Han-Yu Shih

Tcra gene recombination is supported by a Tcra enhancer- and CTCF-dependent chromatin hub

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 Vα-to-Jα 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 (Eα) interacts directly with Vα and Jα gene segments distributed across this domain, specifically in double-positive thymocytes. Moreover, Eα promotes interactions between these Vα and Jα segments that should facilitate their synapsis. We found that the CCCTC-binding factor (CTCF) binds to Eα and to many locus promoters, biases Eα to interact with these promoters, and is required for efficient Vα–Jα recombination. Our data indicate that Eα and CTCF cooperate to create a developmentally regulated chromatin hub that supports Vα–Jα synapsis and recombination.

Distinct contracted conformations of the Tcra/Tcrd locus during Tcra and Tcrd recombination

Studies have suggested that antigen receptor loci adopt contracted conformations to promote long-distance interactions between gene segments during V(D)J recombination. The Tcra/Tcrd locus is unique because it undergoes highly divergent Tcrd and Tcra recombination programs in CD4−CD8− double negative (DN) and CD4+CD8+ double positive (DP) thymocytes, respectively. Using three-dimensional fluorescence in situ hybridization, we asked whether these divergent recombination programs are supported by distinct conformational states of the Tcra/Tcrd locus. We found that the 3′ portion of the locus is contracted in DN and DP thymocytes but not in 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 Vδ gene segments distributed over 1 Mb, whereas the unique 3′-contracted, 5′-decontracted conformation in DP thymocytes biases initial Tcra rearrangements to the most 3′ of the available Vα gene segments. This would maintain a large pool of distal 5′ Vα gene segments for subsequent rounds of recombination. Thus, distinct contracted conformations of the Tcra/Tcrd locus may facilitate a transition from a Tcrd to a Tcra mode of recombination during thymocyte development.

IL-7 coordinates proliferation, differentiation and Tcra recombination during thymocyte β-selection

Signaling via the pre–T cell antigen receptor (pre-TCR) and the receptor Notch1 induces transient self-renewal (β-selection) of TCRβ+ CD4−CD8− double-negative stage 3 (DN3) and DN4 progenitor cells that differentiate into CD4+CD8+ double-positive (DP) thymocytes, which then rearrange the locus encoding the TCR α-chain (Tcra). Interleukin 7 (IL-7) promotes the survival of TCRβ− DN thymocytes by inducing expression of the pro-survival molecule Bcl-2, but the functions of IL-7 during β-selection have remained unclear. Here we found that IL-7 signaled TCRβ+ DN3 and DN4 thymocytes to upregulate genes encoding molecules involved in cell growth and repressed the gene encoding the transcriptional repressor Bcl-6. Accordingly, IL-7-deficient DN4 cells lacked trophic receptors and did not proliferate but rearranged Tcra prematurely and differentiated rapidly. Deletion of Bcl6 partially restored the self-renewal of DN4 cells in the absence of IL-7, but overexpression of BCL2 did not. Thus, IL-7 critically acts cooperatively with signaling via the pre-TCR and Notch1 to coordinate proliferation, differentiation and Tcra recombination during β-selection.

Chromatin Architecture, CCCTC-Binding Factor, and V(D)J Recombination: Managing Long-Distance Relationships at Antigen Receptor Loci

The rearrangement of T and B lymphocyte Ag receptor loci occurs within a highly complex chromosomal environment and is orchestrated through complex mechanisms. During the past decade, a large body of literature has highlighted the significance of chromatin architecture at Ag receptor loci in supporting the genomic assembly process: in preparation for recombination, these loci tend to contract and form multiple loops that shorten the distances between gene segments and facilitate recombination events. CCCTC-binding factor, CTCF, has received much attention in this regard since it has emerged as an important regulator of chromatin organization and transcription. In this review, we summarize recent work outlining conformational dynamics at Ag receptor loci during lymphocyte development and we discuss the role of CTCF in Ag receptor locus conformation and repertoire development.

A discrete chromatin loop in the mouse Tcra-Tcrd locus shapes the TCRδ and TCRα repertoires.

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.

Orchestrating T-cell receptor α gene assembly through changes in chromatin structure and organization.

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.

Regulation of TCRβ Allelic Exclusion by Gene Segment Proximity and Accessibility

Ag receptor loci are regulated to promote allelic exclusion, but the mechanisms are not well understood. Assembly of a functional TCR β-chain gene triggers feedback inhibition of Vβ-to-DJβ recombination in double-positive (DP) thymocytes, which correlates with reduced Vβ chromatin accessibility and a locus conformational change that separates Vβ from DJβ gene segments. We previously generated a Tcrb allele that maintained Vβ 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 Vβ accessibility and increased distance between Vβ and DJβ gene segments both enforce feedback inhibition in DP thymocytes.

Variable Extent of Lineage-Specificity and Developmental Stage-Specificity of Cohesin and CCCTC-Binding Factor Binding Within the Immunoglobulin and T Cell Receptor Loci

CCCTC-binding factor (CTCF) is largely responsible for the 3D architecture of the genome, in concert with the action of cohesin, through the creation of long-range chromatin loops. Cohesin is hypothesized to be the main driver of these long-range chromatin interactions by the process of loop extrusion. Here, we performed ChIP-seq for CTCF and cohesin in two stages each of T and B cell differentiation and examined the binding pattern in all six antigen receptor (AgR) loci in these lymphocyte progenitors and in mature T and B cells, ES cells, and fibroblasts. The four large AgR loci have many bound CTCF sites, most of which are only occupied in lymphocytes, while only the CTCF sites at the end of each locus near the enhancers or J genes tend to be bound in non-lymphoid cells also. However, despite the generalized lymphocyte restriction of CTCF binding in AgR loci, the Igκ locus is the only locus that also shows significant lineage-specificity (T vs. B cells) and developmental stage-specificity (pre-B vs. pro-B) in CTCF binding. We show that cohesin binding shows greater lineage- and stage-specificity than CTCF at most AgR loci, providing more specificity to the loops. We also show that the culture of pro-B cells in IL7, a common practice to expand the number of cells before ChIP-seq, results in a CTCF-binding pattern resembling pre-B cells, as well as other epigenetic and transcriptional characteristics of pre-B cells. Analysis of the orientation of the CTCF sites show that all sites within the large V portions of the Igh and TCRβ loci have the same orientation. This suggests either a lack of requirement for convergent CTCF sites creating loops, or indicates an absence of any loops between CTCF sites within the V region portion of those loci but only loops to the convergent sites at the D-J-enhancer end of each locus. The V region portions of the Igκ and TCRα/δ loci, by contrast, have CTCF sites in both orientations, providing many options for creating CTCF-mediated convergent loops throughout the loci. CTCF/cohesin loops, along with transcription factors, drives contraction of AgR loci to facilitate the creation of a diverse repertoire of antibodies and T cell receptors.

A discrete chromatin loop in the murine Tcra-Tcrd locus shapes the TCRδ and TCRα repertoires

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.

A discrete chromatin loop in the mouse Tcra-Tcrd locus shapes the TCR[delta] and TCR[alpha] repertoires

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.