A Protein Binding Specifically to the IgG2b Switch Region

The Abelson-virus-transformed mouse pre-B-cell line 18-81 switches almost exclusively from μ to γ2b. From nuclear extracts of this cell line, we have isolated a factor that specifically binds to Sγ2b. After an eight-step purification scheme, in which different types of DNA-affinity chromatography were used as key elements, we obtained a preparation with two narrowly spaced bands at approximately 69 kD on a silver-stained SDS gel. Binding specificity of main-peak fractions of affinity-purified proteins was analyzed by gel shift assays in which Sγ2b, but not Sμ, competes. The results are consistent with this factor being part of the switch recombinase

Novel principles of gamma-retroviral insertional transcription activation in murine leukemia virus-induced end-stage tumors

BACKGROUND: Insertional mutagenesis screens of retrovirus-induced mouse tumors have proven valuable in human cancer research and for understanding adverse effects of retroviral-based gene therapies. In previous studies, the assignment of mouse genes to individual retroviral integration sites has been based on close proximity and expression patterns of annotated genes at target positions in the genome. We here employed next-generation RNA sequencing to map retroviral-mouse chimeric junctions genome-wide, and to identify local patterns of transcription activation in T-lymphomas induced by the murine leukemia gamma-retrovirus SL3-3. Moreover, to determine epigenetic integration preferences underlying long-range gene activation by retroviruses, the colocalization propensity with common epigenetic enhancer markers (H3K4Me1 and H3K27Ac) of 6,117 integrations derived from end-stage tumors of more than 2,000 mice was examined. RESULTS: We detected several novel mechanisms of retroviral insertional mutagenesis: bidirectional activation of mouse transcripts on opposite sides of a provirus including transcription of unannotated mouse sequence; sense/antisense-type activation of genes located on opposite DNA strands; tandem-type activation of distal genes that are positioned adjacently on the same DNA strand; activation of genes that are not the direct integration targets; combination-type insertional mutagenesis, in which enhancer activation, alternative chimeric splicing and retroviral promoter insertion are induced by a single retrovirus. We also show that irrespective of the distance to transcription start sites, the far majority of retroviruses in end-stage tumors colocalize with H3K4Me1 and H3K27Ac-enriched regions in murine lymphoid tissues. CONCLUSIONS: We expose novel retrovirus-induced host transcription activation patterns that reach beyond a single and nearest annotated gene target. Awareness of this previously undescribed layer of complexity may prove important for elucidation of adverse effects in retroviral-based gene therapies. We also show that wild-type gamma-retroviruses are frequently positioned at enhancers, suggesting that integration into regulatory regions is specific and also subject to positive selection for sustaining long-range gene activation in end-stage tumors. Altogether, this study should prove useful for extrapolating adverse outcomes of retroviral vector therapies, and for understanding fundamental cellular regulatory principles and retroviral biology

An autoimmune disease prevented by anti-retroviral drugs

<p>Abstract</p> <p>Background</p> <p>Both Aicardi-Goutières syndrome, a Mendelian mimic of congenital infection, and the autoimmune disease systemic lupus erythematosus can result from mutations in the gene encoding the enzyme Trex1. In mice, the absence of Trex1 causes severe myocarditis. The enzyme is thought to degrade endogenous retroelements, thus linking them to autoimmune disease. However, inhibition of reverse transcription by the inhibitor zidovudine (AZT) did not ameliorate the disease, weakening the link to retroelements.</p> <p>Findings</p> <p>Here, we show that two other FDA-approved drugs that inhibit reverse transcriptase can ameliorate the myocarditis in Trex1-null mouse.</p> <p>Conclusions</p> <p>The result suggests that retroelements contribute to this hereditary form of autoimmunity, and that treatment with retroelement inhibitors might ameliorate Aicardi-Goutières syndrome in humans.</p

Slow, stochastic transgene repression with properties of a timer

BACKGROUND: When gene expression varies unpredictably between genetically identical organisms, this is sometimes ascribed as stochastic. With the prevalence of retroviral vectors, stochastic repression is often observed and can complicate the interpretation of outcomes. But it may also faithfully reflect characteristics of sites in the genome. RESULTS: We created and identified several cell clones in which, within a given cell, retroviral transcription of a transgene was repressed heritably and essentially irreversibly. This repression was relatively slow; total repression in all cells took months. We observed the dynamics of repression and found that they were ergodic, that is, tending with a probability to a final state independent of previous conditions. Different positions of the transgene in the genome demonstrated different dynamics. At a position on mouse chromosome 9, repression abided by near perfect first-order kinetics and was highly reproducible, even under conditions where the number of cell generations per day varied. CONCLUSION: We propose that such a cell division independent 'off' mechanism could play a role in endogenous gene expression, potentially providing an epigenetically based timer for extended periods

Two Forms of Activation-Induced Cytidine Deaminase Differing in Their Ability to Bind Agarose

