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

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

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

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

Conditions of host lipid restriction do not impact host cell viability.

A) Primary macrophages were cultured in medium in the presence (+) or absence (-) of exogenous lipids (ExoLipids) supplemented with 4 μM fatostatin, 5 μM FAF albumin plus 20 μM palmitate or oleate, 2 μM A939572 or 100 μM myriocin for 24 hours and cell death based on lactate dehydrogenase (LDH) release was determined. B) Primary macrophages were treated as in A) and cellular respiration based on redox potential was measured. A-B) Data are the mean ± SD of 3 biological replicates, consisting of 2 technical replicates each. (TIF)</p

Inducing host cell lipid storage impacts LCV architecture and integrity.

A) Inducing lipid droplet formation restricts LCV membrane expansion. Primary bone marrow-derived A/J murine macrophages were pre-treated with oleate (OA) or palmitate (PA) for 18 hrs to induce lipid droplet formation, rinsed and then infected with L. pneumophila (Lp) in the absence of exogenous lipids by culturing cells in medium lacking serum. NT, no treatment control macrophages not pre-exposed to fatty acids and cultured in the presence of serum. At 9 hpi, cells were fixed, stained and visualized by fluorescence microscopy (left panel). Compact LCVs were scored as described in Fig 4 and quantified (right panel). B) Treating cells with oleate and palmitate leads to LCV rupture. Primary bone marrow-derived A/J mouse macrophages were infected with L. pneumophila in the presence of oleate or palmitate for 6 hours, then fixed, stained and visualized by fluorescence microscopy (left panel). Colocalization of LCVs with Galectin-3 (Gal3) was scored (right panel). C) Inducing lipid droplet formation leads to host cell death during infection. Primary bone marrow-derived A/J murine macrophages were pre-treated with oleate or palmitate then challenged with L. pneumophila as in A). Cells were fixed, stained and visualized by fluorescence microscopy (left panel), and uninfected (UI) and infected (I) cells were scored for aberrant nuclear morphology at 1 and 9 hpi (right panel). A-C) Data are the mean ± standard deviation of 3 biological replicates consisting of 3 technical replicates each, scoring ∼50–100 LCVs per technical replicate (A,B) or ∼50–100 LCVs for uninfected and infected cells per technical replicate (C). *, Student’s t-test p < 0.05 relative to NT control cells (A,B) or fatty acid-treated cells to NT in uninfected and infected, respectively (C), unless otherwise indicated.</p

Palmitate and oleate similarly modulate host cell <i>de novo</i> lipogenesis.

Since de novo fatty acid synthesis is regulated through SREBP in response to available lipids, one possible explanation for the differential growth of L. pneumophila in macrophages treated with oleate versus palmitate (Fig 1) was that oleate reduced fatty acid biosynthesis to a greater extent than palmitate, thereby having more pronounced effects on cellular fatty acid levels in the absence of exogenous lipid resources. To test this, FASN levels and [3H]acetate incorporation into lipids was examined under these conditions as a measure of de novo lipogenesis. A) Fatty acid treatment does not alter endogenous fatty acid synthase levels in macrophages. Primary A/J mouse macrophages were incubated in the presence (+) or absence (-) of serum (exogenous lipids, ExoLipids) or in medium lacking serum supplemented with either oleate or palmitate for 18 hrs. Cells lysates were then analyzed for FASN by Western analysis (top panel) and quantified (bottom panel), normalizing to total protein. B) Oleate and palmitate treatment similarly reduces de novo lipogenesis. Macrophage as in A) were exposed to [3H]acetate. Lipids were then extracted and examined for radiolabel incorporation, normalizing to total protein. The absence of exogenous lipids resulted in a 5-fold increase in the amount of lipid synthesis when compared to cells cultured in the presence of exogenous lipids, consistent with the response of host cells to activate lipogenesis when exogenous lipids are not available. When either palmitate or oleate was added, [3H]acetate incorporation was significantly reduced, consistent with repression of lipid biosynthetic pathways through SREBP by exogenously available fatty acids. The extent of the effect observed was similar between palmitate and oleate, indicating the differential effects of oleate and palmitate on L. pneumophila replication was not due oleate more extensively limiting de novo fatty acid synthesis in the absence of exogenous lipids. A-B) Data are the mean ± standard deviation of 3 biological replicates, consisting of 1–2 technical replicates each. *, Student’s t test p (TIF)</p

Loss of LegA3/AnkH and/or LegK7 does not attenuate <i>L</i>. <i>pneumophila</i> growth in macrophages impaired for <i>de novo</i> lipogenesis.

Primary bone marrow-derived A/J mouse macrophages were infected with the indicated L. pneumophila strains in the presence (+) or absence (-) of de novo lipid synthesis by treating cells with the SREBP1 inhibitor fatostatin. Bacterial growth was quantified based on recovered colony forming units (cfus) from host cell lysates at 24 hrs, and normalized to wild type (WT) bacteria in untreated host cells by the number of intracellular bacteria at 1 hpi. Data are the mean ± standard deviation of 3 biological replicates consisting of 3 technical replications each. (TIF)</p