Initiation of allelic exclusion by stochastic interaction of Tcrb alleles with repressive nuclear compartments (original) (raw)
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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.
Regulation of TCR Allelic Exclusion by Gene Segment Proximity and Accessibility
The Journal of Immunology, 2011
Ag receptor loci are regulated to promote allelic exclusion, but the mechanisms are not well understood. Assembly of a functional TCR b-chain gene triggers feedback inhibition of V b-to-DJ b recombination in double-positive (DP) thymocytes, which correlates with reduced V b chromatin accessibility and a locus conformational change that separates V b from DJ b gene segments. We previously generated a Tcrb allele that maintained V b accessibility but was still subject to feedback inhibition in DP thymocytes. We have now further analyzed the contributions of chromatin accessibility and locus conformation to feedback inhibition using two novel TCR alleles. We show that reduced V b accessibility and increased distance between V b and DJ b gene segments both enforce feedback inhibition 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.
Both TCRα and TCRδ chain diversity are regulated during thymic ontogeny
The Journal of …, 2001
TCR␣ and TCR␦ chains are coded by a common genetic locus using a single set of V gene segments (ADV segments). This article addresses the question of regulation of the use of the ADV segments by the TCR␣ and TCR␦ chains. Using both qualitative and quantitative analyses we have studied the use of 23 ADV gene families as part of TCR␣ and TCR␦ transcripts. A number of previously undetected rearrangement and transcription events are described, indicating that the intrathymic TCR␦ repertoire is much more diverse than previously supposed. Repertoire analysis at several developmental time points allowed the description of regulated waves of ADV gene use, not only for TCR␦ chains, but also for TCR␣ chains, during thymic ontogeny. Control of these waves appears to be linked directly to the ADV segments and their local chromatin environment, which may change over the course of T cell differentiation.
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...
Journal of Experimental Medicine, 2000
Gene targeting studies have shown that T cell receptor (TCR)- gene expression and recombination are inhibited after deletion of an enhancer (E  ) located at the 3 Ј end of the ف 500-kb TCR- locus. Using knockout mouse models, we have measured, at different regions throughout the TCR- locus, the effects of E  deletion on molecular parameters believed to reflect epigenetic changes associated with the control of gene activation, including restriction endonuclease access to chromosomal DNA, germline transcription, DNA methylation, and histone H3 acetylation. Our results demonstrate that, in early developing thymocytes, E  contributes to major chromatin remodeling directed to an ف 25-kb upstream domain comprised of the D  -J  locus regions. Accordingly, treatment of E  -deleted thymocytes with the histone deacetylase inhibitor trichostatin A relieved the block in TCR- gene expression and promoted recombination within the D  -J  loci. Unexpectedly, however, epigenetic processes at distal V  genes on the 5 Ј side of the locus and at the 3 Ј proximal V  14 gene appear to be less dependent on E  , suggesting that E  activity is confined to a discrete region of the TCR- locus. These findings have implications with respect to the developmental control of TCR- gene recombination, and the process of allelic exclusion at this locus.