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

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 ¹⁸O 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

<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⁺ 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

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

<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

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

<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

Efficiency of UMG-lenti vectors in the transduction of primary human CD34⁺ cells.

<p>CD34⁺ 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

Schematic diagram of IRES-based and dual promoter lentiviral vectors.

<p>The expression cassettes of the lentiviruses used in this study are illustrated. The two IRES-containing vectors, FUIGW and UMG-LV11, differ for the position of the transgene and EGFP cDNA relative to the IRES element. In both viruses the transcription of this bicistronic unit is driven by the UBC promoter. The UMG-LV5 and UMG-LV6 vectors use independent promoters positioned back-to-back: UBC for the transgene and WASP (W) for EGFP. These dual-promoter vectors differ only for the orientation of the expression cassette. A short synthetic polyA signal, based on that of the human growth hormone gene, is downstream of the transcriptional unit in anti-sense orientation and is indicated by a diamond (♦).</p

Transgene expression in transduced, sorted, EGFP⁺ and EGFP⁻ K562 cells.

<p>K562 cells were subjected to one round of transduction with the lentiviruses indicated in the figure. After 5 days the cells were sorted by FACSAriaIII based on EGFP expression (<b>A</b>), and the sorted EGFP-positive (gates 1, 3, 5, 7) and -negative (gates 2, 4, 6, 8) subpopulations were analyzed by western blotting for expression of 3xFLAG-ZNF521 and of EGFP (<b>B</b>). The purity of the sorted populations was subsequently evaluated by flow cytometry and is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114795#pone.0114795.s003" target="_blank">S3 Figure</a>.</p

Transduction with UMG-LV6 carrying the MLL-AF9 fusion oncogene enhances the growth and clonogenicity of human CD34⁺ cells.

<p>CD34⁺ cells purified from cord blood were transduced with void UMG-LV6 vector or UMG-LV6 carrying the MLL-AF9 cDNA (UMG-LV6-MA). (<b>A</b>) FACS analysis of CD34⁺ cells 5 days after transduction. The percentages of EGFP positive cells are indicated. (<b>B</b>) Q-RT-PCR analysis of the expression of MLL-AF9 in CD34⁺ cells transduced with UMG-LV6-MA. The expression level was compared to that of the MLL-AF9-positive THP-1 cells, assumed as 1. (<b>C</b>) 1×10⁴ CD34⁺ cells transduced with void UMG-LV6 vector or with UMG-LV6-MA/well were plated in triplicate in 6-well plates in cytokine-driven cultures in the presence of 100 ng/ml of stem cell factor, FLT3 ligand and thrombopoietin. The culture medium was refreshed weekly, and the cell numbers were determined two weeks after plating. (<b>D</b>) The number of clonogenic progenitors in CD34⁺ cells transduced with void UMG-LV6 vector or with UMG-LV6-MA after two weeks of cytokine-driven culture was determined by clonogenic assays in methylcellulose as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114795#s2" target="_blank">Matherials and Methods</a>.</p

Comparison of the transduction efficiency of FUIGW, UMG-LV5 and UMG-LV6 carrying the ZNF521 cDNA in human hematopoietic cell lines.

<p>The cell lines K562, HL-60, MV4;11, THP-1, Jurkat and DeFew 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-LV6 viruses carrying 3xFLAG-ZNF521 cDNA as a transgene and EGFP cDNA as a reporter gene. As a control, void FUIGW vector without transgene cDNA 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 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> and analyzed by Western blotting for FLAG-ZNF521 and EGFP expression respectively. HDAC1 was used as a control for the amounts of extract loaded.</p

UMG-LVs efficiency in non hematopoietic cells.

<p>(<b>A</b>) Non-hematopoietic cell lines, HEK293T, MS-5, NIH-3T3 and DAOY, were transduced with FUIGW, FUIGW-ZNF521, UMG-LV6-ZNF521 and UMG-LV11-ZNF521 and analyzed by FACS to assess the percentage of EGFP positive cells. (<b>B</b>) Nuclear and cytosolic extracts were assayed with anti-FLAG and anti-EGFP antibodies as described above. HDAC1 was used as loading control.</p