Abbas Hawwari

Regulation of the TCRalpha repertoire by the survival window of CD4(+)CD8(+) thymocytes.

T cell receptor (TCR) α alleles undergo primary and secondary rearrangement in double-positive (DP) thymocytes. By analyzing TCRα rearrangement in orphan nuclear receptor RORγ-deficient mice, in which the DP lifespan is shorter, and in Bcl-xL–transgenic mice, in which the DP lifespan is extended, we show that the progression of secondary Vα to Jα rearrangements is controlled by DP thymocyte survival. In addition, because Bcl-xL induces a bias towards 3′ Jα usage in peripheral T cells, we conclude that the programmed cell death of DP thymocytes is not simply a consequence of failed positive selection. Rather, it limits the progression of rearrangement along the Jα locus and the opportunities for positive selection, thereby regulating the TCRα repertoire.

Enforcing order within a complex locus: current perspectives on the control of V(D)J recombination at the murine T-cell receptor α/δ locus

Summary:  V(D)J recombination proceeds according to defined developmental programs at T‐cell receptor (TCR) and immunoglobulin loci as a function of cell lineage and stage of differentiation. Although the molecular details are still lacking, such regulation is thought to occur at the level of accessibility of chromosomal recombination signal sequences to the recombinase. The unique and complex organization of the TCRα/δ locus poses intriguing regulatory challenges in this regard: embedded TCRα and TCRδ gene segments rearrange at distinct stages of thymocyte development, there is a highly regulated progression of primary followed by secondary rearrangements involving Jα segments, and there are important developmental constraints on V gene segment usage. The locus therefore provides a fascinating laboratory in which to explore the basic mechanisms underlying developmental control. We provide here a current view of cis‐acting mechanisms that enforce the TCRα/δ locus developmental program, and we emphasize the unresolved issues that command the attention of our and other laboratories.

Regulation of T cell receptor |[alpha]| gene assembly by a complex hierarchy of germline J|[alpha]| promoters

Assembly of the gene encoding T cell receptor α (Tcra) is characterized by an orderly progression of primary and secondary Vα-to-Jα recombination events across the Jα array, but the targeting mechanisms responsible for this progression are mostly unknown. Studies have shown that the T early-α promoter is important in targeting primary Tcra rearrangements. We found that T early-α and a previously unknown promoter associated with Jα49 targeted primary recombination to discrete sets of constant α region (Cα)–distal Jα segments and together directed nearly all normal primary recombination events. Furthermore, deletion of the T early-α promoter activated previously suppressed downstream promoters and stimulated primary rearrangement to centrally located Jα segments. Central promoter derepression also occurred after primary rearrangement, thereby providing a mechanism to target secondary recombination events.

Regulation of TCR δ and α repertoires by local and long-distance control of variable gene segment chromatin structure

Murine Tcrd and Tcra gene segments reside in a single genetic locus and undergo recombination in CD4−CD8− (double negative [DN]) and CD4+CD8+ (double positive [DP]) thymocytes, respectively. TcraTcrd locus variable gene segments are subject to complex regulation. Only a small subset of ∼100 variable gene segments contributes substantially to the adult TCRδ repertoire. Moreover, although most contribute to the TCRα repertoire, variable gene segments that are Jα proximal are preferentially used during primary Tcra recombination. We investigate the role of local chromatin accessibility in determining the developmental pattern of TcraTcrd locus variable gene segment recombination. We find variable gene segments to be heterogeneous with respect to acetylation of histones H3 and H4. Those that dominate the adult TCRδ repertoire are hyperacetylated in DN thymocytes, independent of their position in the locus. Moreover, proximal variable gene segments show dramatic increases in histone acetylation and germline transcription in DP thymocytes, a result of super long-distance regulation by the Tcra enhancer. Our results imply that differences in chromatin accessibility contribute to biases in TcraTcrd locus variable gene segment recombination in DN and DP thymocytes and extend the distance over which the Tcra enhancer can regulate chromatin structure to a remarkable 525 kb.

The Human IL-3 Locus Is Regulated Cooperatively by Two NFAT-Dependent Enhancers That Have Distinct Tissue-Specific Activities

The human IL-3 gene is expressed by activated T cells, mast cells, and eosinophils. We previously identified an enhancer 14 kb upstream of the IL-3 gene, but this element only functioned in a subset of T cells and not in mast cells. To identify additional mechanisms governing IL-3 gene expression, we mapped DNase I hypersensitive (DH) sites and evolutionarily conserved DNA sequences in the IL-3 locus. The most conserved sequence lies 4.5 kb upstream of the IL-3 gene and it encompassed an inducible cyclosporin A-sensitive DH site. A 245-bp fragment spanning this DH site functioned as a cyclosporin A-sensitive enhancer, and was induced by calcium and kinase signaling pathways in both T cells and mast cells via an array of three NFAT sites. The enhancer also encompassed AML1, AP-1, and Sp1 binding sites that potentially mediate function in both T and myeloid lineage cells, but these sites were not required for in vitro enhancer function in T cells. In stably transfected T cells, the −4.5-kb enhancer cooperated with the −14-kb enhancer to activate the IL-3 promoter. Hence, the IL-3 gene is regulated by two enhancers that have distinct but overlapping tissue specificities. We also identified a prominent constitutive DH site at −4.1 kb in T cells, mast cells, and CD34+ myeloid cells. This element lacked in vitro enhancer function, but may have a developmental role because it appears to be the first DH site to exist upstream of the IL-3 gene during hemopoietic development before IL-3 expression.

