Ching-Yu Huang

ATM stabilizes DNA double-strand-break complexes during V(D)J recombination

The ATM (ataxia-telangiectasia mutated) protein kinase mediates early cellular responses to DNA double-strand breaks (DSBs) generated during metabolic processes or by DNA-damaging agents. ATM deficiency leads to ataxia-telangiectasia, a disease marked by lymphopenia, genomic instability and an increased predisposition to lymphoid malignancies with chromosomal translocations involving lymphocyte antigen receptor loci. ATM activates cell-cycle checkpoints and can induce apoptosis in response to DNA DSBs. However, defects in these pathways of the DNA damage response cannot fully account for the phenotypes of ATM deficiency. Here, we show that ATM also functions directly in the repair of chromosomal DNA DSBs by maintaining DNA ends in repair complexes generated during lymphocyte antigen receptor gene assembly. When coupled with the cell-cycle checkpoint and pro-apoptotic activities of ATM, these findings provide a molecular explanation for the increase in lymphoid tumours with translocations involving antigen receptor loci associated with ataxia-telangiectasia.

53BP1 facilitates long-range DNA end-joining during V(D)J recombination.

Variable, diversity and joining (V(D)J) recombination and class-switch recombination use overlapping but distinct non-homologous end joining pathways to repair DNA double-strand-break intermediates. 53BP1 is a DNA-damage-response protein that is rapidly recruited to sites of chromosomal double-strand breaks, where it seems to function in a subset of ataxia telangiectasia mutated (ATM) kinase-, H2A histone family member X (H2AX, also known as H2AFX)- and mediator of DNA damage checkpoint 1 (MDC1)-dependent events. A 53BP1-dependent end-joining pathway has been described that is dispensable for V(D)J recombination but essential for class-switch recombination. Here we report a previously unrecognized defect in the joining phase of V(D)J recombination in 53BP1-deficient lymphocytes that is distinct from that found in classical non-homologous-end-joining-, H2ax-, Mdc1- and Atm-deficient mice. Absence of 53BP1 leads to impairment of distal V–DJ joining with extensive degradation of unrepaired coding ends and episomal signal joint reintegration at V(D)J junctions. This results in apoptosis, loss of T-cell receptor α locus integrity and lymphopenia. Further impairment of the apoptotic checkpoint causes propagation of lymphocytes that have antigen receptor breaks. These data suggest a more general role for 53BP1 in maintaining genomic stability during long-range joining of DNA breaks.

Dynamic regulation of c-Myc proto-oncogene expression during lymphocyte development revealed by a GFP-c-Myc knock-in mouse

c‐Myc induces widely varying cellular effects, including cell proliferation and cell death. These different cellular effects are determined, in part, by c‐Myc protein expression levels, which are regulated through several transcriptional and post‐transcriptional pathways. c‐Myc transcripts can be detected in cells at all stages of B and T lymphocyte development. However, little is known about c‐Myc protein expression, and how it varies, in developing lymphocytes. Here mice have been generated in which the endogenous c‐Myc locus has been modified (c‐MycG) so that it encodes a GFP‐c‐Myc fusion protein. c‐MycG/G mice are viable, appear normal and exhibit grossly normal lymphocyte development. Flow cytometric analyses revealed significant heterogeneity in c‐Myc protein expression levels in developing c‐MycG/G B and T lymphocytes. GFP‐c‐Myc expression levels were highest in proliferating lymphocytes, suggesting that c‐Myc up‐regulation is important for promoting lymphocyte cell division, and demonstrating that GFP‐c‐Myc expression is a marker of proliferating lymphocytes in vivo.

Proteasome activator PA200 is required for normal spermatogenesis.

ABSTRACT The PA200 proteasome activator is a broadly expressed nuclear protein. Although how PA200 normally functions is not fully understood, it has been suggested to be involved in the repair of DNA double-strand breaks (DSBs). The PA200 gene (Psme4) is composed of 45 coding exons spanning 108 kb on mouse chromosome 11. We generated a PA200 null allele (PA200Δ) through Cre-loxP-mediated interchromosomal recombination after targeting loxP sites at either end of the locus. PA200Δ/Δ mice are viable and have no obvious developmental abnormalities. Both lymphocyte development and immunoglobulin class switching, which rely on the generation and repair of DNA DSBs, are unperturbed in PA200Δ/Δ mice. Additionally, PA200Δ/Δ embryonic stem cells do not exhibit increased sensitivity to either ionizing radiation or bleomycin. Thus, PA200 is not essential for the repair of DNA DSBs generated in these settings. Notably, loss of PA200 led to a marked reduction in male, but not female, fertility. This was due to defects in spermatogenesis observed in meiotic spermatocytes and during the maturation of postmeiotic haploid spermatids. Thus, PA200 serves an important nonredundant function during spermatogenesis, suggesting that the efficient generation of male gametes has distinct protein metabolic requirements.

Ordered and Coordinated Rearrangement of the TCR α Locus: Role of Secondary Rearrangement in Thymic Selection

The Ag receptor of the T lymphocyte is composed of an αβ heterodimer. Both α- and β-chains are products of the somatic rearrangement of V(D)J segments encoded on the respective loci. During T cell development, β-chain rearrangement precedes α-chain rearrangement. The mechanism of allelic exclusion ensures the expression of a single β-chain in each T cell, whereas a large number of T cells express two functional α-chains. Here we demonstrate evidence that TCR α rearrangement is initiated by rearranging a 3′ Vα segment and a 5′ Jα segment on both chromosomes. Rearrangement then proceeds by using upstream Vα and downstream Jα segments until it is terminated by successful positive selection. This ordered and coordinated rearrangement allows a single thymocyte to sequentially express multiple TCRs with different specificities to optimize the efficiency of positive selection. Thus, the lack of allelic exclusion and TCR α secondary rearrangement play a key role in the formation of a functional T cell repertoire.

