd locus by using three-dimensional fluorescence in situ hybridization (3D-FISH) and chromosome conformation capture (3C). Using 3D-FISH, we found the 3' portion of the locus is contracted in both DN and DP thymocytes as compared to B cells. Remarkably, the 5' portion of the locus is contracted in DN thymocytes, but is decontracted in DP thymocytes. We propose that the fully contracted conformation in DN thymocytes allows Tcrd rearrangements involving V δ gene segments distributed over one megabase, whereas the unique 3'-contracted, 5'decontracted conformation in DP thymocytes biases initial Tcra rearrangements to the most 3' of the available V α gene segments. This would maintain a large pool of distal V α gene segments for subsequent rounds of recombination. To study the conformational changes at the molecular level, we used 3C to detect interactions between different sites spanning 400kb in the contracted 3' portion of the locus. The Tcra enhancer (E α) is known to activate V α and J α segment promoters and to v stimulate V α-to-J α recombination in DP thymocytes. We detected various pair-wise interactions between elements essential for initial Tcra recombination, including proximal V α segments, TEA promoter, 5' J α array and E α. Notably, these interactions occur specifically in DP thymocytes and all are E α-dependent. We proposed that in addition to regulating transcriptional activity, E α promotes synapsis of RSSs by tethering proximal V α and 5'J α segments together to facilitate initial Tcra recombination. We also asked whether a known chromatin organizer, CTCF, regulates the formation of the DP stage-specific, E α-dependent chromatin hub. Using ChIP-seq, we identified CTCF binding sites at E α , TEA promoter, and many V α promoters in DN and DP thymocytes. Loss of CTCF in DP thymocytes resulted in impaired primary V α-to-J α recombination, reduced V α and TEA germline transcription, and reduced interactions between E α and Tcra genes. Strikingly, we also observed aberrantly increased Tcrd gene transcription and interactions between E α and Tcrd gene segments in CTCF-deficient DP thymocytes. Our data suggest that CTCF helps E α to organize a DP stage-specific chromatin hub that sets the stage for synapsis and recombination of proximal V α and 5' J α segments in DP thymocytes.
Reversible contraction by looping of the Tcra and Tcrb loci in rearranging thymocytes
Nature Immunology, 2007
Reversible contraction of immunoglobulin loci juxtaposes the variable (V) genes next to the (diversity)-joining-constant ((D)JC) gene domain, thus facilitating V-(D)J recombination. Here we show that the T cell receptor b (Tcrb) and T cell receptor ad (Tcra-Tcrd ) loci also underwent long-range interactions by looping in double-negative and double-positive thymocytes, respectively. Contraction of the Tcrb and Tcra loci occurred in rearranging thymocytes and was reversed at the next developmental stage. Decontraction of the Tcrb locus probably prevented further V b -DJ b rearrangements in double-positive thymocytes by separating the V b genes from the DJC b domain. In most double-negative cells, one Tcrb allele was recruited to pericentromeric heterochromatin. Such allelic positioning may facilitate asynchronous V b -DJ b recombination. Hence, pericentromeric recruitment and locus 'decontraction' seem to contribute to the initiation and maintenance of allelic exclusion at the Tcrb locus.
Receptor-Specific Allelic Exclusion of TCRVα-Chains During Development
The Journal of Immunology, 1998
Expression of a single Ag receptor on lymphocytes is maintained via allelic exclusion that generates cells with a clonal receptor repertoire. We show in normal mice and mice expressing functionally rearranged TCR␣ transgenes that allelic exclusion at the TCR␣ locus is not operational in immature thymocytes, whereas most mature T cells express a single TCRV␣-chain. TCRV␣ allelic exclusion in mature thymocytes is regulated through a CD45 tyrosine phosphatase-mediated signal during positive selection. Using functional and genetic systems for selection of immature double TCRV␣ ؉ thymocytes, we show that peptide-specific ligand recognition provides the signal for allelic exclusion, i.e., mature T cells maintain expression of the ligand-specific TCRV␣-chain, but lose the nonfunctional receptor. Whereas activation of TCRV-chains or CD3⑀ leads to receptor internalization, TCRV␣ ligation promotes retention of the TCR on the cell surface. Although both TCRV␣-and TCRV-chains trigger phosphotyrosine signaling, only the TCRV-chain mediates membrane recruitment of the GTPase dynamin. These data indicate that TCRV␣directed signals for positive selection control allelic exclusion in T cells, and that developmental signals can select for single receptor usage.
