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

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

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

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

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

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

Figure 2

<p>(a) Representative textured pattern mimicking the surface of legs of the wharf roach <i>Ligia exotica</i>. The right and left paths mimic the central and edge regions, while the middle path is a hybrid of central and edge textures. The length of “podite” is designed to be mm (or 3.4 mm), which is comparable to a typical size of podite in real legs. The sizes in the illustrations are expressed in the unit mm. (b) A snapshot of capillary rise on the reference surface. The imbibition fronts are shown by the horizontal arrows. The vertical arrows indicate velocities with reflecting relative magnitudes. (c) Height of the rise renormalized by vs elapsed time obtained from the reference surface. The labels, -, correspond to the labels in (b). (d) vs , obtained from the reference surface. The solid and thin lines with Labels “init.” and “final” are those fitting the data in the initial and final regions of the front in the central region of the HT path labeled in (b). (e) and (f): the same plots as the plot in (d) but for substrates different from the reference.</p

Figure 3

<p>(1a)–(1d) Texture-parameter dependences of the imbibition dynamics on the CT paths, with the insets giving those on the ET paths. (1a) The coefficient , a measure of the rising speed, vs the length at for CT and for ET. (1b) vs at for CT and for ET. (1c) vs at for CT and for ET. (1d) Comparison of the data with the theory. All the data collected from (1a) - (1c), together with other three data, are well on a straight line as predicted in Eq. (6). The straight line shown in the plot is obtained by numerical fitting. The curved lines in (1a)–(1c) and those in the inset are based on the theory in Eq. (6) with and determined by the straight line in (1d). (2a)–(2d) Texture-parameter dependences of the “initial” imbibition dynamics in the central region of the HT paths with the edge region specified by the set . (2a) vs at . (2b) vs at . (2c) vs at . (2d) Comparison of the data with the theory. All the data collected from (2a)–(2c) are well on the straight solid line, obtained by numerical fitting. The curved solid lines in (2a)–(2c) are based on the theory in Eq. (6) with and determined by the straight solid line in (2d). The insets to (2a)–(2c) quantify speed-up by the edge effect, showing the ratios of average values of in the main plots of (2a)–(2c) to those in (1a)–(1c), demonstrating the dynamics in the central region on the HT paths are clearly faster than those on the corresponding CT paths, except for two exceptional cases. The dashed lines in (2a)–(2d) are the solid lines in (1a)–(1d), respectively, confirming the edge effect in different ways.</p

Figure 1

<p>(a)–(b) SEM images of a leg of the wharf roach <i>Ligia exotica</i>. A path for water transport on the leg seen in (a) is covered with small blades. In a simplified view, wide blades cover the central-texture (CT) region and narrow blades the edge-texture (ET) region, as seen in the image in (b) showing the boundary between the central and edge regions with a higher magnification. The height of the blades are about 50 m. (c) A leg of the wharf roach used for the imbibition experiment. (d) Height of the rising water front, renormalized by the length of the lowest podite in (c), as a function of the elapsed time . The labels in the plot correspond to those in (c). Label in (c) corresponds to the origin of the plot.</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