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 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

Luciferase activity driven by the human pIII promoter in CAL-1 cells.

<p><b>A-C.</b> Luciferase assays performed with various lengths of the human pIII promoters (A), the pIII promoters in the presence of PU.1 siRNA (open bar) or control siRNA (closed bar) (B), and the mutant promoters lacking Ets-motif(s) (C). Data represent means ± SEMs of three independent experiments. Each experiment was performed with triplicate samples. *, <i>p</i> < 0.05.</p

PU.1 knockdown cancels GM-CSF-mediated increase of mRNAs.

<p><b>A-D.</b> Quantitative real-time PCR analysis of the mRNA expression of PU.1 (A), HLA-DRα (B), total CIITA (C), and CIITA driven by pIII (D) in PU.1 siRNA (PU.1)- or control siRNA (cont.)-introduced CAL-1 cells. Cells were incubated for 24h after siRNA transfection and cultured for additional 72h with GM-CSF stimulation. The results are expressed as means ± SEMs of three independent experiments. Each experiment was performed with duplicate samples. *, <i>p</i> < 0.05.</p

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

Quantitative analysis of PU.1 binding to the pIII in CAL-1 and murine splenic pDCs.

<p><b>A and B.</b> The amount of chromosomal DNA immunprecipitated with anti-PU.1 Ab (open bar, anti-PU.1) or control IgG Ab (closed bar, IgG) in CAL-1 cells (A) and murine splenic pDCs (B). Binding degree is expressed as a percentage of the input for each ChIP assay. <b>C.</b> The amount of PU.1 binding to the pIII in PU.1 siRNA-introduced CAL-1 cells (PU.1) or control siRNA-introduced cells (cont.). Cells were harvested at 48h after siRNA transfection. Binding level of PU.1 (open bar) is expressed as fold change against that of control IgG (closed bar). Data represent means ± SEMs of three independent experiments. Each experiment was performed with duplicate samples. *, <i>p</i> < 0.05.</p