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

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

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

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

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

Pyk3 and Phg2 are required for full transcriptional activation of STATc-dependent genes.

<p>Real time PCR analysis of selected genes was carried out with cDNAs from untreated cells and from cells treated for 15 minutes with either 200(A), 20 mM 8-Br-cGMP (B) or 30 µM BHQ (C). The differential expression was investigated in Ax2 wild-type, pyk3⁻, phg2⁻, pyk3⁻/phg2⁻, and STATc⁻ cells upon treatment with sorbitol and 8-Br-cGMP (A, B) or in Ax2 wild-type, pyk3⁻, phg2⁻, and pyk3⁻/phg2⁻ cells upon treatment with BHQ (C). The data are expressed as means of fold change in comparison to untreated cells. A students t-test was performed and significance values were calculated: p-value <0.05 = <b>*/^▪</b>; p-value <0.01 = <b>**/^▪▪</b>_; p-value <0.001 = <b>***/^▪▪▪</b>_. Fold changes and standard deviations of six measurements from three independent experiments are shown. <b>*</b>Statistically significant changes, compared with AX2 cells. <b>^▪</b>Statistically significant changes, compared with pyk3⁻ cells. N/A: <i>not applicable.</i></p

Selection of STATc regulated genes.

<p>Ax2 and STATc⁻ cells were treated for 15 min with 200 mM sorbitol or left untreated and differentially regulated genes were identified by microarray analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090025#pone.0090025-Na1" target="_blank">[2]</a>. Depicted are <i>dstc</i>, <i>ptpc</i> and five genes encoding TKL proteins.</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