Extensive Gene-Specific Translational Reprogramming in a Model of B Cell Differentiation and Abl-Dependent Transformation

To what extent might the regulation of translation contribute to differentiation programs, or to the molecular pathogenesis of cancer? Pre-B cells transformed with the viral oncogene v-Abl are suspended in an immortalized, cycling state that mimics leukemias with a BCR-ABL1 translocation, such as Chronic Myelogenous Leukemia (CML) and Acute Lymphoblastic Leukemia (ALL). Inhibition of the oncogenic Abl kinase with imatinib reverses transformation, allowing progression to the next stage of B cell development. We employed a genome-wide polysome profiling assay called Gradient Encoding to investigate the extent and potential contribution of translational regulation to transformation and differentiation in v-Abl-transformed pre-B cells. Over half of the significantly translationally regulated genes did not change significantly at the level of mRNA abundance, revealing biology that might have been missed by measuring changes in transcript abundance alone. We found extensive, gene-specific changes in translation affecting genes with known roles in B cell signaling and differentiation, cancerous transformation, and cytoskeletal reorganization potentially affecting adhesion. These results highlight a major role for gene-specific translational regulation in remodeling the gene expression program in differentiation and malignant transformation

Initiation of V(D)J Recombination by Dβ-Associated Recombination Signal Sequences: A Critical Control Point in TCRβ Gene Assembly

T cell receptor (TCR) β gene assembly by V(D)J recombination proceeds via successive Dβ-to-Jβ and Vβ-to-DJβ rearrangements. This two-step process is enforced by a constraint, termed beyond (B)12/23, which prohibits direct Vβ-to-Jβ rearrangements. However the B12/23 restriction does not explain the order of TCRβ assembly for which the regulation remains an unresolved issue. The initiation of V(D)J recombination consists of the introduction of single-strand DNA nicks at recombination signal sequences (RSSs) containing a 12 base-pairs spacer. An RSS containing a 23 base-pairs spacer is then captured to form a 12/23 RSSs synapse leading to coupled DNA cleavage. Herein, we probed RSS nicks at the TCRβ locus and found that nicks were only detectable at Dβ-associated RSSs. This pattern implies that Dβ 12RSS and, unexpectedly, Dβ 23RSS initiate V(D)J recombination and capture their respective Vβ or Jβ RSS partner. Using both in vitro and in vivo assays, we further demonstrate that the Dβ1 23RSS impedes cleavage at the adjacent Dβ1 12RSS and consequently Vβ-to-Dβ1 rearrangement first requires the Dβ1 23RSS excision. Altogether, our results provide the molecular explanation to the B12/23 constraint and also uncover a ‘Dβ1 23RSS-mediated’ restriction operating beyond chromatin accessibility, which directs Dβ1 ordered rearrangements

Haplotypes of DNA repair and cell cycle control genes, X-ray exposure, and risk of childhood acute lymphoblastic leukemia

[[abstract]]Background: Acute leukemias of childhood are a heterogeneous group of malignancies characterized by cytogenetic abnormalities, such as translocations and changes in ploidy. These abnormalities may be influenced by altered DNA repair and cell cycle control processes. Methods: We examined the association between childhood acute lymphoblastic leukemia (ALL) and 32 genes in DNA repair and cell cycle pathways using a haplotype-based approach, among 377 childhood ALL cases and 448 controls enrolled during 1995-2002. Results: We found that haplotypes in APEX1, BRCA2, ERCC2, and RAD51 were significantly associated with total ALL, while haplotypes in NBN and XRCC4, and CDKN2A were associated with structural and numerical change subtypes, respectively. In addition, we observed statistically significant interaction between exposure to 3 or more diagnostic X-rays and haplotypes of XRCC4 on risk of structural abnormality-positive childhood ALL. Conclusions: These results support a role of altered DNA repair and cell cycle processes in the risk of childhood ALL, and show that this genetic susceptibility can differ by cytogenetic subtype and may be modified by exposure to ionizing radiation. To our knowledge, our study is the first to broadly examine the DNA repair and cell cycle pathways using a haplotype approach in conjunction with X-ray exposures in childhood ALL risk. If confirmed, future studies are needed to identify specific functional SNPs in the regions of interest identified in this analysis

Silencing and Nuclear Repositioning of the λ5 Gene Locus at the Pre-B Cell Stage Requires Aiolos and OBF-1

The chromatin regulator Aiolos and the transcriptional coactivator OBF-1 have been implicated in regulating aspects of B cell maturation and activation. Mice lacking either of these factors have a largely normal early B cell development. However, when both factors are eliminated simultaneously a block is uncovered at the transition between pre-B and immature B cells, indicating that these proteins exert a critical function in developing B lymphocytes. In mice deficient for Aiolos and OBF-1, the numbers of immature B cells are reduced, small pre-BII cells are increased and a significant impairment in immunoglobulin light chain DNA rearrangement is observed. We identified genes whose expression is deregulated in the pre-B cell compartment of these mice. In particular, we found that components of the pre-BCR, such as the surrogate light chain genes λ5 and VpreB, fail to be efficiently silenced in double-mutant mice. Strikingly, developmentally regulated nuclear repositioning of the λ5 gene is impaired in pre-B cells lacking OBF-1 and Aiolos. These studies uncover a novel role for OBF-1 and Aiolos in controlling the transcription and nuclear organization of genes involved in pre-BCR function

Transcriptomic analysis supports similar functional roles for the two thymuses of the tammar wallaby

Background: The thymus plays a critical role in the development and maturation of T-cells. Humans have a single thoracic thymus and presence of a second thymus is considered an anomaly. However, many vertebrates have multiple thymuses. The tammar wallaby has two thymuses: a thoracic thymus (typically found in all mammals) and a dominant cervical thymus. Researchers have known about the presence of the two wallaby thymuses since the 1800s, but no genome-wide research has been carried out into possible functional differences between the two thymic tissues. Here, we used pyrosequencing to compare the transcriptomes of a cervical and thoracic thymus from a single 178 day old tammar wallaby.Results: We show that both the tammar thoracic and the cervical thymuses displayed gene expression profiles consistent with roles in T-cell development. Both thymuses expressed genes that mediate distinct phases of T-cells differentiation, including the initial commitment of blood stem cells to the T-lineage, the generation of T-cell receptor diversity and development of thymic epithelial cells. Crucial immune genes, such as chemokines were also present. Comparable patterns of expression of non-coding RNAs were seen. 67 genes differentially expressed between the two thymuses were detected, and the possible significance of these results are discussed.Conclusion: This is the first study comparing the transcriptomes of two thymuses from a single individual. Our finding supports that both thymuses are functionally equivalent and drive T-cell development. These results are an important first step in the understanding of the genetic processes that govern marsupial immunity, and also allow us to begin to trace the evolution of the mammalian immune system

Distinct and opposite diversifying activities of terminal transferase splice variants

The short splice variant of mouse terminal deoxynucleotidyl transferase (TdTS) catalyzes the addition of nontemplated nucleotides (N addition) at the coding joins of B cell and T cell antigen receptor genes. However, the activity and function of the long isoform of TdT (TdTL) have not been determined. We show here, in vitro and in vivo, that TdTL is a 3'-->5' exonuclease that catalyzes the deletion of nucleotides at coding joins. These findings suggest that the two TdT isoforms may act in concert to preserve the integrity of the variable region of antigen receptors while generating diversity