Genome-wide visualization of chip-on-ChIP identified (magenta), Gene Expression Analysis (GEA) and chip-on-ChIP overlap (green) and select (blue) putative STAT5 binding sites chosen for validation.

<p>Visualization of the results obtained from the genome-wide identification of IL-2 induced genes and STAT5 binding sites (as described in Fig. 1) using the IGV.</p

Validation of STAT5-dependent, IL-2-mediated gene expression changes.

<p>Transcript level changes of 57 intersect genes were measured using qRT²PCR arrays in PHA activated, quiescent and IL-2 stimulated (3 and 6 h) PBMCs (72 h activated, 48 h quiescent, from 3 independent donors). Fold response and the p-value to the un-stimulated control samples are shown. Bold letters indicate significantly up-regulated, while italic letters represent significantly down-regulated genes. Stars specify known target genes. Genomic locations are marked as follows: Pp, Proximal Promoter; ID, Immediate Downstream; En, Enhancer; E, Exon. Unlabeled boxes contain genes that were identified by GEA but not chip-on-ChIP.</p

STAT5 binds PDE4B in an IL-2 inducible manner in hPBMC.

<p>ChIP assays performed with STAT5 antibodies or control sera (IgG) were carried out in quiescent (−) or IL-2 stimulated (30 min, +) hPBMCs isolated from three independent donors to measure the enrichment of the PDE4B putative STAT5 regulated region (<b>A</b>) or the IL2RA enhancer PRR III (<b>B</b>) identified by chip-on-ChIP. Inputs represent 1% of chromatin used in ChIP assays and error bars represent standard deviations. * represents statistically significant differences (p<0.05).</p

Genomic location of IL-2 regulated STAT5 binding sites.

<p>(<b>A</b>) Shown are known STAT5 target genes including SOCS2, SOCS3, CISH and those also identified by chip-on-ChIP (IL2RA, BCL2, BCL6 and CDK6) and (<b>B</b>) 18 newly identified promoter located genes visualized by the IGV using hg19.</p

IL-2 mediated enrichment of the STAT5 binding site in the IL-2RA enhancer (A) before and (B) after amplification.

<p>(A) Kit225 IL-2-dependent human leukemia cells were made quiescent and then stimulated with medium (−) or IL-2 (+) for 30 min at 37°C, fixed with 1% formaldehyde for 10 min at room temperature and then chromatin immunoprecipitated with antibodies to C-terminal STAT5A/B mix or control IgG. The eluted DNA was amplified with primers corresponding to the human IL2RA PRR III. Representative data from three independent experiments are shown. Input material represents 5% of immunoprecipitated chromatin. Beads control represents samples in which immunoprecipitation was performed without any antibodies but otherwise was handled identically. (B) Cells were treated as described above and then for the microarray experiments the ChIP-ed DNA was randomly amplified following ligation of linkers as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057326#s3" target="_blank">Materials and Methods</a> section from three independent experiments. The amplified DNA was then used as template in qPCR reactions to measure the enrichment of the IL2RA PRR III.</p

Cis-Regulatory Element Annotation System (CEAS) analysis of STAT5 binding elements.

<p>(<b>A</b>) <b>Genome-wide distribution of STAT5 binding sites (percent of total number of sites).</b> The chip-on-ChIP identified elements that fell within 300 kb from coding regions were analyzed based on their distance from nearest genes using CEAS. The pie chart represents “%” distribution. (<b>B</b>) <b>Enriched transcription factor binding motifs with highest fold-change (upper panel) and highest significance (lower panel).</b> Enriched TF binding sites and their matrices within the chip-on-ChIP identified, CEAS analyzed regulatory elements are shown.</p

Measurement of siRNA expression with small RNA specific UPL-based quantitative PCR assays.

<p>Sequence specificity of PRMT1 specific siRNA for exon 3 of PRMT1 (A). PRMT1 specific siRNA levels as detected by qPCR and the corresponding mPRMT1 mRNA as well as protein levels detected by qPCR and Western blot analysis (B).</p

Sequences of matured small RNA molecules investigated. All sequences are in written in 5′-3′ direction.

<p>Sequences of matured small RNA molecules investigated. All sequences are in written in 5′-3′ direction.</p

Sensitivity and specificity of miRNA specific UPL-based quantitative PCR system.

<p>Amplification plot of mmu-mir-1 in range from 10 ng to 10^–3 ng input mouse heart total RNA (A). Sequence similarities and differences between mir-181a, b, and c (B). Amplification plot of synthetic mir-181a miRNA ranging from 10 pM to 10^–4 pM input mir-181a amplicon (C). Standard curve of synthetic mir-181a miRNA (D). Specificity and relative detection capacity of mir-181 specific UPL-based qPCR assays. Numbers represent the percentage of the signals measured on the synthetic amplicons. 100% is always the signal measured by an assay on its specific synthetic amplicon, like mir-180a assay on the mir-181a synthetic amplicon. In brackets the corresponding Cp values are shown.(E).</p

Schematic description of small RNA specific UPL-based quantitative PCR assay and our oligo design system.

<p>Two steps small RNA specific UPL-based quantitative PCR assay relies on reverse transcription using small RNA specific stem-loop RT primer and real-time quantitative PCR reaction using small RNA specific forward primer, UPL21 probe and universal reverse primer (A). Workflow of our oligo design system (B). Primers and probe for the designed hsa-mir-181a specific UPL-based quantitative PCR assay (C).</p