TEA regulates local TCR-Jalpha accessibility through histone acetylation (original) (raw)
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TEA regulates local TCR-Jα accessibility through histone acetylation
European Journal of Immunology, 2003
Enhancer §-dependent histone acetylation has been proposed as a molecular mechanism underlying the control of accessibility of recombination signal sequences along the TCR § locus. Here we show that chromatin acetylation along the first J § segments is under the dependence of the T early § element (TEA), located upstream of TCRJ § locus. The targeted deletion of TEA leads to an absence of histones H3 and H4 tail acetylation, while maintaining histone acetylation in the region spanning downstream J § segments. During thymocyte maturation, TEA-dependent histone acetylation appears at immature single-positive stage, known to represent the stage of V § J § initiation. TEA-dependent histone acetylation of the most upstream J § segments leads to enhanced DNA accessibility thus optimizing TCRJ § usage and increasing Ag receptor diversity potential.
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.
Transcription-Dependent Generation of a Specialized Chromatin Structure at the TCRβ Locus
Journal of immunology (Baltimore, Md. : 1950), 2015
V(D)J recombination assembles Ag receptor genes during lymphocyte development. Enhancers at AR loci are known to control V(D)J recombination at associated alleles, in part by increasing chromatin accessibility of the locus, to allow the recombination machinery to gain access to its chromosomal substrates. However, whether there is a specific mechanism to induce chromatin accessibility at AR loci is still unclear. In this article, we highlight a specialized epigenetic marking characterized by high and extended H3K4me3 levels throughout the Dβ-Jβ-Cβ gene segments. We show that extended H3K4 trimethylation at the Tcrb locus depends on RNA polymerase II (Pol II)-mediated transcription. Furthermore, we found that the genomic regions encompassing the two DJCβ clusters are highly enriched for Ser(5)-phosphorylated Pol II and short-RNA transcripts, two hallmarks of transcription initiation and early transcription. Of interest, these features are shared with few other tissue-specific genes. ...
T early alpha (TEA) regulates initial TCRVAJA rearrangements and leads to TCRJA coincidence
European Journal of Immunology, 2001
Both TCRA alleles are rearranged in mature T lymphocytes, as a result of a lack of allelic exclusion at the TRCA locus. We show in a series of T cell clones that the two TCRJA segments are not randomly, but rather coincidentally, rearranged in a given T cell. The TCRJA coincidence relies, in part, on the presence of "T early alpha" (TEA), a cis-regulatory genetic element located upstream of the TCRJA cluster. TEA promotes specific recombinational accessibility that targets primary TCRVAJA rearrangements on the 5' side of the TCRA locus. In a model of multiple waves of TCRVAJA recombination, this cis-regulatory effect of TEA allows for the scanning of the entire TCRJA cluster, thereby increasing the TCR § / g diversity potential.
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.
Immunologic Research, 2010
V(D)J recombination is regulated through changes in chromatin structure that allow recombinase proteins access to recombination signal sequences and through changes in three-dimensional chromatin organization that bring pairs of distant recombination signal sequences into proximity. The Tcra/Tcrd locus is complex and undergoes distinct recombination programs in double negative and double positive thymocytes that lead to the assembly of Tcrd and Tcra genes, respectively. Our studies provide insights into how locus chromatin structure is regulated and how changes in locus chromatin structure can target and then retarget the recombinase to create developmental progressions of recombination events. Our studies also reveal distinct locus conformations in double negative and double positive thymocytes and suggest how these conformations may support the distinct recombination programs in the two compartments.
Journal of Experimental Medicine
Cell differentiation is accompanied by epigenetic changes leading to precise lineage definition and cell identity. Here we present a comprehensive resource of epigenomic data of human T cell precursors along with an integrative analysis of other hematopoietic populations. Although T cell commitment is accompanied by large scale epigenetic changes, we observed that the majority of distal regulatory elements are constitutively unmethylated throughout T cell differentiation, irrespective of their activation status. Among these, the TCRA gene enhancer (Eα) is in an open and unmethylated chromatin structure well before activation. Integrative analyses revealed that the HOXA5-9 transcription factors repress the Eα enhancer at early stages of T cell differentiation, while their decommission is required for TCRA locus activation and enforced αβ T lineage differentiation. Remarkably, the HOXA-mediated repression of Eα is paralleled by the ectopic expression of homeodomain-related oncogenes i...
Rearrangement V{{beta}} Segments Before Gene Regulation of Individual TCR
The promoter sequences of individual murine TCR V segments are dissimilar, but any functional differences between them are masked after productive gene rearrangement by the dominance of the TCR 3 enhancer. However, thymocytes of recombinationactivating gene-2 (Rag2)-deficient mice allow the transcriptional activity of V promoters to be studied before rearrangement. Here we report that many V segments are detectably transcribed in Rag2 ؊/؊ thymocytes and that there are significant differences in expression among different V segments. Primer extension and characterization of cDNA clones from SCID thymocytes suggest that these germline V transcripts generally use the same start sites as those previously determined in mature T cells. The strength of expression before rearrangement does not correlate with proximity to the known enhancer, because members of the most distal V cluster (V2.1, V1.1, V4.1) are relatively strongly expressed and more proximal V segments (V14.1, V3.1, V7.1, V6.1) are only weakly expressed. Different V segments also show different developmental programs of activation in different thymocyte subsets, with the V5.1(L)-8.2(V) spliced transcript expressed earliest as well as most strongly overall. Comparison with Rag ؉ MHC class I ؊/؊ and class II ؊/؊ thymocytes confirms that many of these expression differences are leveled by rearrangement and/or by  selection, before MHC-dependent selection. However, the expression pattern of V2.1 is highly distinctive and includes cell types apparently outside the T lineage, suggesting potential acquisition of specialized roles.
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.
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...