Distribution of PBX1, RNA polymerase II, H3K4me3, and H3K27me3 binding events at gene promoters.

<p>Histograms show the distribution of the peak binding frequency with respect to their position on promoters (x axis). Binding frequency as shown in the y axis was determined by the number of peak binding events at specific promoter locations. TSS: transcription start site.</p

Transcription factor motifs enriched in ChIP-chip target sequences.

<p>Selected examples of transcription factor binding motifs that are statistically enriched in the PBX1 immunoprecipitated DNA sequences.</p

Validation of promoters bound by PBX1 and its cofactors.

<p><b>A.</b> Seven genes were chosen from the PBX1 ChIP target gene list, and occupancy of each promoter by PBX1 was validated by ChIP-qPCR analysis. ChIP was performed using either IgG or anti-PBX1 antibody, and the immunoprecipitated DNA was subjected to qPCR analysis using primers flanking the peak of the PBX1 bound region. <b>B.</b> Transcriptional regulation of these target genes by PBX1 or MEOX1 was assessed using siRNA and RT-qPCR analysis. Expression of all seven target genes are significantly down-regulated by PBX1 siRNA as compared to control siRNA (Student's <i>t</i>-test, <i>p</i><0.01). Except for ZHX2, MEOX1 siRNA significantly downregulated expression of the tested genes (Student's <i>t</i>-test, <i>p</i><0.01). <b>C.</b> ChIP-qPCR validation of GATA1 and MEOX1 binding near the PBX1-bound sequences. ChIP was performed using IgG, anti-GATA1, or anti-MEOX1 antibody and the immunoprecipitated DNA was subjected to qPCR using same primers as in <b>A</b>. Except for binding of MEOX1 to the <i>ZHX2</i> promoter, binding of GATA1 or MEOX1 to gene promoters is significantly higher than binding of IgG to the same promoters (Student's <i>t</i>-test, <i>p</i><0.01).</p

MEOX1 interacts with PBX1 and mediates its growth effect.

<p><b>A.</b> HEK293 cells were transfected with PBX1-V5 and/or MEOX1-FLAG expression vectors. Immunoprecipitation and Western blot were performed using epitope tag-specific antibodies. <b>B.</b> Left panel: OVCAR3 cells were transfected with MEOX1 expression vector or control vector. Western blot was performed to test MEOX1 expression (top). The same cells were transfected with PBX1 siRNA or control siRNA and Western blot was performed to test the down-regulation of PBX1 protein (middle). Detection of GAPDH protein was used as a loading control (bottom). Right panel: Relative cell numbers were measured in OVCAR3 cells transfected with MEOX1 cDNA, PBX1 siRNA, control plasmid (pLPC), and control siRNA (siLuc). Student's <i>t</i>-test was used to determine the significance between the MEOX1 over-expressed group and the control group.</p

PBX1 binds to a specific set of genes and controls their transcription in ovarian cancer.

<p><b>A:</b> Venn diagram of PBX1 ChIP-chip target genes and PBX1 transcriptome reveal a set of overlapping genes that are direct target genes of PBX1. The full list of the overlapping genes is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036054#pone.0036054.s011" target="_blank">Table S5</a>. Experiments were performed in OVCAR3 cells. <b>B:</b> Gene set enrichment analysis was performed to determine if PBX1 ChIP targets are enriched in the PBX1 transcriptome. Genes down-regulated by PBX1 siRNA are significantly enriched in the PBX1 ChIP target set.</p

PBX1 and several of its direct target genes are over-expressed in ovarian cancer tissues.

<p>Oncomine meta-analysis was performed on a previously published gene expression microarray dataset containing nine different tumor types. Genes were ranked according to their over-expression in ovarian cancer and the top 18 genes were plotted.</p

Genomic spectrum of acquired driver alterations.

A) The circle graph represents for each case (n = 74) the proportion of driver mutations detected in primary and/or metastatic tumor samples. Outer numbers represent mutations of eBC, inner numbers represent mutations of mBC. B) Cumulative frequency of the difference (Δ) between number of mutations in metastatic vs. primary tumor samples (Δ vs. metastatic tumor; Δ > 0, number of driver mutations in the primary sample lower than in the metastatic sample. C) Non-linear relationship between the difference of driver mutations in metastasis/primary pair (Δ, x-axis), and DRFS hazard ratio of Schoenfeld residuals (y-axis). The analysis is adjusted for T/N status, Ki67, menopausal status and tumor grade. The solid line represents a penalized spline fit of the predicting variables, while the dashed lines show 95% confidence intervals. D) Functional analysis of Gene Ontology (GO) terms associated to cell cycle, DDR, epigenetic regulation, androgen receptor activity and WNT signaling pathway. The size of the dots is inversely proportional to the p values of estimated hazard ratio (x-axis) displayed in log10 scale. P values are reported in S5 Table.</p

Association between <i>MAP3K</i> alterations and clinical outcome.

Kaplan Meier curves displaying distant relapse-free survival (DRFS) (A) and overall survival (OS) (B) in patients with MAP3K gene alteration (red curves) as compared with patients with wild-type MAP3K status (blue curves). Forest plots indicating the hazard ratios for DRFS (C) and OS (D), and the corresponding confidence intervals, in MAP3K-altered and MAP3K-wild type patients. Multivariable Cox analysis is adjusted for tumor size, lymph node involvement, Ki67, menopausal status and tumor grade of the primary tumor.</p

Repertoire of genomic alterations in primary and metastatic HR+ HER2- breast cancer (BC).

A) Recurrent driver somatic mutations and copy number variations identified in matched primary and metastatic HR+ HER2- BC specimens (n = 74) subjected to targeted sequencing (n = 148). Cases are shown in columns, whereas genes are shown in rows. Mutation types are color-coded according to the legend. The total number of alterations detected in individual genes is displayed on the bar plot (right). For graphical purpose, only the top 25 genes are shown. B) The scatter plot reports the mutational frequencies in matched primary (n = 74) and metastatic (n = 74) tumor samples. Color indicates statistical significance (p-value adjusted by false discovery rate ≤ 0.1), the shape of the points reflects the difference between mutational frequency in metastatic vs. primary tumor samples (triangle = frequency difference ≥ 5%, circle = frequency difference ESR1, MAP3K1 and MAP3K13 and of the somatic mutations in matched primary and metastatic HR+ HER2- BC specimens (n  =  74). Mutations are color-coded according to the legend, and their overall occurrence is represented on the y-axis.</p

(GO terms).

(XLSX)</p