Defects in coding joint formation in vivo in developing ATM-deficient B and T lymphocytes

Ataxia-telangiectasia mutated (ATM)–deficient lymphocytes exhibit defects in coding joint formation during V(D)J recombination in vitro. Similar defects in vivo should affect both T and B cell development, yet the lymphoid phenotypes of ATM deficiency are more pronounced in the T cell compartment. In this regard, ATM-deficient mice exhibit a preferential T lymphopenia and have an increased incidence of nontransformed and transformed T cells with T cell receptor α/δ locus translocations. We demonstrate that there is an increase in the accumulation of unrepaired coding ends during different steps of antigen receptor gene assembly at both the immunoglobulin and T cell receptor loci in developing ATM-deficient B and T lymphocytes. Furthermore, we show that the frequency of ATM-deficient αβ T cells with translocations involving the T cell receptor α/δ locus is directly related to the number of T cell receptor α rearrangements that these cells can make during development. Collectively, these findings demonstrate that ATM deficiency leads to broad defects in coding joint formation in developing B and T lymphocytes in vivo, and they provide a potential molecular explanation as to why the developmental impact of these defects could be more pronounced in the T cell compartment.

T cell receptor CDR3 loop length repertoire is determined primarily by features of the V(D)J recombination reaction.

The third complementarity‐determining region (CDR) of the TCR α and β chains forms loops that engage amino acid residues of peptides complexed with MHC. This interaction is central to the specific discrimination of antigenic‐peptide–MHC complexes by the TCR. The TCRβ chain CDR3 loop is encoded by the Dβ gene segment and flanking portions of the Vβ and Jβ gene segments. The joining of these gene segments is imprecise, leading to significant variability in the TCRβ chain CDR3 loop length and amino acid composition. In marked contrast to other pairing antigen‐receptor chains, the TCR β and α chain CDR3 loop size distributions are relatively narrow and closely matched. Thus, pairing of TCR α and β chains with relatively similar CDR3 loop sizes may be important for generating a functional repertoire of α β TCR. Here we show that the TCRβ chain CDR3 loop size distribution is minimally impacted by TCRβ chain or α β TCR selection during thymocyte development. Rather, this distribution is determined primarily at the level of variable‐region gene assembly, and is critically dependent on unique features of the V(D)J recombination reaction that ensure Dβ gene segment utilization.

Aberrant V(D)J Recombination in Ataxia Telangiectasia Mutated-Deficient Lymphocytes Is Dependent on Nonhomologous DNA End Joining

During lymphocyte Ag receptor gene assembly, DNA cleavage by the Rag proteins generates pairs of coding and signal ends that are normally joined into coding joints and signal joints, respectively, by the classical nonhomologous end-joining (NHEJ) pathway of DNA double strand break repair. Coding and signal ends can also be aberrantly joined to each other, generating hybrid joints, through NHEJ or through NHEJ-independent pathways, such as Rag-mediated transposition. Hybrid joints do not participate in the formation of functional Ag receptor genes and can alter the configuration of Ag receptor loci in ways that limit subsequent productive rearrangements. The formation of these nonfunctional hybrid joints occurs rarely in wild type lymphocytes, demonstrating that mechanisms exist to limit both the NHEJ-dependent and the NHEJ-independent joining of a signal end to a coding end. In contrast to wild-type cells, hybrid joint formation occurs at high levels in ataxia telangiectasia mutated (Atm)-deficient lymphocytes, suggesting that Atm functions to limit the formation of these aberrant joints. In this study, we show that hybrid joint formation in Atm-deficient cells requires the NHEJ proteins Artemis, DNA-PKcs, and Ku70, demonstrating that Atm functions primarily by modulating the NHEJ-dependent, and not the NHEJ-independent, joining of coding ends to signal ends.

Superantigen-Induced TCR α Locus Secondary Rearrangement: Role in Tolerance Induction

Immunization with superantigen in vivo induces transient activation of superantigen-specific T cells, followed by a superantigen-nonresponsive state. In this study, using a TCR α knock-in mouse in which the knock-in α-chain can be replaced with endogenous α-chain through secondary rearrangement, we show that immunization of superantigen changes the TCR α-chain expression on peripheral superantigen-specific T cells, induces expression of recombination-activating genes, and generates DNA double-strand breaks at the TCR α-chain locus. These results suggest that viral superantigens are capable of inducing peripheral TCR revision. Our findings thus provide a new perspective on pathogen-immune system interaction.

Impact of early expression of TCR α chain on thymocyte development

CD4–CD8– thymocytes expressing a transgenic T cell receptor (TCR) α  chain have decreased capacity to give rise to CD4+CD8+ thymocytes when compared with wild‐type thymocytes. This inefficient CD4–CD8– to CD4+CD8+ maturation is mediated by the transgenic TCR α  chain pairing with endogenous TCR β  chain but not with endogenous TCR γ  chain. Comparison between TCR α  chain‐transgenic mice with or without a functional pre‐TCR α  (pTα ) chain reveals that the formation of transgenic α /endogenous β  TCR on CD4–CD8– thymocytes inhibits the formation of pre‐TCR, but at the same time mediates CD4–CD8– to CD4+CD8+ maturation in the absence of pre‐TCR, albeit inefficiently. These results indicate that α β  TCR and pre‐TCR provide different signals for thymocyte development. They also suggest that the precise regulation of the sequential rearrangements of TCR β  and α  loci and the cellular expansion induced by the pre‐TCR may both be evolved to ensure the efficient generation of mature α β  T cells.