Background: Activation-induced cytidine deaminase (AID) is a B-cell-specific DNA mutator that plays a key role in the formation of the secondary antibody repertoire in germinal center B cells. In the search for binding partners, protein coimmunoprecipitation assays are often performed, generally with agarose beads. Methodology/Principal Findings: We found that, regardless of whether cell lysates containing exogenous or endogenous AID were examined, one of two mouse AID forms bound to agarose alone. Conclusions/Significance: These binding characteristics may be due to the known post-translational modifications of AID; they may also need to be considered in coimmunoprecipitation experiments to avoid false-positive results

Identification of novel Bach2 transcripts and protein isoforms through tagging analysis of retroviral integrations in B-cell lymphomas

BACKGROUND: The Bach2 gene functions as a transcriptional repressor in B-cells, showing high expression level only before the plasma cell stage. Several lines of evidence indicate that Bach2 is a B-cell specific tumor suppressor. We here address patterns of insertional mutagenesis and expression of Bach2 is a murine retroviral model of B-cell lymphoma induction. RESULTS: We report that the Bach2 gene is a target of proviral integrations in B-cell lymphomas induced by murine leukemia virus. An alternative Bach2 promoter was identified within intron 2 and this promoter was activated in one of the tumors harboring proviral integration. The alternative promoter was active in both normal and tumor tissue and the tissue specificity of the two Bach2 promoters was similar. Three different alternatively used Bach2 terminal exons were identified to be located in intron 4. The inclusion of these exons resulted in the generation of Bach2 mRNA with open reading frames lacking the bZIP DNA binding domain present in the normal Bach2 protein, but retaining a partial BTB protein dimerization domain. Such Bach2 protein was excluded from the cell nucleus. CONCLUSION: We have identified an alternative promoter and new protein isoforms of Bach2. Our data imply that activation of an alternative promoter by proviral integration serves as a possible mechanism of up-regulation of the Bach2 gene with a potential role in B-cell lymphomagenesis. The finding of novel Bach2 transcripts and protein isoforms will facilitate a better insight into the normal and pathophysiological regulation of the Bach2 gene

Retroviral activation of the mir-106a microRNA cistron in T lymphoma

Retroviral insertion into a host genome is a powerful tool not only for the discovery of cancer genes, but also for the discovery of potential oncogenic noncoding RNAs. In a large-scale mouse T lymphocyte tumor screen we found a high density of integrations upstream of the mir-106a microRNA cistron. In tumors containing an integration, the primary transcript encoding the mir-106a cistron was overexpressed five to 20-fold compared with that of control tumors; concomitantly, the mature mir-106a and mir-363 microRNAs were highly overexpressed as well. These findings suggest the mir-106a cistron plays an important role in T cell tumorigenesis

A Mouse with a Monoclonal Primary lmmunoglobulin Repertoire not Further Diversified by V-Gene Replacement

We have generated a monoclonal B-cell mouse by introducing homozygous, nonfunctional RAG-2 alleles and a λ1 light-chain transgene into the quasi-monoclonal (QM) mouse, which contains a “knocked-in” VHDJH rearrangement. Thus, this mouse, which we call MonoB, is devoid of T cells and contains preformed heavy- and light-chain genes encoding immunoglobulin with an anti-NP specificity. The MonoB mouse allows us to examine immunoglobulin diversity in the absence of processes mediated by V(D)J recombination and T cells. Here we report that not only is the MonoB's primary immunoglobulin repertoire monoclonal, but also that its secondary repertoire is not further diversified by V-gene replacement or gene conversion. Among 99 heavy-chain and 41 λ light-chain genes from peripheral B cells of the MonoB mouse, there were no V-gene replacements. When compared to the QM mouse, which has RAG activity, and for which V-gene replacement is the major diversifying mechanism, these data suggest that V-gene replacement is mediated by V(D)J recombination and not by other recombination systems

Pvt1-encoded microRNAs in oncogenesis

<p>Abstract</p> <p>Background</p> <p>The functional significance of the <it>Pvt1 </it>locus in the oncogenesis of Burkitt's lymphoma and plasmacytomas has remained a puzzle. In these tumors, <it>Pvt1 </it>is the site of reciprocal translocations to immunoglobulin loci. Although the locus encodes a number of alternative transcripts, no protein or regulatory RNA products were found. The recent identification of non-coding microRNAs encoded within the <it>PVT1 </it>region has suggested a regulatory role for this locus.</p> <p>Results</p> <p>The mouse <it>Pvt1 </it>locus encodes several microRNAs. In mouse T cell lymphomas induced by retroviral insertions into the locus, the <it>Pvt1 </it>transcripts, and at least one of their microRNA products, mmu-miR-1204 are overexpressed. Whereas up to seven co-mutations can be found in a single tumor, in over 2,000 tumors none had insertions into both the <it>Myc </it>and <it>Pvt1 </it>loci.</p> <p>Conclusion</p> <p>Judging from the large number of integrations into the <it>Pvt1 </it>locus – more than in the nearby <it>Myc </it>locus – <it>Pvt1 </it>and the microRNAs encoded by it are as important as <it>Myc </it>in T lymphomagenesis, and, presumably, in T cell activation. An analysis of the co-mutations in the lymphomas likely place <it>Pvt1 </it>and <it>Myc </it>into the same pathway.</p