Expanded combined analysis of direct and functional E2A-PBX1 targets.

<p>The analysis was performed using the 102 direct targets identified by ChIP-chip analysis and the data set of the functional targets including the top 2000 genes up and down regulated after siRNA silencing of E2A-PBX1. Venn diagrams were generated and the interfaces of direct and functional E2A-PBX1 targets are depicted. 10 genes that were both direct targets and functionally down-regulated <b>(</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087602#pone-0087602-g005" target="_blank"><b>Figure 5A</b></a><b>)</b> and 10 that were both direct targets and functionally up-regulated <b>(</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087602#pone-0087602-g005" target="_blank"><b>Figure 5B</b></a><b>)</b> dependent on expression of E2A-PBX1 were identified. We are not able to address E2A-PBX1 as a functional repressor (for the 10 down regulated genes) or whether its depletion allowed accessibility to another transcriptional activator. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087602#pone-0087602-g005" target="_blank"><b>Figure 5C</b></a> Selected direct and functional E2A-PBX1 targets are highly expressed in primary E2A-PBX1 leukemias. Pediatric Cancer Genome Project. <a href="http://www.pediatriccancergenomeproject.org/site/accessed" target="_blank">http://www.pediatriccancergenomeproject.org/site/accessed</a> 14 Oct 2013. The expression of four genes UGT2B15, ASNS, WNT16 and HK2, that were among the direct and functional E2A-PBX1 targets in primary leukemia’s are shown. The expression of these genes was analyzed in Hyperdiploid leukemia’s (H), in E2A-PBX1 leukemia’s (E), TEL-AML1 (T) leukemia’s and BCR-ABL1 (B) leukemia’s are shown. The analysis of expression was performed using the gene expression tool and data available at St. Jude Children’s Research Hospital – (Washington University).</p

E2A-PBX1 ChIP binding.

<p><b>A. Peak promoter sequence analysis</b>. Chromatin immunoprecipitation assays were performed to identify direct targets E2A-PBX1 using the human promoter array; HG18; (NCBI Build 36, Roche Nimblegen, Madison WI). A search was performed using the software WebMOTIFS and de novo motif finding a search for Transcription Factors that are likely to contact and regulate the binding sites identified by ChIP-chip. The 3 most significant transcription factor families (classes of transcription factors most likely to regulate the input sequences) are PBX, POU and STAT. <b>B. Positional binding of E2A-PBX1</b>. Among the 108 top hit ChIP genes, 76 genes have a single annotated transcription start site. The average intensity of ChIP pull-down is graphed along the promoter region in a smoothed plot. This histogram of the positions of maximal peak intensities (mean of 3 replicates) relative to the transcription start site (TSS) in target genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087602#pone.0087602.s007" target="_blank">Table S3</a>). Most target genes show a maximum probe intensity around −500 bp upstream of the TSS. Probe regions with multiple TSS were excluded from this analysis. The promoter coverage area was complete for all genes between −2200 and +500 by the TSS (solid line) with some additional coverage for some genes outside of that region (dotted line).</p

The 9p21 deletion is not always targeting CDKN2A.

<p>Copy number alterations in a group of 22 t(1;19)⁺<i>E2A-PBX1</i> bone marrow samples are shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087602#pone-0087602-g003" target="_blank"><b>Figure 3A</b></a>. Patient samples were subjected to hierarchical clustering analysis; we found the 9p21 deletion to exist in four of the 22 patients we have analyzed, with one patient exhibiting a homozygous deletion, white color refers to no changes, red to gain and blue to loss. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087602#pone-0087602-g003" target="_blank"><b>Figure 3B</b></a>. Fine map of deletions in the 9p21 region showing the deletion endpoints among 8 patients. The commonly deleted region among 10 patients (with 45%, including 7 patients in this figure and the other 3 others with complete arm loss) is the interferon gene cluster telomeric to the <i>CDKN2A/B</i> locus.</p

Combined KEGG Pathway analysis of direct and functional E2A-PBX1 targets.

<p>The analysis was performed using Exploratory Gene Association Networks (EGAN) software tool. E2A-PBX1 direct (108 direct targets identified by ChIP-chip) and functional targets (122 significant differentially expressed genes between E2a-Pbx1 silenced samples and controls) and Pathways that might be regulated by them were visualized. Interfaces of direct and functional E2A-PBX1 targets are depicted. Magenta lines depict the connection of the genes to the direct target and/or functional target group; blue lines show the participation of the genes in KEGG pathways and brown lines show known interaction between genes connected.</p

Identified ChIP-chip targets on expression array in E2A-PBX1 silenced vs control cells.

