PU.1 binds to the <i>Gata3-1b</i> promoter.

<p>(A) Upper carton: Schematic drawing of the mouse <i>Gata3</i> gene. Ex, exon; the arrows over exons 1a/1b and that under exon 2, indicate forward and reverse PCR primers, respectively. Lower graph: BMDCs were transfected with either control siRNA (siCTRL) or PU.1 siRNA (siPU.1). Relative mRNA levels (1a/GAPDH or 1b/GAPDH) were determined by quantitative RT-PCR after normalization to GAPDH mRNA and are expressed as the ratio of the expression level of GATA3-1b in control siRNA-introduced cells. (B, C) Quantification of PU.1 or control goat IgG binding to the <i>Gata3-1b</i> promoter in BMDCs (B) or in splenic DCs (C) was performed using a ChIP assay with the series of primers described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137699#pone.0137699.s001" target="_blank">S1 Table</a>. Binding is expressed as a percentage of the input for each ChIP assay. All results are means ± S.E.s (<i>n</i> = 3). Similar results were obtained in three separate experiments. *, <i>p</i> < 0.05 in a two-tailed paired Student’s <i>t</i> test.</p

Effects of PU.1 knockdown on expression of GATAs, 1, 2, and 3.

<p>(A) BMDCs were transfected with either control (siCTRL; open bars) or PU.1 (siPU.1; closed bars) siRNA. After 48 h incubation, relative mRNA levels were determined by quantitative RT-PCR after normalization to GAPDH mRNA. Data are expressed as the ratio of the expression level of respective control siRNA-introduced cells. Results are means ± S.E.s (<i>n</i> = 3). Similar results were obtained in three separate experiments. *, <i>p</i> < 0.05 in two-tailed paired Student’s <i>t</i> test. (B) Aliquots of total proteins (15 μg/lane) of the indicated cells were subjected to SDS-PAGE and immunoblot analysis using the indicated antibodies. Similar results were obtained in three separate experiments.</p

Effects of PU.1 knockdown on the histone acetylation and on HDAC recruitment at the <i>Gata3-1b</i> promoter.

<p>Histone acetylation status of the <i>Gata3-1b</i> promoter in BMDCs transfected with either PU.1 siRNA (siPU.1) or its control siRNA (siCTRL) (A), and the acetylation status in BMDCs cultured without (CTRL) or with 10 nM the histone deacetylation inhibitor trichostatin A (TSA) for 3 h (B). Quantification of acetyl-histone H3 at the <i>Gata3-1b</i> promoter was performed by ChIP assay. (C, D) GATA3 mRNA levels in BMDCs cultured with 10 nM trichostatin A for the indicated times (C) or with the indicated trichostatin A concentration for 3 h (D). Effects of HDAC inhibitors (E) and knockdown of HDACs (F) on GATA3 mRNA levels. BMDCs were treated with MS-275 (1 μM, 10 μM), Droxinostat (20 μM, 50 μM), or MC1568 (5 μM, 20 μM) for 6 h (E). Relative mRNA levels (GATA3-1b/GAPDH) were determined by quantitative RT-PCR after normalizing to GAPDH mRNA. (G) The binding degree of HDAC3 on the <i>Gata3-1b</i> promoter was analyzed by a ChIP assay. Open circles, control rabbit IgG binding in control siRNA-transfected cells; closed circles, anti-HDAC3 antibody binding in control cells; open squares, control IgG binding in PU.1 siRNA-transfected cells; closed squares, anti-HDAC3 antibody binding in PU.1 siRNA-transfected cells. All results are means ± S.E.s (<i>n</i> = 3). Similar results were obtained in three separate experiments. *, <i>p</i> < 0.05 in a two-tailed paired Student’s <i>t</i> test.</p

Increased expression of GATA3 is involved in the LPS-inducible IL-13 upregulation in PU.1 knockdown cells.

<p>BMDCs were transfected with the indicated siRNAs. After 48 h incubation, the cells were stimulated with (black bars) or without (white bars) of 1 μg/ml LPS for 6 h. (A, B) Relative mRNA levels of the indicated cytokines were determined by quantitative RT-PCR after normalizing to GAPDH mRNA. Data are expressed as the ratio of the expression level of respective control siRNA-introduced cells without LPS stimulation. (C) IL-13 protein concentrations in the supernatant were measured using ELISA. All results are means ± S.E.s (<i>n</i> = 3). Similar results were obtained in three separate experiments. *, <i>p</i> < 0.05 in a two-tailed paired Student’s <i>t</i> test.</p

PU.1 knockdown enables GATA3 to transactivate the <i>Il13</i> promoter via affecting histone H3 modification of CGRE.

<p>(A) BMDCs from wild type (WT) or CGRE deletion (ΔCGRE) mice were transfected with either control siRNA (siCTRL) or PU.1 siRNA (siPU.1). After 48 h incubation, the cells were stimulated or not with 1 μg/ml LPS for 6 h. Relative mRNA levels (IL-13/GAPDH) were determined by quantitative RT-PCR after normalizing to GAPDH mRNA. *, <i>p</i> < 0.05 in a two-tailed paired Student’s <i>t</i> test. (B) Quantification of the H3K4me3 degree around CGRE on the <i>Il13</i> promoter was performed by a ChIP assay with anti-H3K4me3 antibody or its control antibody, and a primer set described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137699#pone.0137699.s001" target="_blank">S1 Table</a>. All results are means ± S.E.s (<i>n</i> = 3). Similar results were obtained in another experiment. *, <i>p</i> < 0.05 in a two-tailed paired Student’s <i>t</i> test.</p

Effects of PU.1 knockdown on Th2 cytokine expression.

