15 research outputs found

    Comparison between experimental expression profiling and computed attractors.

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    <p>Each condition, for both experimental and model results, is normalized with respect to the given maximum value. It is possible to note a good agreement, in terms of bistable behavior, between the model prediction, the measured expression of the key factors involved in the commitment switch and the markers of the multipotent and the committed stage (FLT3 and CD19), respectively. Blue bars correspond to the LMPP stage, green bars to the LRP in the pro-B phase.</p

    Computational Modeling of a Transcriptional Switch Underlying B-Lymphocyte Lineage Commitment of Hematopoietic Multipotent Cells

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    <div><p>Despite progresses in identifying the cellular mechanisms at the basis of the differentiation of hematopoietic stem/progenitor cells, little is known about the regulatory circuitry at the basis of lineage commitment of hematopoietic multipotent progenitors. To address this issue, we propose a computational approach to give further insights in the comprehension of this genetic mechanism. Differently from T lymphopoiesis, however, there is at present no mathematical model describing lineage restriction of multipotent progenitors to early B-cell precursors. Here, we provide a first model—constructed on the basis of current experimental evidence from literature and of publicly available microarray datasets—of the genetic regulatory network driving the cellular fate determination at the stage of lymphoid lineage commitment, with particular regard to the multipotent-B-cell progenitor transition. By applying multistability analysis methods, we are able to assess the capability of the model to capture the experimentally observed switch-like commitment behavior. These methods allow us to confirm the central role of zinc finger protein 521 (ZNF521) in this process, that we had previously reported, and to identify a novel putative functional interaction for <i>ZNF521</i>, which is essential to realize such characteristic behavior. Moreover, using the devised model, we are able to rigorously analyze the mechanisms underpinning irreversibility of the physiological commitment step and to devise a possible reprogramming strategy, based on the combined modification of the expression of <i>ZNF521</i> and <i>EBF1</i>.</p></div

    EBF1-dependence of the B-cell development bifurcation diagram w.r.t the ZNF521 transcriptional activation.

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    <p><b>(a)</b> EBF1 versus external factor <i>T</i><sub><i>EBF</i>1</sub> with no activation of ZNF521, <i>T</i><sub><i>ZNF</i>521</sub> = 0. Overexpression of EBF1 is sufficient to switch the system from the LMPP state (low EBF1) to the pro-B (high EBF1) state, as indicated by the arrows. <b>(b)</b> EBF1 versus external factor <i>T</i><sub><i>EBF</i>1</sub> with transcriptional activation of ZNF521, <i>T</i><sub><i>ZNF</i>521</sub> = 0.12. A couple of limit points (LPs) defines a region of bistability for the proposed network. In this case, transcriptional activation of ZNF521 may convert the system from an irreversible to a reversible bistable switch. <b>(c)</b> EBF1 versus external factor <i>T</i><sub><i>EBF</i>1</sub> with transcriptional activation of ZNF521, <i>T</i><sub><i>ZNF</i>521</sub> = 0.3. Consistently, higher values of transcriptional activation of ZNF521 entailing a sharp delay in B-cell development, showing a more ultrasensitive response to higher values of <i>T</i><sub><i>EBF</i>1</sub>.</p

    Expression programs characterizing the commitment of LMPPs toward B-lymphoid LRPs.

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    <p>Each state is guaranteed by a distinctive expression program of regulatory factors. In red (green), are depicted the factors up (down) regulated in each condition, LMPP and LRP, respectively. Values are taken in arbitrary units from the experimental gene profiling datasets, reported in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132208#pone.0132208.ref020" target="_blank">20</a>].</p

    Gene level differential expression analysis.