Allelic Exclusion of the TCR -Chain Is an Active Process Requiring TCR-Mediated Signaling and c-Cbl
The Journal of Immunology, 2003
Phenotypic allelic exclusion at the TCR␣ locus is developmentally regulated in thymocytes. Many immature thymocytes express two cell surface ␣-chain species. Following positive selection, the vast majority of mature thymocytes and peripheral T cells display a single cell surface ␣-chain. A posttranslational mechanism occurring at the same time as positive selection and TCR upregulation leads to this phenotypic allelic exclusion. Different models have been proposed to explain the posttranslational regulation of the ␣-chain allelic exclusion. In this study, we report that allelic exclusion is not regulated by competition between distinct ␣-chains for a single -chain, as proposed by the dueling ␣-chain model, nor by limiting CD3-chain in mature TCR high thymocytes. Our data instead favor the selective retention model where the positive selection signal through the TCR leads to phenotypic allelic exclusion by specifically maintaining cell surface expression of the selected ␣-chain while the nonselected ␣-chain is internalized. The use of inhibitors specific for Lck and/or other Src kinases indicates a role for these protein tyrosine kinases in the signaling events leading to the down-regulation of the nonselectable ␣-chain. Loss of the ubiquitin ligase/TCR signaling adapter molecule c-Cbl, which is important in TCR down-modulation and is a negative regulator of T cell signaling, leads to increased dual ␣-chain expression on the cell surface of double-positive thymocytes. Thus, not only is there an important role for TCR signaling in causing ␣-chain allelic exclusion, but differential ubiquitination by c-Cbl may be an important factor in causing only the nonselected ␣-chain to be down-modulated.
A discrete chromatin loop in the mouse Tcra-Tcrd locus shapes the TCRδ and TCRα repertoires
Nature immunology, 2015
The locus encoding the T cell antigen receptor (TCR) α-chain and δ-chain (Tcra-Tcrd) undergoes recombination of its variable-diversity-joining (V(D)J) segments in CD4(-)CD8(-) double-negative thymocytes and CD4(+)CD8(+) double-positive thymocytes to generate diverse TCRδ repertoires and TCRα repertoires, respectively. Here we identified a chromatin-interaction network in the Tcra-Tcrd locus in double-negative thymocytes that was formed by interactions between binding elements for the transcription factor CTCF. Disruption of a discrete chromatin loop encompassing the D, J and constant (C) segments of Tcrd allowed a single V segment to frequently contact and rearrange to D and J segments and dominate the adult TCRδ repertoire. Disruption of this loop also narrowed the TCRα repertoire, which, we believe, followed as a consequence of the restricted TCRδ repertoire. Hence, a single CTCF-mediated chromatin loop directly regulated TCRδ diversity and indirectly regulated TCRα diversity.
Tcra gene recombination is supported by a Tcra enhancer- and CTCF-dependent chromatin hub
Proceedings of the National Academy of Sciences, 2012
Antigen receptor locus V(D)J recombination requires interactions between widely separated variable (V), diversity (D), and joining (J) gene segments, but the mechanisms that generate these interactions are not well understood. Here we assessed mechanisms that direct developmental stage-specific long-distance interactions at the Tcra/Tcrd locus. The Tcra/Tcrd locus recombines Tcrd gene segments in CD4 − CD8 − double-negative thymocytes and Tcra gene segments in CD4 + CD8 + double-positive thymocytes. Initial V α -to-J α recombination occurs within a chromosomal domain that displays a contracted conformation in both thymocyte subsets. We used chromosome conformation capture to demonstrate that the Tcra enhancer (E α ) interacts directly with V α and J α gene segments distributed across this domain, specifically in double-positive thymocytes. Moreover, E α promotes interactions between these V α and J α segments that should facilitate their synapsis. We found that the CCCTC-binding fac...