Locus folding mechanisms determine modes of antigen receptor gene assembly.

The dynamic folding of genomes regulates numerous biological processes, including antigen receptor (AgR) gene assembly. We show that, unlike other AgR loci, homotypic chromatin interactions and bidirectional chromosome looping both contribute to structuring Tcrb for efficient long-range V(D)J recombination. Inactivation of the CTCF binding element (CBE) or promoter at the most 5'Vβ segment (Trbv1) impaired loop extrusion originating locally and extending to DβJβ CBEs at the opposite end of Tcrb.

The Cyclin D3 Protein Enforces Monogenic TCRβ Expression by Mediating TCRβ Protein-Signaled Feedback Inhibition of Vβ Recombination.

In jawed vertebrates, adaptive immunity depends on the process of V(D)J recombination creating vast numbers of T and B lymphocytes that each expresses unique Ag receptors of uniform specificity. The asynchronous initiation of V-to-(D)J rearrangement between alleles and the resulting protein from one allele signaling feedback inhibition of V recombination on the other allele ensures homogeneous receptor specificity of individual cells. Upon productive Vβ-to-DβJβ rearrangements in noncycling double-negative thymocytes, TCRβ protein signals induction of the cyclin D3 protein to accelerate cell cycle entry, thereby driving proliferative expansion of developing αβ T cells.

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.

The RAG1 Ubiquitin Ligase Domain Stimulates Recombination of TCRβ and TCRα Genes and Influences Development of αβ T Cell Lineages.

RAG1/RAG2 (RAG) endonuclease-mediated assembly of diverse lymphocyte Ag receptor genes by V(D)J recombination is critical for the development and immune function of T and B cells. The RAG1 protein contains a ubiquitin ligase domain that stabilizes RAG1 and stimulates RAG endonuclease activity in vitro. We report in this study that mice with a mutation that inactivates the Rag1 ubiquitin ligase in vitro exhibit decreased rearrangements and altered repertoires of TCRβ and TCRα genes in thymocytes and impaired thymocyte developmental transitions that require the assembly and selection of functional TCRβ and/or TCRα genes.

Monogenic TCRβ Assembly and Expression Are Paramount for Uniform Antigen Receptor Specificity of Individual αβ T Lymphocytes.

The ability of individual T and B cells to display Ag receptors of unique uniform specificity is the molecular basis of adaptive immunity. Most αβ T cells achieve uniform specificity by assembling in-frame genes on only one allelic copy of TCRβ and TCRα loci, while others prevent incorporation of TCRα protein from both alleles into TCRs. Analysis of mice expressing TCR proteins from a restricted combination of transgenes showed that TCR protein pairing restrictions achieve uniform specificity of cells expressing two types of TCRβ protein.

Poor-Quality Vβ Recombination Signal Sequences and the DNA Damage Response ATM Kinase Collaborate to Establish TCRβ Gene Repertoire and Allelic Exclusion.

The monoallelic expression (allelic exclusion) of diverse lymphocyte Ag receptor genes enables specific immune responses. Allelic exclusion is achieved by asynchronous initiation of V(D)J recombination between alleles and protein encoded by successful rearrangement on the first allele signaling permanent inhibition of V rearrangement on the other allele. The ATM kinase that guides DNA repair and transiently suppresses V(D)J recombination also helps impose allelic exclusion through undetermined mechanisms.

Nemo-Dependent, ATM-Mediated Signals from RAG DNA Breaks at Feedback Inhibit Recombination to Enforce Igκ Allelic Exclusion.

Monoallelic AgR gene expression underlies specific adaptive immune responses. AgR allelic exclusion is achieved by sequential initiation of V(D)J recombination between alleles and resultant protein from one allele signaling to prevent recombination of the other. The ATM kinase, a regulator of the DNA double-strand break (DSB) response, helps enforce allelic exclusion through undetermined mechanisms.

The RAG1 N-terminal region regulates the efficiency and pathways of synapsis for V(D)J recombination.

