Human platelets express 2 thrombin receptors: protease-activated receptor (PAR)-1 and PAR4. Recently, we reported 3.7-fold increased PAR4-mediated aggregation kinetics in platelets from black ...subjects compared with white subjects. We now show that platelets from blacks (n = 70) express 14% more PAR4 protein than those from whites (n = 84), but this difference is not associated with platelet PAR4 function. Quantitative trait locus analysis identified 3 common single nucleotide polymorphisms in the PAR4 gene (F2RL3) associated with PAR4-induced platelet aggregation. Among these single nucleotide polymorphisms, rs773902 determines whether residue 120 in transmembrane domain 2 is an alanine (Ala) or threonine (Thr). Compared with the Ala120 variant, Thr120 was more common in black subjects than in white subjects (63% vs 19%), was associated with higher PAR4-induced human platelet aggregation and Ca2+ flux, and generated greater inositol 1,4,5-triphosphate in transfected cells. A second, less frequent F2RL3 variant, Phe296Val, was only observed in blacks and abolished the enhanced PAR4-induced platelet aggregation and 1,4,5-triphosphate generation associated with PAR4-Thr120. PAR4 genotype did not affect vorapaxar inhibition of platelet PAR1 function, but a strong pharmacogenetic effect was observed with the PAR4-specific antagonist YD-3 1-benzyl-3(ethoxycarbonylphenyl)-indazole. These findings may have an important pharmacogenetic effect on the development of new PAR antagonists.
•White individuals have a high frequency of the common PAR4 gene (F2RL3) variant Ala120; blacks have a high frequency of Thr120.•PAR4 Thr120 induces greater signaling and is associated with greater platelet aggregation and reduced inhibition by a PAR4 antagonist.
Genome-wide analysis of thymic lymphomas from Tp53−/− mice with wild-type or C-terminally truncated Rag2 revealed numerous off-target, RAG-mediated DNA rearrangements. A significantly higher fraction ...of these errors mutated known and suspected oncogenes/tumor suppressor genes than did sporadic rearrangements (p < 0.0001). This tractable mouse model recapitulates recent findings in human pre-B ALL and allows comparison of wild-type and mutant RAG2. Recurrent, RAG-mediated deletions affected Notch1, Pten, Ikzf1, Jak1, Phlda1, Trat1, and Agpat9. Rag2 truncation substantially increased the frequency of off-target V(D)J recombination. The data suggest that interactions between Rag2 and a specific chromatin modification, H3K4me3, support V(D)J recombination fidelity. Oncogenic effects of off-target rearrangements created by this highly regulated recombinase may need to be considered in design of site-specific nucleases engineered for genome modification.
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•V(D)J recombination errors are investigated in lymphomas from p53-deficient mice•Numerous off-target rearrangements are found scattered across tumor genomes•These rearrangements target known and suspected oncogenes and tumor suppressors•Loss of Rag2 C terminus increases the frequency of off-target V(D)J recombination
To investigate the role of V(D)J recombination errors in oncogenesis, Mijuskovic et al. analyzed whole genomes of lymphomas spontaneously arising in p53-deficient mice. The systematic analysis revealed numerous off-target, RAG-mediated rearrangements in known and suspected cancer genes, which were escalated by the loss of Rag2 C terminus.
V(D)J recombination-associated DNA double-strand breaks (DSBs) are normally repaired by the high-fidelity classical nonhomologous end-joining (cNHEJ) machinery. Previous studies implicated the ...recombination-activating gene (RAG)/DNA postcleavage complex (PCC) in regulating pathway choice by preventing access to inappropriate repair mechanisms such as homologous recombination (HR) and alternative NHEJ (aNHEJ). Here, we report that RAG2’s “acidic hinge,” previously of unknown function, is critical for several key steps. Mutations that reduce the hinge’s negative charge destabilize the PCC, disrupt pathway choice, permit repair of RAG-mediated DSBs by the translocation-prone aNHEJ machinery, and reduce genomic stability in developing lymphocytes. Structural predictions and experimental results support our hypothesis that reduced flexibility of the hinge underlies these outcomes. Furthermore, sequence variants present in the human population reduce the hinge’s negative charge, permit aNHEJ, and diminish genomic integrity.
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•The acidic hinge in RAG2 prevents aberrant repair of RAG-mediated DNA breaks•Acidic residues strengthen the RAG/DNA postcleavage complex and enhance flexibility•Acidic residues preserve genomic stability in lymphocytes during V(D)J recombination•Experiments with identified sequence variants suggest similar mechanism in humans
The pathway chosen to mend genomic lesions can have a significant impact on the repaired product. Lymphocytes employ the classical NHEJ machinery for repairing V(D)J breaks during development, allowing for the safe generation of an effective immune repertoire. Roth and colleagues now examine specific residues in a flexible acidic hinge within the V(D)J recombinase, and look at the effects of changes, some of which are present in the human population. These amino acid alterations affect postcleavage handling of DNA breaks, permit alternative joining mechanisms to occur, and increase genomic instability.
