Katherine S. Yang-Iott

Formation of Dynamic γ-H2AX Domains along Broken DNA Strands Is Distinctly Regulated by ATM and MDC1 and Dependent upon H2AX Densities in Chromatin

A hallmark of the cellular response to DNA double-strand breaks (DSBs) is histone H2AX phosphorylation in chromatin to generate gamma-H2AX. Here, we demonstrate that gamma-H2AX densities increase transiently along DNA strands as they are broken and repaired in G1 phase cells. The region across which gamma-H2AX forms does not spread as DSBs persist; rather, gamma-H2AX densities equilibrate at distinct levels within a fixed distance from DNA ends. Although both ATM and DNA-PKcs generate gamma-H2AX, only ATM promotes gamma-H2AX formation to maximal distance and maintains gamma-H2AX densities. MDC1 is essential for gamma-H2AX formation at high densities near DSBs, but not for generation of gamma-H2AX over distal sequences. Reduced H2AX levels in chromatin impair the density, but not the distance, of gamma-H2AX formed. Our data suggest that H2AX fuels a gamma-H2AX self-reinforcing mechanism that retains MDC1 and activated ATM in chromatin near DSBs and promotes continued local phosphorylation of H2AX.

Histone H2AX stabilizes broken DNA strands to suppress chromosome breaks and translocations during V(D)J recombination

The H2AX core histone variant is phosphorylated in chromatin around DNA double strand breaks (DSBs) and functions through unknown mechanisms to suppress antigen receptor locus translocations during V(D)J recombination. Formation of chromosomal coding joins and suppression of translocations involves the ataxia telangiectasia mutated and DNA-dependent protein kinase catalytic subunit serine/threonine kinases, each of which phosphorylates H2AX along cleaved antigen receptor loci. Using Abelson transformed pre-B cell lines, we find that H2AX is not required for coding join formation within chromosomal V(D)J recombination substrates. Yet we show that H2AX is phosphorylated along cleaved Igkappa DNA strands and prevents their separation in G1 phase cells and their progression into chromosome breaks and translocations after cellular proliferation. We also show that H2AX prevents chromosome breaks emanating from unrepaired RAG endonuclease-generated TCR-alpha/delta locus coding ends in primary thymocytes. Our data indicate that histone H2AX suppresses translocations during V(D)J recombination by creating chromatin modifications that stabilize disrupted antigen receptor locus DNA strands to prevent their irreversible dissociation. We propose that such H2AX-dependent mechanisms could function at additional chromosomal locations to facilitate the joining of DNA ends generated by other types of DSBs

Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element

Majumder et al. explore the large-scale looping architecture of the Tcrb locus early in murine thymocyte development during the generation of TCRβ diversity. They dissect novel DNA regulatory elements controlling V to D-J recombination and identify within an insulator region a distally located CTCF-containing element functioning as a tether, which facilitates looping of distal Vβ to Dβ-Jβ regions and promotes locus contraction. A second CTCF-containing element, proximal to the Dβ-Jβ region, acts as a boundary, preventing the spread of active chromatin associated with Dβ-Jβ regions. Removal of the proximal boundary element impairs the locus contraction capabilities of the tethering element.

The Ataxia Telangiectasia mutated kinase controls Igκ allelic exclusion by inhibiting secondary Vκ-to-Jκ rearrangements

DNA double-strand breaks induced during Igκ recombination signal through ATM to suppress the initiation of additional Vκ-to-Jκ rearrangements.

Tp53 deletion in B lineage cells predisposes mice to lymphomas with oncogenic translocations.

Inactivating Tp53 mutations are frequent genetic lesions in human tumors that harbor genomic instability, including B lineage lymphomas with IG translocations. Antigen receptor genes are assembled and modified in developing lymphocytes by RAG/AID-initiated genomic rearrangements that involve the induction of DNA double strand breaks (DSBs). Although TP53 inhibits the persistence of DSBs and induces apoptosis to protect cells from genomic instability and transformation, the development of spontaneous tumors harboring clonal translocations has not been reported in mice that only lack wild-type Tp53 protein or express Tp53 mutants. Tp53-deficient (Tp53−/−) mice succumb to T lineage lymphomas lacking clonal translocations but develop B lymphoid tumors containing immunoglobulin (Ig) translocations upon combined inactivation of DSB repair factors, RAG mutation or AID overexpression; mice expressing apoptosis-defective Tp53 mutants develop B cell lymphomas that have not been characterized for potential genomic instability. As somatic rather than germline inactivating mutations of TP53 are typically associated with human cancers and Tp53 deletion has cellular context dependent effects upon lymphocyte transformation, we generated mice with conditional Tp53 deletion in lineage-committed B lymphocytes to avoid complications associated with defective Tp53 responses during embryogenesis and/or in multi-lineage potential cells and, thereby, directly evaluate the potential physiological role of Tp53 in suppressing translocations in differentiated cells. These mb1-cre:Tp53flox/flox mice succumbed to lymphoid tumors containing Ig gene rearrangements and immunophenotypes characteristic of B cells from various developmental stages. Most mb1-cre:Tp53flox/flox tumors harbored clonal translocations, including Igh/c-myc or other oncogenic translocations generated by the aberrant repair of RAG/AID-generated DSBs. Our data indicate that Tp53 serves critical functions in B lineage lymphocytes to prevent transformation caused by translocations in cell populations experiencing physiological levels of RAG/AID-initiated DSB intermediates, and provide evidence that the somatic TP53 mutations found in diffuse large B-cell lymphoma and Burkitts lymphoma may contribute to the development of these human malignancies.

