RORγt up-regulates RAG gene expression in DP thymocytes to expand the repertoire.

Recombination activating gene (RAG) expression increases as thymocytes transition from the CD4CD8 double-negative (DN) to the CD4CD8 double-positive (DP) stage, but the physiological importance and mechanism of transcriptional up-regulation are unknown. Here, we show that a DP-specific component of the recombination activating genes antisilencer (DPASE) provokes elevated RAG expression in DP thymocytes. Mouse DP thymocytes lacking the DPASE display RAG expression equivalent to that in DN thymocytes, but this supports only a partial repertoire due to inefficient secondary Vα-Jα rearrangement.

E protein binding at the enhancer promotes repertoire diversity.

V(D)J recombination of antigen receptor loci is a highly developmentally regulated process. During T lymphocyte development, recombination of the gene occurs in CD4CD8 double positive (DP) thymocytes and requires the enhancer (Eα). E proteins are known regulators of DP thymocyte development and have three identified binding sites in Eα.

Methods for Study of Mouse T Cell Receptor α and β Gene Rearrangements.

Quantitative real-time PCR and next-generation sequencing (NGS) are invaluable techniques to analyze T cell receptor (Tcr) gene rearrangements in mouse lymphocyte populations. Although these approaches are powerful, they also have limitations that must be accounted for in experimental design and data interpretation. Here, we provide relevant background required for understanding these limitations and then outline established quantitative real-time PCR and NGS methods that can be used for analysis of mouse Tcra and Tcrb gene rearrangements in mice.

Trav15-dv6 family Tcrd rearrangements diversify the Tcra repertoire.

The Tcra repertoire is generated by multiple rounds of Vα-Jα rearrangement. However, Tcrd recombination precedes Tcra recombination within the complex Tcra-Tcrd locus. Here, by ablating Tcrd recombination, we report that Tcrd rearrangement broadens primary Vα use to diversify the Tcra repertoire in mice.

The order and logic of CD4 versus CD8 lineage choice and differentiation in mouse thymus.

CD4 and CD8 mark helper and cytotoxic T cell lineages, respectively, and serve as coreceptors for MHC-restricted TCR recognition. How coreceptor expression is matched with TCR specificity is central to understanding CD4/CD8 lineage choice, but visualising coreceptor gene activity in individual selection intermediates has been technically challenging. It therefore remains unclear whether the sequence of coreceptor gene expression in selection intermediates follows a stereotypic pattern, or is responsive to signaling.

A role of the CTCF binding site at enhancer Eα in the dynamic chromatin organization of the Tcra-Tcrd locus.

The regulation of T cell receptor Tcra gene rearrangement has been extensively studied. The enhancer Eα plays an essential role in Tcra rearrangement by establishing a recombination centre in the Jα array and a chromatin hub for interactions between Vα and Jα genes. But the mechanism of the Eα and its downstream CTCF binding site (here named EACBE) in dynamic chromatin regulation is unknown.

RSSs set the odds for exclusion.

In this issue of JEM, Wu et al. (https://doi.org/10.

Diversification of the TCR β Locus V Repertoire by CTCF.

The architectural protein CTCF regulates the conformation and recombination of antigen receptor loci. To study the importance of CTCF in locus repertoire formation, we created a conditional knockout mouse line that deletes early during thymocyte development. We observed an incomplete block in thymocyte development at the double-negative to double-positive transition, resulting in greatly lowered thymic cellularity.

Hierarchical assembly and disassembly of a transcriptionally active RAG locus in CD4CD8 thymocytes.

Expression of and is tightly regulated in developing T cells to mediate TCR gene assembly. Here we have investigated the molecular mechanisms governing the assembly and disassembly of a transcriptionally active RAG locus chromatin hub in CD4CD8 thymocytes. and gene expression in CD4CD8 thymocytes depends on and promoter activation by a distant antisilencer element (ASE).

A Lamina-Associated Domain Border Governs Nuclear Lamina Interactions, Transcription, and Recombination of the Tcrb Locus.

Tcrb locus V(D)J recombination is regulated by positioning at the nuclear periphery. Here, we used DamID to profile Tcrb locus interactions with the nuclear lamina at high resolution. We identified a lamina-associated domain (LAD) border composed of several CTCF-binding elements that segregates active non-LAD from inactive LAD regions of the locus.

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.

Tcrd Rearrangement Redirects a Processive Tcra Recombination Program to Expand the Tcra Repertoire.

Adaptive immunity depends on diverse T cell receptor repertoires generated by variable, diversity, and joining (V[D]J) recombination. Here, we define the principles by which combinatorial diversity is generated in the murine Tcra repertoire. Tcra and Tcrd gene segments share the Tcra-Tcrd locus, with interspersed V and V segments undergoing V-D-J rearrangement in CD4CD8 thymocytes and then multiple rounds of V-J rearrangement in CD4CD8 thymocytes.