Immunoglobulin and T cell receptor gene assembly depends on V(D)J recombination initiated by the RAG1-RAG2 recombinase. The RAG1 N-terminal region (NTR; aa 1-383) has been implicated in regulatory functions whose influence on V(D)J recombination and lymphocyte development in vivo is poorly understood. We generated mice in which RAG1 lacks ubiquitin ligase activity (P326G), the major site of autoubiquitination (K233R), or its first 215 residues (Δ215).

Inefficient V(D)J recombination underlies monogenic T cell receptor β expression.

The assembly of T cell receptor (TCR) and immunoglobulin (Ig) genes by V(D)J recombination generates the antigen receptor (AgR) diversity that is vital for adaptive immunity. At most AgR loci, V(D)J recombination is regulated so that only one allele assembles a functional gene, ensuring that nearly every T and B cell expresses a single type, or specificity, of AgR. The genomic organizations of some AgR loci permit the assembly and expression of two distinct genes on each allele; however, this is prevented by undetermined mechanisms.

DNA double-strand breaks induce H2Ax phosphorylation domains in a contact-dependent manner.

Efficient repair of DNA double-strand breaks (DSBs) requires a coordinated DNA Damage Response (DDR), which includes phosphorylation of histone H2Ax, forming γH2Ax. This histone modification spreads beyond the DSB into neighboring chromatin, generating a DDR platform that protects against end disassociation and degradation, minimizing chromosomal rearrangements. However, mechanisms that determine the breadth and intensity of γH2Ax domains remain unclear.

Poor quality Vβ recombination signal sequences stochastically enforce TCRβ allelic exclusion.

The monoallelic expression of antigen receptor (AgR) genes, called allelic exclusion, is fundamental for highly specific immune responses to pathogens. This cardinal feature of adaptive immunity is achieved by the assembly of a functional AgR gene on one allele, with subsequent feedback inhibition of V(D)J recombination on the other allele. A range of epigenetic mechanisms have been implicated in sequential recombination of AgR alleles; however, we now demonstrate that a genetic mechanism controls this process for Tcrb.

Genome Topology Control of Antigen Receptor Gene Assembly.

The past decade has increased our understanding of how genome topology controls RAG endonuclease-mediated assembly of lymphocyte AgR genes. New technologies have illuminated how the large IgH, Igκ, TCRα/δ, and TCRβ loci fold into compact structures that place their numerous V gene segments in similar three-dimensional proximity to their distal recombination center composed of RAG-bound (D)J gene segments. Many studies have shown that CTCF and cohesin protein-mediated chromosome looping have fundamental roles in lymphocyte lineage- and developmental stage-specific locus compaction as well as broad usage of V segments.

A Spontaneous RAG1 Nonsense Mutation Unveils Naturally Occurring N-Terminal Truncated RAG1 Isoforms.

The RAG1 and RAG2 proteins are essential for the assembly of Ag receptor genes in the process known as VDJ recombination, allowing for an immense diversity of lymphocyte Ag receptors. Congruent with their importance, RAG1 and RAG2 have been a focus of intense study for decades. To date, RAG1 has been studied as a single isoform; however, our identification of a spontaneous nonsense mutation in the 5' region of the mouse Rag1 gene lead us to discover N-truncated RAG1 isoforms made from internal translation initiation.

Two Successive Inversional Vβ Rearrangements on a Single Allele Can Contribute to the TCRβ Repertoire.

Mammalian TCRβ loci contain 30 Vβ gene segments upstream and in the same transcriptional orientation as two DJCβ clusters, and a downstream Vβ (TRBV31) in the opposite orientation. The textbook view is upstream Vβs rearrange only by deletion and TRBV31 rearranges only by inversion to create VβDJCβ genes. In this study, we show in mice that upstream Vβs recombine through inversion to the DJCβ2 cluster on alleles carrying a preassembled -DJCβ1 gene.

V(D)J Recombination Exploits DNA Damage Responses to Promote Immunity.

It has been recognized for 40 years that the variable (diversity) joining [V(D)J] recombination-mediated assembly of diverse B and T lymphocyte antigen receptor (AgR) genes is not only essential for adaptive immunity, but also a risk for autoimmunity and lymphoid malignancies. Over the past few years, several studies have revealed that recombination-activating gene (RAG) endonuclease-induced DNA double-strand breaks (DSBs) transcend hazardous intermediates during antigen receptor gene assembly. RAG cleavage within the genomes of lymphocyte progenitors and immature lymphocytes regulates the expression of ubiquitous and lymphocyte-specific gene transcripts to control the differentiation and function of both adaptive and innate immune cell lineages.