<p>BMDCs were transfected with either control (siCTRL) or PU.1 (siPU.1) siRNA and were then incubated for 48 h prior to stimulation with (closed bars) or without (open bars) 1 μg/ml LPS for 6 h. (A) Relative mRNA levels were determined by quantitative RT-PCR after normalization to GAPDH mRNA. Data are expressed as the ratio of the expression level of respective control siRNA-introduced cells without LPS stimulation. (B) IL-13 protein concentrations in the supernatant were measured using ELISA. All results are means ± S.E.s (<i>n</i> = 3). Similar results were obtained in three separate experiments. *, <i>p</i> < 0.05 in a two-tailed paired Student’s <i>t</i> test.</p

Role of PU.1 in MHC Class II Expression via CIITA Transcription in Plasmacytoid Dendritic Cells

<div><p>The cofactor CIITA is a master regulator of MHC class II expression and several transcription factors regulating the cell type-specific expression of CIITA have been identified. Although the MHC class II expression in plasmacytoid dendritic cells (pDCs) is also mediated by CIITA, the transcription factors involved in the CIITA expression in pDCs are largely unknown. In the present study, we analyzed the role of a hematopoietic lineage-specific transcription factor, PU.1, in CIITA transcription in pDCs. The introduction of PU.1 siRNA into mouse pDCs and a human pDC cell line, CAL-1, reduced the mRNA levels of MHC class II and CIITA. When the binding of PU.1 to the 3rd promoter of CIITA (pIII) in CAL-1 and mouse pDCs was analyzed by a chromatin immunoprecipitation assay, a significant amount of PU.1 binding to the pIII was detected, which was definitely decreased in PU.1 siRNA-transfected cells. Reporter assays showed that PU.1 knockdown reduced the pIII promoter activity and that three Ets-motifs in the human pIII promoter were candidates of <i>cis</i>-enhancing elements. By electrophoretic mobility shift assays, it was confirmed that two Ets-motifs, GGAA (-181/-178) and AGAA (-114/-111), among three candidates, were directly bound with PU.1. When mouse pDCs and CAL-1 cells were stimulated by GM-CSF, mRNA levels of PU.1, pIII-driven CIITA, total CIITA, MHC class II, and the amount of PU.1 binding to pIII were significantly increased. The GM-CSF-mediated up-regulation of these mRNAs was canceled in PU.1 siRNA-introduced cells. Taking these results together, we conclude that PU.1 transactivates the pIII through direct binding to Ets-motifs in the promoter in pDCs.</p></div

Effect of GM-CSF stimulation on the expression of PU.1, CIITA, and MHC class II, and on the recruitment of PU.1 to the pIII.

<p><b>A-H.</b> Quantitative real-time PCR analysis of the mRNA expression of PU.1 (A, E), MHC class II (B, F), total CIITA (C, G), and CIITA driven by pIII (D, H) in mouse splenic pDCs (A-D) or CAL-1 cells (E-H) with (+) or without (-) GM-CSF stimulation. Mouse pDCs and CAL-1 cells were stimulated with 20 ng/ml mGM-CSF for 24h and 10 ng/ml hGM-CSF for 72h, respectively. <b>I.</b> The amount of PU.1 binding to the pIII in GM-CSF-stimulated CAL-1 cells (+) or control cells (-) was determined by a ChIP assay. Binding level of PU.1 (open bar) is expressed as fold change against that of control IgG (closed bar). In E-I, data represent means ± SEMs of three independent experiments performed with duplicate samples. In A-E, the results are expressed as means ± SEMs of triplicate samples, and similar result was obtained in another experiment. *, <i>p</i> < 0.05.</p

Direct binding of PU.1 to Ets-motifs in the human pIII promoter.

<p><b>A.</b> Nucleotide sequences of probes and competitive oligonucleotides used for EMSAs. <b>B and C.</b> EMSA profiles. Anti-PU.1 Ab (P) or control Ab (C) was used to identify the specific band composed of PU.1 and probe DNA (B). EMSA with an excess amount of competitors (a 1- or 10-fold molar concentration of probe 1 or 2) was performed to identify PU.1-binding sites (C). λB, which is previously published element bound with PU.1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154094#pone.0154094.ref023" target="_blank">23</a>], was mixed with a 15- or 30-fold concentration of competitors. Specific bands corresponding to complexes of PU.1 and the probe are marked with an asterisk. Super-shift band corresponding to complexes of PU.1, the probe, and anti-PU.1 Ab are marked with double asterisks. n.s., non-specific bands detected in protein mixture without probe DNA.</p

Effect of PU.1 knockdown by siRNA on the mRNA levels of CIITA and MHC class II in mouse pDCs and human pDC cell line.

<p>The mRNA expression levels of PU.1 (A, E), MHC class II (B, F), CIITA (C, G), and CIITA specifically driven from pIII (D, H) in mouse PU.1 siRNA-introduced BMpDCs (A-D), or human PU.1 siRNA (Spi1-HSS186060)-introduced CAL-1 cells (E-H) are displayed as the ratio of mRNA levels versus those seen in control siRNA-introduced cells. The mRNA levels of PU.1 (I) and CIITA (J) in CAL-1 cells transfected with another PU.1 siRNA (Spi1-HSS144058) are also displayed. Cells were harvested at 24h (A-D) or 96h (E-J) after siRNA transfection. Open bar, PU.1 siRNA; closed bar, control siRNA. In E-J, data represent means ± SEMs of three independent experiments, and each experiment was performed with triplicate samples. In A-E, the results are expressed as means ± SEMs of triplicate samples, and similar result was obtained in another experiment. *, <i>p</i> < 0.05.</p