Reconstitution of T Cell-Specific Transcription Directed by Composite NFAT/Oct Elements

The complex nature of most promoters and enhancers makes it difficult to identify key determinants of tissue-specific gene expression. Furthermore, most tissue-specific genes are regulated by transcription factors that have expression profiles more widespread than the genes they control. NFAT is an example of a widely expressed transcription factor that contributes to several distinct patterns of cytokine gene expression within the immune system and where its role in directing specificity remains undefined. To investigate distinct combinatorial mechanisms employed by NFAT to regulate tissue-specific transcription, we examined a composite NFAT/AP-1 element from the widely active GM-CSF enhancer and a composite NFAT/Oct element from the T cell-specific IL-3 enhancer. The NFAT/AP-1 element was active in the numerous cell types that express NFAT, but NFAT/Oct enhancer activity was T cell specific even though Oct-1 is ubiquitous. Conversion of the single Oct site in the IL-3 enhancer to an AP-1 enabled activation outside of the T cell lineage. By reconstituting the activities of both the IL-3 enhancer and its NFAT/Oct element in a variety of cell types, we demonstrated that their T cell-specific activation required the lymphoid cofactors NIP45 and OCA-B in addition to NFAT and Oct family proteins. Furthermore, the Oct family protein Brn-2, which cannot recruit OCA-B, repressed NFAT/Oct enhancer activity. Significantly, the two patterns of combinatorial regulation identified in this study mirror the cell-type specificities of the cytokine genes that they govern. We have thus established that simple composite transcription factor binding sites can indeed establish highly specific patterns of gene expression.

Role for rearranged variable gene segments in directing secondary T cell receptor α recombination

During the recombination of variable (V) and joining (J) gene segments at the T cell receptor α locus, a VαJα joint resulting from primary rearrangement can be replaced by subsequent rounds of secondary rearrangement that use progressively more 5′ Vα segments and progressively more 3′ Jα segments. To understand the mechanisms that target secondary T cell receptor α recombination, we studied the behavior of a T cell receptor α allele (HYα) engineered to mimic a natural primary rearrangement of TRAV17 to Jα57. The introduced VαJα segment was shown to provide chromatin accessibility to Jα segments situated within several kilobases downstream and to suppress germ-line Jα promoter activity and accessibility at greater distances. As a consequence, the VαJα segment directed secondary recombination events to a subset of Jα segments immediately downstream from the primary rearrangement. The data provide the mechanistic basis for a model of primary and secondary T cell receptor α recombination in which recombination events progress in multiple small steps down the Jα array.

Multiple Constraints at the Level of TCRα Rearrangement Impact Vα14i NKT Cell Development

CD1d-restricted NKT cells that express an invariant Vα14 TCR represent a subset of T cells implicated in the regulation of several immune responses, including autoimmunity, infectious disease, and cancer. Proper rearrangement of Vα14 with the Jα18 gene segment in immature thymocytes is a prerequisite to the production of a TCR that can be subsequently positively selected by CD1d/self-ligand complexes in the thymus and gives rise to the NKT cell population. We show here that Vα14 to Jα rearrangements are temporally regulated during ontogeny providing a molecular explanation to their late appearance in the thymus. Using mice deficient for the transcription factor RORγ and the germline promoters T early-α and Jα49, we show that developmental constraints on both Vα and Jα usage impact NKT cell development. Finally, we demonstrate that rearrangements using Vα14 and Jα18 occur normally in the absence of FynT, arguing that the effect of FynT on NKT cell development occurs subsequent to α-chain rearrangement. Altogether, this study provides evidence that there is no directed rearrangement of Vα14 to Jα18 segments and supports the instructive selection model for NKT cell selection.

Specification of Vδ and Vα Usage by Tcra/Tcrd Locus V Gene Segment Promoters

The Tcra/Tcrd locus undergoes V-Dδ-Jδ rearrangement in CD4−CD8− thymocytes to form the TCRδ chain of the γδ TCR and V-Jα rearrangement in CD4+CD8+ thymocytes to form the TCRα-chain of the αβ TCR. Most V segments in the locus participate in V-Jα rearrangement, but only a small and partially overlapping subset participates in V-Dδ-Jδ rearrangement. What specifies any particular Tcra/Tcrd locus V gene segment as a Vδ, a Vα, or both is currently unknown. We tested the hypothesis that V segment usage is specified by V segment promoter-dependent chromatin accessibility in developing thymocytes. TRAV15/DV6 family V gene segments contribute to both the Tcrd and the Tcra repertoires, whereas TRAV12 family V gene segments contribute almost exclusively to the Tcra repertoire. To understand whether the TRAV15/DV6 promoter region specifies TRAV15/DV6 as a Vδ, we used gene targeting to replace the promoter region of a TRAV12 family member with one from a TRAV15/DV6 family member. The TRAV15/DV6 promoter region conferred increased germline transcription and histone modifications to TRAV12 in double-negative thymocytes and caused a substantial increase in usage of TRAV12 in Tcrd recombination events. Our results demonstrate that usage of TRAV15/DV6 family V gene segments for Tcrd recombination in double-negative thymocytes is regulated, at least in part, by intrinsic features of TRAV15/DV6 promoters, and argue that Tcra/Tcrd locus Vδ gene segments are defined by their local chromatin accessibility in CD4−CD8− thymocytes.