An Ectopic CTCF Binding Element Inhibits Tcrd Rearrangement by Limiting Contact between Vδ and Dδ Gene Segments.

Chromatin looping mediated by the CCCTC binding factor (CTCF) regulates V(D)J recombination at Ag receptor loci. CTCF-mediated looping can influence recombination signal sequence (RSS) accessibility by regulating enhancer activation of germline promoters. CTCF-mediated looping has also been shown to limit directional tracking of the RAG recombinase along chromatin, and to regulate long-distance interactions between RSSs, independent of the RAG recombinase.

Orientation-specific RAG activity in chromosomal loop domains contributes to Tcrd V(D)J recombination during T cell development.

T cell antigen receptor δ (Tcrd) variable region exons are assembled by RAG-initiated V(D)J recombination events in developing γδ thymocytes. Here, we use linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) to map hundreds of thousands of RAG-initiated Tcrd D segment (Trdd1 and Trdd2) rearrangements in CD4(-)CD8(-) double-negative thymocyte progenitors differentiated in vitro from bone marrow-derived hematopoietic stem cells. We find that Trdd2 joins directly to Trdv, Trdd1, and Trdj segments, whereas Trdd1 joining is ordered with joining to Trdd2, a prerequisite for further rearrangement.

The Ties that Bind (the Igh Locus).

Immunoglobulin heavy-chain locus V(D)J recombination requires a 3D chromatin organization which permits widely distributed variable (V) gene segments to contact distant diversity (D) and joining (J) gene segments. A recent study has identified key nodes in the locus interactome, paving the way for new molecular insights into how the locus is configured for recombination.

Yin Yang 1 Promotes Thymocyte Survival by Downregulating p53.

Yin Yang 1 (YY1) is a zinc finger protein that functions as a transcriptional activator or repressor and participates in multiple biological processes, including development and tumorigenesis. To investigate the role of YY1 in developing T cells, we used mouse models that depleted YY1 at two distinct stages of thymocyte development. When YY1 was depleted in CD4(-)CD8(-) double-negative thymocytes, development to the CD4(+)CD8(+) double-positive stage was impaired, due to increased apoptosis that prevented expansion of post-β-selection thymocytes.

Inactivation of nuclear GSK3β by Ser(389) phosphorylation promotes lymphocyte fitness during DNA double-strand break response.

Variable, diversity and joining (V(D)J) recombination and immunoglobulin class switch recombination (CSR) are key processes in adaptive immune responses that naturally generate DNA double-strand breaks (DSBs) and trigger a DNA repair response. It is unclear whether this response is associated with distinct survival signals that protect T and B cells. Glycogen synthase kinase 3β (GSK3β) is a constitutively active kinase known to promote cell death.

Chromatin Dynamics and the Development of the TCRα and TCRδ Repertoires.

The adaptive immune system allows vertebrates to orchestrate highly specific responses to a virtually unlimited milieu of antigens. Effective adaptive immune responses depend on the capacity of T and B lymphocytes to generate diverse repertoires of antigen receptors through the recombination of variable (V), diversity (D), and joining (J) gene segments at antigen receptor loci. V(D)J recombination must be carefully regulated during the early stages of T and B lymphocyte development to ensure the proper development of lymphocyte subsets and to maximize antigen receptor combinatorial diversity.

Molecular Analysis of Mouse T Cell Receptor α and β Gene Rearrangements.

PCR on genomic DNA isolated from lymphocyte populations is an invaluable technique to analyze T cell receptor (TCR) α and β gene rearrangements. Although this approach is powerful, it also has limitations that must be accounted for in experimental design and data interpretation. Here, we provide background required for understanding these limitations, and then outline standard PCR methods that can be used for analysis of TCRα and β gene rearrangements in mice.

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.

An anti-silencer- and SATB1-dependent chromatin hub regulates Rag1 and Rag2 gene expression during thymocyte development.

Rag1 and Rag2 gene expression in CD4(+)CD8(+) double-positive (DP) thymocytes depends on the activity of a distant anti-silencer element (ASE) that counteracts the activity of an intergenic silencer. However, the mechanistic basis for ASE activity is unknown. Here, we show that the ASE physically interacts with the distant Rag1 and Rag2 gene promoters in DP thymocytes, bringing the two promoters together to form an active chromatin hub.

T-cell receptor α enhancer is inactivated in αβ T lymphocytes.

The Tcra enhancer (Eα) is essential for Tcra locus germ-line transcription and primary Vα-to-Jα recombination during thymocyte development. We found that Eα is inhibited late during thymocyte differentiation and in αβ T lymphocytes, indicating that it is not required to drive transcription of rearranged Tcra genes. Eα inactivation resulted in the disruption of functional long-range enhancer-promoter interactions and was associated with loss of Eα-dependent histone modifications at promoter and enhancer regions, and reduced expression and recruitment of E2A to the Eα enhanceosome in T cells.