A scheme for the regulation of pspA gene expression.

<p>The scheme hypothesises a general activator of transcription (yellow-boxed) that has the potential to direct transcription in all cells in the slug. However, DimB acts in pstO cells in its repressor form (red-boxed) to prevent the activator functioning. Not shown here is a proposed functionally redundant repressor that can subsume the role of DimB as a repressor of pspA in a dimB- strain. The <i>ecmA</i> promoter is hyper-active in pstO cells of the DimB null strain, so is shown as being co-repressed by the DimB repressor form. In pstB cells the ecmB gene is directly induced by the activating form (green-boxed) of DimB.</p

DIF repression of <i>pspA</i> expression.

<p>Disaggregated cells at the mound stage were incubated in the presence or absence of DIF-1. Q-PCR analysis of RNA samples was performed and the data is plotted as the mean of 3 independent biological repeats each performed in triplicate. The data is normalized to the expression level of <i>Ig7</i>, a constitutively expressed gene and that for each strain is normalized to the value without DIF. The mean results are shown with their standard deviations.</p

DimB binding to the pspA promoter <i>in vivo</i>.

<p>Cells were incubated with or without DIF and subjected to ChIP analysis. The absolute recoveries from the procedure varied from experiment to experiment, (three independent experiments with triplicate Q-PCR analyses in each). Therefore values are normalized to the induced signal for the <i>ecmA</i> positive control and are shown with their Standard Deviations. Student's paired T test was applied to the <i>pspA</i> analysis with and without DIF-1 and in samples immuno-precipitated from GFP-DimB transformant cells. As indicated by the asterisk the induction by DIF is significant with a P<0.05.</p

Mass spectrometry scores for selected of the proteins purified by affinity chromatography using region A.

<p>Only known proteins identified as binding to region A (A entire), or the cap-site proximal tandem dimer (A dimer, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029895#pone-0029895-g001" target="_blank">Fig. 1B</a>), and with a “Mowse” score of 50 or over are presented.</p

Mapping DimB binding sites in region A by gel retardation analysis.

<p>A) Alignment of regions S and W, the proposed DimB binding sites in region A of the <i>pspA</i> promoter, with the known DimB binding sites within the <i>ecmA</i> promoter: R2 and R1. Also indicated, above the sequence, are the positions of the point mutations used in scanning analysis of DimB binding. B) Total nuclear extracts obtained from Ax-2 and dimB- slug cells used in gel retardation with a region A probe. The competitors are the R2 and R2M sequences from within the <i>ecmA</i> promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029895#pone.0029895-Zhukovskaya1" target="_blank">[8]</a> C) Total nuclear extracts obtained from Ax-2 slug cells used in gel retardation with a region A probe. The competitors are region A itself and scanning mutants M1 to M6. D) Gel retardation with recombinant DimB using an A region probe. Competitors are: A itself, and oligonucleotide M145, containing region A with mutations M1, M4 and M5 that collectively mutate the S and W DimB binding sites. Again, the control competitors are the R2 and R2M sequences from within the <i>ecmA</i> promoter.</p

Identification of proteins that bind to the pspA promoter.

<p>A representation of the minimal promoter sequence required for <i>pspA</i> expression (thick line) showing the sequence of the region used in affinity chromatography, with a proposed DimB binding site underlined. (B) The combined peptide coverage for DimB in the two different purifications, described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029895#pone-0029895-t001" target="_blank">Table 1</a>. is shown in red. (C) Identification of proteins bound to a 16nt tandem dimer containing the proposed DimB site. Only those proteins with a deducible function are indicated and their scores in the mass spectrometry analysis are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029895#pone-0029895-t001" target="_blank">Table 1</a>.</p

Expression patterns of <i>pspA</i> reporter fusions.

<p>The <i>pspA</i> promoter region −990 to −122 (pspA:lacZ), and versions of the same region containing a mutation of either the W (pspA-M1:lacZ) or S (pspA-M456:lacZ) sites. Expression patterns were established in standing slugs stained with X-gal.</p

<i>D. discoideum</i> mutant strains used in this study.