TCRβ Feedback Signals Inhibit the Coupling of Recombinationally Accessible Vβ14 Segments with DJβ Complexes

Ag receptor allelic exclusion is thought to occur through monoallelic initiation and subsequent feedback inhibition of recombinational accessibility. However, our previous analysis of mice containing a V(D)J recombination reporter inserted into Vβ14 (Vβ14Rep) indicated that Vβ14 chromatin accessibility is biallelic. To determine whether Vβ14 recombinational accessibility is subject to feedback inhibition, we analyzed TCRβ rearrangements in Vβ14Rep mice containing a preassembled in-frame transgenic Vβ8.2Dβ1Jβ1.1 or an endogenous Vβ14Dβ1Jβ1.4 rearrangement on the homologous chromosome. Expression of either preassembled VβDJβC β-chain accelerated thymocyte development because of enhanced cellular selection, demonstrating that the rate-limiting step in early αβ T cell development is the assembly of an in-frame VβDJβ rearrangement. Expression of these preassembled VβDJβ rearrangements inhibited endogenous Vβ14-to-DJβ rearrangements as expected. However, in contrast to results predicted by the accepted model of TCRβ feedback inhibition, we found that expression of these preassembled TCR β-chains did not downregulate recombinational accessibility of Vβ14 chromatin. Our findings suggest that TCRβ-mediated feedback inhibition of Vβ14 rearrangements depends on inherent properties of Vβ14, Dβ, and Jβ recombination signal sequences.

The Ataxia Telangiectasia Mutated and Cyclin D3 Proteins Cooperate To Help Enforce TCRβ and IgH Allelic Exclusion

Coordination of V rearrangements between loci on homologous chromosomes is critical for Ig and TCR allelic exclusion. The Ataxia Telangietasia mutated (ATM) protein kinase promotes DNA repair and activates checkpoints to suppress aberrant Ig and TCR rearrangements. In response to RAG cleavage of Igκ loci, ATM inhibits RAG expression and suppresses further Vκ-to-Jκ rearrangements to enforce Igκ allelic exclusion. Because V recombination between alleles is more strictly regulated for TCRβ and IgH loci, we evaluated the ability of ATM to restrict biallelic expression and V-to-DJ recombination of TCRβ and IgH genes. We detected greater frequencies of lymphocytes with biallelic expression or aberrant V-to-DJ rearrangement of TCRβ or IgH loci in mice lacking ATM. A preassembled DJβ complex that decreases the number of TCRβ rearrangements needed for a productive TCRβ gene further increased frequencies of ATM-deficient cells with biallelic TCRβ expression. IgH and TCRβ proteins drive proliferation of prolymphocytes through cyclin D3 (Ccnd3), which also inhibits VH transcription. We show that inactivation of Ccnd3 leads to increased frequencies of lymphocytes with biallelic expression of IgH or TCRβ genes. We also show that Ccnd3 inactivation cooperates with ATM deficiency to increase the frequencies of cells with biallelic TCRβ or IgH expression while decreasing the frequency of ATM-deficient lymphocytes with aberrant V-to-DJ recombination. Our data demonstrate that core components of the DNA damage response and cell cycle machinery cooperate to help enforce IgH and TCRβ allelic exclusion and indicate that control of V-to-DJ rearrangements between alleles is important to maintain genomic stability.