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    <p>Every point in the scatter plots shows the expression of a gene in the two conditions CD34<sup>+</sup>/lin<sup>−</sup> vs. pro-B. <b>HG-U133A</b> Chip. Total number of genes evaluated: 22645, with 550 genes differentially expressed. CD34<sup>+</sup>/lin<sup>−</sup> vs. pro-B analysis shows that 167 genes are up-regulated and 383 genes are down-regulated. <b>HG-U133B</b> Chip. Total number of genes evaluated: 22283 with 1080 genes differentially expressed CD34<sup>+</sup>/lin<sup>−</sup> vs. pro-B analysis shows that 553 genes are up-regulated and 527 genes are down-regulated. According with the TAC default filter criteria, we setted a fold change (linear) cut-off of ±2 (all that points having a fold-change less than 2 are shown in gray) and p-value < 0,05. Red: up in CD34<sup>+</sup>/lin<sup>−</sup> vs pro-B; green: down in CD34<sup>+</sup>/lin<sup>−</sup> vs pro-B. Highlighted are shown the factors involved in the proposed model.</p

    Validation of a Novel Shotgun Proteomic Workflow for the Discovery of Protein–Protein Interactions: Focus on ZNF521

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    The study of protein–protein interactions is increasingly relying on mass spectrometry (MS). The classical approach of separating immunoprecipitated proteins by SDS-PAGE followed by in-gel digestion is long and labor-intensive. Besides, it is difficult to integrate it with most quantitative MS-based workflows, except for stable isotopic labeling of amino acids in cell culture (SILAC). This work describes a fast, flexible and quantitative workflow for the discovery of novel protein–protein interactions. A cleavable cross-linker, dithiobis­[succinimidyl propionate] (DSP), is utilized to stabilize protein complexes before immunoprecipitation. Protein complex detachment from the antibody is achieved by limited proteolysis. Finally, protein quantitation is performed via <sup>18</sup>O labeling. The workflow has been optimized concerning (i) DSP concentration and (ii) incubation times for limited proteolysis, using the stem cell-associated transcription cofactor ZNF521 as a model target. The interaction of ZNF521 with the core components of the nuclear remodelling and histone deacetylase (NuRD) complex, already reported in the literature, was confirmed. Additionally, interactions with newly discovered molecular partners of potentially relevant functional role, such as ZNF423, Spt16, Spt5, were discovered and validated by Western blotting

    UMG Lenti: Novel Lentiviral Vectors for Efficient Transgene- and Reporter Gene Expression in Human Early Hematopoietic Progenitors

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    <div><p>Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and –LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34<sup>+</sup> cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG–LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and progenitor cells, as well as in non-hematopoietic cells.</p></div

    Efficiency of UMG-lenti vectors in the transduction of primary human CD34<sup>+</sup> cells.

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    <p>CD34<sup>+</sup> cells purified from cord blood were transduced with FUIGW, UMG-LV6 or UMG-LV11 viruses carrying the cDNAs for 3xFLAG-ZNF521 and EGFP. (<b>A</b>) FACS analysis of the transduced cells 5 days after transduction. The percentages of EGFP positive cells are indicated. (<b>B</b>) Western blotting analysis of FLAG-ZNF521 and EGFP expression was performed as described above on nuclear and cytosolic extracts. HDAC1 was used as a control for the amounts of extract loaded.</p

    UMG-LV11 promotes efficient transgene- and reporter gene expression in human hematopoietic cell lines.

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    <p>The cell lines indicated were infected as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114795#s2" target="_blank">materials and methods</a> with FUIGW, UMG-LV5 or UMG-LV11 viruses carrying the cDNAs for 3xFLAG-ZNF521. As a control, void FUIGW vector was used. (<b>A</b>) Flow-cytometric analysis of EGFP expression in cells exposed to the relevant vectors. The percentages of EGFP-positive cells are indicated. (<b>B</b>) Nuclear and cytosolic extracts were analyzed by Western blotting for FLAG-ZNF521 and EGFP expression respectively. HDAC1 was used as a control for the amounts of extract loaded.</p

    Comparison of the transduction efficiency of FUIGW, UMG-LV5 and UMG-LV6 carrying the MSI2 cDNA in human hematopoietic cell lines.

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    <p>The cell lines K562, HL-60, MV4;11 and Jurkat were infected with FUIGW, UMG-LV5 or UMG-LV6 viruses carrying 3xFLAG-MSI2 cDNA as a transgene. As a control, void FUIGW vector was used. (<b>A</b>) Flow-cytometric analysis of EGFP expression in cells exposed to the relevant vectors. The percentages of EGFP-positive cells are indicated. (<b>B</b>) Whole-cell extracts, prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114795#s2" target="_blank">materials and methods</a>, were analyzed by Western blotting for FLAG-MSI2 and EGFP expression. Actin was used as a control for the amounts of extract loaded.</p