<p><i>D. discoideum</i> mutant strains used in this study.</p

Phg2 but not Pyk3 acts upstream of PTP3.

<p>(A) Upon hyperosmotic conditions, PTP3 becomes phosphorylated on two serine residues, S448 and S747, resulting in an inhibition of its phosphatase activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090025#pone.0090025-Araki2" target="_blank">[7]</a>. (B–E) Ax2/Myc-PTP3, pyk3⁻/Myc-PTP3 and phg2⁻/Myc-PTP3 cells were left untreated (−) or treated (+) with 200 mM sorbitol for 5 min (B, C) or 10 min (D, E). Myc-PTP3 was immunoprecipitated, separated by SDS-PAGE, transferred to nitrocellulose, and phosphorylated Myc-PTP3 was detected with PTP3 antibodies specific for phospho-serine 448 (B, D) and phospho-serine 747 (C, E). Total Myc-PTP3 was used as loading control and detected with an anti-Myc antibody (mAb 9E10). (F, G): Quantification of PTP3 phospho-serine 448 (F) and 747 (G) in either treated or untreated cells. Band intensities were determined densitometrically and normalised using the values for total Myc-PTP3. The amount of serine phosphorylated PTP3 of treated Ax2/Myc-PTP3 cells was set to 1 and relative values were calculated for untreated Ax2/Myc-PTP3 cells as well as for treated and untreated phg2^−/Myc-PTP3 cells. The error bars depict standard deviations of three independent experiments. (H, I): Pull-down assay to investigate the binding of Myc-PTP3 to bacterially expressed GST-Phg2. In the first approach (H) Myc-PTP3 was expressed in phg2⁻ cells, immunoprecipitated with anti-Myc Dynabeads, and eluted from the beads via a pH shift for binding to GST-Phg2 bound to glutathione beads. Lane 1: Total cell lysate of Myc-PTP3 expressing phg2⁻ cells; lane 2: Anti-Myc Dynabeads after immunoprecipitation of Myc-PTP3; lane 3: Myc-PTP3 after elution from anti-Myc Dynabeads; lane 4: GST, purified from bacteria and bound to glutathione-beads (GST-beads); lane 5: GST-Phg2, purified from bacteria and bound to glutathione-beads (GST-Phg2-beads); lane 6: supernatant after incubation of Myc-PTP3 with GST-beads; lane 7: pellet after incubation of Myc-PTP3 with GST-beads; lane 8: supernatant after incubation of Myc-PTP3 with GST-Phg2-beads; lane 9: pellet after incubation of Myc-PTP3 with GST-Phg2-beads. In the reverse approach (I) the binding of bacterially expressed GST-Phg2, which was eluted from glutathione-beads, to Myc-PTP3 bound to anti-Myc Dynabeads (Myc-PTP3-beads) was investigated. Lane 1: Total cell lysate of Myc-PTP3 expressing phg2⁻ cells; lane 2: lysate after immunoprecipitation of Myc-PTP3 with anti-Myc Dynabeads; lane 3: Anti-Myc Dynabeads with bound Myc-PTP3; lane 4: GST, purified from bacteria and eluted from glutathione-beads; lane 5: GST-Phg2, purified from bacteria and eluted from glutathione-beads; lane 6: supernatant after incubation of GST with Myc-PTP3-beads; lane 7: pellet after incubation of GST with Myc-PTP3-beads; lane 8: supernatant after incubation of GST-Phg2 with Myc-PTP3-beads; lane 9: pellet after incubation of GST-Phg2 with Myc-PTP3-beads. The order of lines from the same immunoblot were digitally re-arranged for illustration purposes to omit dispensable lines.</p

Real time PCR confirms STATc-dependent transcriptional activation of selected genes.

<p>The differential expression of seven selected genes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090025#pone-0090025-t003" target="_blank">Table 3</a>) in Ax2 wild-type (black bars) and STATc⁻ (white bars) cells in response to treatment with 200 mM sorbitol for 15 minutes was analysed by quantitative real time PCR. The data are expressed as means of fold change in comparison to untreated cells. Fold changes and standard deviations of six measurements from three independent experiments are shown. N/A: <i>not applicable.</i></p