Genomic Alterations of Non-Coding Regions Underlie Human Cancer: Lessons from T-ALL.

It has been appreciated for decades that somatic genomic alterations that change coding sequences of proto-oncogenes, translocate enhancers/promoters near proto-oncogenes, or create fusion oncogenes can drive cancer by inducing oncogenic activities. An explosion of genome-wide technologies over the past decade has fueled discoveries of the roles of three-dimensional chromosome structure and powerful cis-acting elements (super-enhancers) in regulating gene transcription. In recent years, studies of human T cell acute lymphoblastic leukemia (T-ALL) using genome-wide technologies have provided paradigms for how non-coding genomic region alterations can disrupt 3D chromosome architecture or establish super-enhancers to activate oncogenic transcription of proto-oncogenes.

Immature Lymphocytes Inhibit and Transcription and V(D)J Recombination in Response to DNA Double-Strand Breaks.

Mammalian cells have evolved a common DNA damage response (DDR) that sustains cellular function, maintains genomic integrity, and suppresses malignant transformation. In pre-B cells, DNA double-strand breaks (DSBs) induced at loci by the Rag1/Rag2 (RAG) endonuclease engage this DDR to modulate transcription of genes that regulate lymphocyte-specific processes. We previously reported that RAG DSBs induced at one allele signal through the ataxia telangiectasia mutated (ATM) kinase to feedback-inhibit RAG expression and RAG cleavage of the other allele.

Lymphocyte lineage-specific and developmental stage specific mechanisms suppress cyclin D3 expression in response to DNA double strand breaks.

Mammalian cells are thought to protect themselves and their host organisms from DNA double strand breaks (DSBs) through universal mechanisms that restrain cellular proliferation until DNA is repaired. The Cyclin D3 protein drives G1-to-S cell cycle progression and is required for proliferation of immature T and B cells and of mature B cells during a T cell-dependent immune response. We demonstrate that mouse thymocytes and pre-B cells, but not mature B cells, repress Cyclin D3 protein levels in response to DSBs.

RAG-mediated DNA double-strand breaks activate a cell type-specific checkpoint to inhibit pre-B cell receptor signals.

DNA double-strand breaks (DSBs) activate a canonical DNA damage response, including highly conserved cell cycle checkpoint pathways that prevent cells with DSBs from progressing through the cell cycle. In developing B cells, pre-B cell receptor (pre-BCR) signals initiate immunoglobulin light (Igl) chain gene assembly, leading to RAG-mediated DNA DSBs. The pre-BCR also promotes cell cycle entry, which could cause aberrant DSB repair and genome instability in pre-B cells.

Defining ATM-Independent Functions of the Mre11 Complex with a Novel Mouse Model.

Unlabelled: The Mre11 complex (Mre11, Rad50, and Nbs1) occupies a central node of the DNA damage response (DDR) network and is required for ATM activation in response to DNA damage. Hypomorphic alleles of MRE11 and NBS1 confer embryonic lethality in ATM-deficient mice, indicating that the complex exerts ATM-independent functions that are essential when ATM is absent. To delineate those functions, a conditional ATM allele (ATM(flox)) was crossed to hypomorphic NBS1 mutants (Nbs1(ΔB/ΔB) mice).

Regulation of Tcrb Gene Assembly by Genetic, Epigenetic, and Topological Mechanisms.

The adaptive immune system endows mammals with an ability to recognize nearly any foreign invader through antigen receptors that are expressed on the surface of all lymphocytes. This defense network is generated by V(D)J recombination, a set of sequentially controlled DNA cleavage and repair events that assemble antigen receptor genes from physically separated variable (V), joining (J), and sometimes diversity (D) gene segments. The recombination process itself must be stringently regulated to minimize oncogenic translocations involving chromosomes that harbor immunoglobulin and T cell receptor loci.