Defining the architecture of a specific cancer genome, including its structural variants, is essential for understanding tumor biology, mechanisms of oncogenesis, and for designing effective ...personalized therapies. Short read paired-end sequencing is currently the most sensitive method for detecting somatic mutations that arise during tumor development. However, mapping structural variants using this method leads to a large number of false positive calls, mostly due to the repetitive nature of the genome and the difficulty of assigning correct mapping positions to short reads. This study describes a method to efficiently identify large tumor-specific deletions, inversions, duplications and translocations from low coverage data using SVDetect or BreakDancer software and a set of novel filtering procedures designed to reduce false positive calls. Applying our method to a spontaneous T cell lymphoma arising in a core RAG2/p53-deficient mouse, we identified 40 validated tumor-specific structural rearrangements supported by as few as 2 independent read pairs.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Proteins that combine PML NBs (ND10) can be divided into two groups: "transient" (that accumulate at PML NBs upon over-expression, interferon-induced up-regulation, block of proteosomal degradation, ...environmental stress or viral infection) and "constitutive" that co-localize with PML in the majority of cultured cells. One of the few "constitutive" components of PML NBs is the death domain-associated protein Daxx. While PML NBs are the most obvious depositories of Daxx, there are multiple alternative localization of this protein in the nucleus and cytoplasm, suggesting differential functionality of Daxx at different cellular compartments and stages of the cell cycle. The purpose of this review is to analyze Daxx spatiotemporal behavior within and outside of PML NBs and to discuss functions attributed to these localizations. We suggest that Daxx can participate in numerous cellular functions as a mediator of protein interactions, thus acting as a fine tuning instrument in highly orchestrated cellular processes; we also envision PML NBs accumulation of Daxx as an "out of action" storage depot.
The double membrane nuclear envelope (NE), which is contiguous with the ER, contains nuclear pore complexes (NPCs) - the channels for nucleocytoplasmic transport, and the nuclear lamina (NL) - ...a scaffold for NE and chromatin organization. Since numerous human diseases linked to NE proteins occur in mesenchyme-derived cells, we used proteomics to characterize NE and other subcellular fractions isolated from mesenchymal stem cells and from adipocytes and myocytes. Based on spectral abundance, we calculated enrichment scores for proteins in the NE fractions. We demonstrated by quantitative immunofluorescence microscopy that five little-characterized proteins with high enrichment scores are substantially concentrated at the NE, with Itprip exposed at the outer nuclear membrane, Smpd4 enriched at the NPC, and Mfsd10, Tmx4, and Arl6ip6 likely residing in the inner nuclear membrane. These proteins provide new focal points for studying the functions of the NE. Moreover, our datasets provide a resource for evaluating additional potential NE proteins.
The intracellular translocation of Daxx to the cytoplasm is a phenomenon often attributed to cells undergoing stress, opposite to predominant nuclear localization of this protein under normal ...homeostatic conditions. Moreover, a number of reports have suggested that export to the cytosol upon several stress conditions, including oxidative stress, glucose deprivation and beta-amyloid peptide treatment, is indispensable for the proper execution of Daxx-induced apoptosis. On the contrary, other studies have described translocation of Daxx from cytoplasm to nucleus upon stress application. Here, we examined cellular distribution of Daxx by sub-cellular fractionation and immunofluorescent localization of endogenous protein, using SH-SY5Y neuroblastoma cell line previously reported to exhibit cytoplasmic translocation of Daxx after oxidative stress and beta-amyloid exposure. In control conditions, Daxx is an exclusively nuclear protein in SH-SY5Y cells. Short treatment by either H2O2 or beta-amyloid did not show any significant change in nuclear localization of Daxx. Prolonged exposure of cells to stress compounds did not alter the intracellular deposition of Daxx that remains exclusively in the nucleus. A cohort of other cell lines, including human prostate cancer cell line DU-145, previously reported to exhibit stress-induced cytosol translocation was examined for Daxx distribution and none were confirmed to show re-localization of Daxx to the cytoplasm after either short or long stress. Time-lapse visualization of Daxx-GFP upon H2O2 treatment or glucose deprivation did not show cytoplasmic translocation either. Thus, while several Daxx-dependent apoptotic mechanisms have been described, the cytosolic association and function of this protein is questionable in light of these findings.