Novel Mechanism of Tumor Suppression by Polarity Gene Discs Large 1 (DLG1) Revealed in a Murine Model of Pediatric B-ALL

Using two different murine models of pre–B-cell leukemia, Sandoval and colleagues delineate the mechanism of tumor suppression by the PDZ domain polarity gene DLG1, which interacts with and stabilizes the PTEN protein in early-stage B cells. Drosophila melanogaster discs large (dlg) is an essential tumor suppressor gene (TSG) controlling epithelial cell growth and polarity of the fly imaginal discs in pupal development. A mammalian ortholog, Dlg1, is involved in embryonic urogenital morphogenesis, postsynaptic densities in neurons, and immune synapses in lymphocytes. However, a potential role for Dlg1 as a mammalian TSG is unknown. Here, we present evidence that loss of Dlg1 confers strong predisposition to the development of malignancies in a murine model of pediatric B-cell acute lymphoblastic leukemia (B-ALL). Using mice with conditionally deleted Dlg1 alleles, we identify a novel “pre-leukemic” stage of developmentally arrested early B-lineage cells marked by preeminent c-Myc expression. Mechanistically, we show that in B-lineage progenitors Dlg1 interacts with and stabilizes the PTEN protein, regulating its half-life and steady-state abundance. The loss of Dlg1 does not affect the level of PTEN mRNAs but results in a dramatic decrease in PTEN protein, leading to excessive phosphoinositide 3-kinase signaling and proliferation. Our data suggest a novel model of tumor suppression by a PDZ domain-containing polarity gene in hematopoietic cancers. Cancer Immunol Res; 1(6); 426–37. ©2013 AACR.

Position-Dependent Silencing of Germline Vβ Segments on TCRβ Alleles Containing Preassembled VβDJβCβ1 Genes

The genomic organization of TCRβ loci enables Vβ-to-DJβ2 rearrangements on alleles with assembled VβDJβCβ1 genes, which could have deleterious physiologic consequences. To determine whether such Vβ rearrangements occur and, if so, how they might be regulated, we analyzed mice with TCRβ alleles containing preassembled functional VβDJβCβ1 genes. Vβ10 segments were transcribed, rearranged, and expressed in thymocytes when located immediately upstream of a Vβ1DJβCβ1 gene, but not on alleles with a Vβ14DJβCβ1 gene. Germline Vβ10 transcription was silenced in mature αβ T cells. This allele-dependent and developmental stage-specific silencing of Vβ10 correlated with increased CpG methylation and decreased histone acetylation over the Vβ10 promoter and coding region. Transcription, rearrangement, and expression of the Vβ4 and Vβ16 segments located upstream of Vβ10 were silenced on alleles containing either VβDJβCβ1 gene; sequences within Vβ4, Vβ16, and the Vβ4/Vβ16-Vβ10 intergenic region exhibited constitutive high CpG methylation and low histone acetylation. Collectively, our data indicate that the position of Vβ segments relative to assembled VβDJβCβ1 genes influences their rearrangement and suggest that DNA sequences between Vβ segments may form boundaries between active and inactive Vβ chromatin domains upstream of VβDJβCβ genes.

Assembled DJβ Complexes Influence TCRβ Chain Selection and Peripheral Vβ Repertoire

TCRβ chain repertoire of peripheral αβ T cells is generated through the stepwise assembly and subsequent selection of TCRβ V region exons during thymocyte development. To evaluate the influence of a two-step recombination process on Vβ rearrangement and selection, we generated mice with a preassembled Dβ1Jβ1.1 complex on the Jβ1ω allele, an endogenous TCRβ allele that lacks the Dβ2-Jβ2 cluster, creating the Jβ1DJβ allele. As compared with Jβ1ω/ω mice, both Jβ1DJβ/ω and Jβ1DJβ/DJβ mice exhibited grossly normal thymocyte development and TCRβ allelic exclusion. In addition, Vβ rearrangements on Jβ1DJβ and Jβ1ω alleles were similarly regulated by TCRβ-mediated feedback regulation. However, in-frame VβDJβ rearrangements were present at a higher level on the Jβ1DJβ alleles of Jβ1DJβ/ω αβ T cell hybridomas, as compared with on the Jβ1ω alleles. This bias was most likely due to both an increased frequency of Vβ-to-DJβ rearrangements on Jβ1DJβ alleles and a preferential selection of cells with in-frame VβDJβ exons assembled on Jβ1DJβ alleles during the development of Jβ1DJβ/ω αβ T cells. Consistent with the differential selection of in-frame VβDJβ rearrangements on Jβ1DJβ alleles, the Vβ repertoire of αβ T cells was significantly altered during αβ TCR selection in Jβ1DJβ/ω and Jβ1DJβ/DJβ mice, as compared with in Jβ1ω/ω mice. Our data indicate that the diversity of DJβ complexes assembled during thymocyte development influences TCRβ chain selection and peripheral Vβ repertoire.