Resistance to anti-neoplastic drug paclitaxel is frequent in breast cancer patients. Most
studies of paclitaxel resistance have focused on pathways that elicit cellular response,
while little is ...known about players involved in the acquirement of taxane resistance. By
screening a cohort of breast cancer cell lines, we observed a correlation between level of
protein Daxx and response to paclitaxel. Cells lines expressing increased level of Daxx
displayed a robust paclitaxel response with nearly all cells undergoing micronucleation,
while cell lines with low amount of Daxx showed a decrease in micronucleation, and
accumulation in mitosis. At used paclitaxel concentrations, apoptotic levels were
negligible in all cell lines tested. Human cell lines expressing anti-Daxx siRNA as well as
Daxx-/- mouse fibroblasts showed similar cellular response to paclitaxel. Importantly,
absence or depletion of Daxx resulted in cell survival after paclitaxel treatment, as
measured by colony formation assay. We conclude that Daxx may be an important
predictive factor in cellular response to paclitaxel, which emphasizes a critical but
unknown function of this protein in mitotic progression, which, when disabled, leads to
survival advantages upon paclitaxel treatment.
We describe a method for detecting and validating genomic aberrations arising from cell lines exposed to zinc finger nucleases (ZFNs), an important reagent used for targeted genome modifications. ...This method makes use of cloned cell lines, an approach that adds power when testing variables that may affect gene correction efficiency and evaluating potential side effects on a genome-wide scale. After cell treatment, the genomic DNA isolation method, as described, is ideal for high-resolution array comparative genomic hybridization (aCGH) and quantitative PCR. Guidelines for aCGH analysis and calling significant copy number variations (CNVs) for validation by qPCR are also discussed. Using this method, we describe a novel ZFN-associated chromosome 4 copy number variation (CNV) attributable to a predicted ZFN off-target cleavage site found within the CNV.
A fundamental understanding of platelet biology requires functional assessment of megakaryocyte-platelet expressed genes and transcripts. The need for gene functional studies has recently increased ...with unbiased genome wide associations with platelet phenotypes. Animal models with an over-expressed or knocked-out gene have been particularly valuable systems, although species differences may preclude optimal evaluation. Assessing protein function in human platelets can be accomplished with pharmacologic inhibitors, although inhibitor availability and specificity can plague this approach. Studies with primary human megakaryocytes (MKs) are greatly challenged by inaccessibility, low yields and contamination with other cells. Cultured human megakaryocyte-like cell lines (e.g., Meg-01, HEL and others) have proven valuable, but these cells poorly recapitulate platelet physiology. Increasingly, investigators are using cultured human MKs to study gene function. CD34+ hematopoietic stem cells from human cord blood are routinely used to generate megakaryocytes in culture. This system has advantages of being human, relatively easy to obtain, cultured from primary cells and able to be genetically manipulated. Although cord blood-derived megakaryocytes (CBDMs) have proven useful for studying aspects of megakaryocyte/platelet biology, there is little information about the heterogeneous cell populations observed in CBDMs. In the course of our studies with CBDMs we observed multiple distinct cell populations by flow cytometry. Similar populations were also observed in the megakaryocytic Meg-01 cell line. The largest cells (called P1) were the most abundant (consisting of nearly 100% of cells) at day 3 in culture. P2 cells, which are smaller and more granular than P1, appeared at day 6 and by day 13 were ~50% of the total. P3 appeared at day 6 and are the smallest, with size and granularity roughly similar to platelets; by day 13 these were ~30% of the total. P1 but not P2 nor P3 became CD61/CD41/CD42 positive and CD34 negative over 13 days in culture. Ninety-seven percent and 93% of P2 and P3 cells, respectively, were phosphatidyl serine (PS) positive, whereas 93% of P1 cells were PS negative. Electron microscopy revealed many typical features of bone marrow MKs in the PS negative (P1) cells, including large size, polyploid nucleus, mitochondria and immature granules. However, the demarcation membrane system was poorly developed. Virtually all of the PS positive (P2) cells were apoptotic, lacked granules and had no discernable nuclei. Purification of P1 and P2 populations followed by re-culturing revealed that P1 gives rise to both the P2 and P3 populations, whereas P2 gave rise to no other population. CD34+ cells cultured with the pan-caspase inhibitor Z-VAD-FMK developed a much smaller proportion of P2 cells with a corresponding increase in P1 cells. Stimulation of these 3 populations with collagen related peptide, thrombin, protease activated receptor 1-activating peptide (PAR1-AP) and PAR4-AP showed strong integrin activation in P1 cells, but not in P2 nor P3 cells. To assess which population was susceptible to genetic manipulation, day 3 cultures were infected with a lentiviral construct containing a shRNA to talin. Talin knockdown prevented agonist-induced integrin activation in P1 cells and as expected, had no effect on the P2 or P3 population. In summary, these data indicate that only a portion of CBDMs are functional and only the P1 population should be used to assess MK gene function. Over time, the majority of CBDMs become apoptotic and the smaller P3, platelet-like particles have minimal response to agonist compared to peripheral blood platelets. These results may partially explain the difficulties in generating functional platelets in vitro and are consistent with the need to restrain apoptosis for successful megakaryocytopoiesis.
No relevant conflicts of interest to declare.
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