<p><b>A.</b> ChIP-chip analysis data and expression analysis data upon E2A-PBX1 silencing were combined to get an overview of the direct and functional targets of E2A-PBX1. siRNA to E2A-PBX1 was employed to silence E2A-PBX1. The MA plot depicts the expression array changes upon silencing E2A-PBX1. Genes are ordered on the x-axis on the basis of their expression in untreated 697 cells. The Y-axis displays the change in expression upon E2A-PBX1 silencing. Those genes that are direct hits of E2A-PBX1 by chromatin immunoprecipitation assays (108 genes) were plotted against the expression array changes upon silencing and are shown in red color and marked with a blue dot; grey dots indicate all other genes assessed by the array. <b>B.</b> Here we compare the changes in expression of the 108 E2A-PBX1 direct targets to the expression change of all the other genes (non-direct E2A-PBX1 targets) upon E2A-PBX1 silencing. The set of identified ChIP-chip targets show collective down-regulation trend in the E2A-PBX1 silenced samples compared to controls (p<1.63e-06).</p

Risk allele proportions at genomic loci with somatic gain (<i>i</i>.<i>e</i>. hyperdiploid chromosomes).

<p>Allelic copy number was measured in constitutional DNA and leukemia bone marrow (tumor) DNA from HeH ALL patients heterozygous for ALL-associated SNPs on chromosomes frequently gained in HeH ALL: <i>CEBPE</i> SNP rs2239633 (A), <i>ARID5B</i> SNP rs7089424 (B), <i>PIP4K2A</i> SNP rs10764338 (C), and <i>GATA3</i> SNP rs3824662 (D). Risk allele proportions are displayed as a fraction of the total allelic copy number measured in each patient using ddPCR. Each subject was assayed in duplicate, and error bars represent the standard error of the mean (some error bars not visible due to their range falling within boundaries of the data point). Upper/lower thresholds of allelic imbalance (AI) were +/- 3 SDs from the mean allelic proportion from repeat measurements in constitutional DNA samples (white squares). For rs2239633, 19 tumor samples showed AI favoring the risk allele versus 13 patients with AI favoring the protective allele (P = 0.19). For rs7089424, 20 tumor samples showed AI favoring the risk allele versus 15 patients with AI favoring the protective allele (P = 0.25). For rs10764338, 4 tumor samples showed AI favoring the risk allele versus 5 patients with AI favoring the protective allele (P = 0.50). For rs3824662, 10 tumor samples showed AI favoring the risk allele versus 9 patients with AI favoring the protective allele (P = 0.50). Data points clustering at ~0.66 and ~0.33 represent a 3:2 or 2:3 risk:protective allele proportion due to chromosomal copy number shifting from diploid (n = 2) to triploid (n = 3). Data points at ~0.75 represents a 3:1 risk:protective allele proportion due to a diploid to tetraploid (n = 4) shift in chromosome ploidy. Data points at 1 and 0 likely represent HeH ALL that has arisen via near-haploidy, leading to chromosomal LOH (Paulsson <i>et al</i>. 2005) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143343#pone.0143343.ref031" target="_blank">31</a>].</p

Candidates for tumor PAI: recurrent SCNA loci from TCGA that overlap cancer-associated SNPs (NHGRI GWAS Catalog) identified in matching tumor types.

<p>SNP associations and tumor types highlighted in bold indicate those whereby cancer type of SNP associations matches tumor type in which recurrent SCNAs were identified.</p><p>* Chromosomal locations based on human genome build hg19.</p><p>** Cancer type of SNP association loci that overlap SCNA regions (SNPs retrieved from January 2015 version of NHGRI GWAS catalog).</p><p>‡ Tumor type in which recurrent SCNAs were detected in TCGA.</p><p>ALL = acute lymphoblastic leukemia; BLCA = bladder; BRCA = breast; CLL = chronic lymphoblastic leukemia; CRC = colorectal; GBM = glioblastoma multiforme; HNSC = head and neck squamous cell carcinoma; KIRC = kidney renal cell carcinoma; LAML = acute myeloid leukemia; LUAD = lung adenocarcinoma; LUSC = lung squamous cell carcinoma; OV = serous ovarian carcinoma; UCEC = endometrial (uterine).</p><p>Candidates for tumor PAI: recurrent SCNA loci from TCGA that overlap cancer-associated SNPs (NHGRI GWAS Catalog) identified in matching tumor types.</p

Comparison between deletion gene copy number measurements made by SMART-ddPCR and MLPA.

<p>Copy number measurements were available from ddPCR and MLPA assays for SNPs at the two deletion genes <i>CDKN2A</i> (SNP rs3731249) and <i>IKZF1</i> (SNP rs4132601) in 27 and 75 tumor DNA samples respectively. (A) High correlation (R2 = 0.91) between the combined deletion gene copy number measurements made by ddPCR and MLPA. (B) Bland-Altman plot displaying the difference between measurements made in the same individual against their mean, as measured by two different methodologies (<i>i</i>.<i>e</i>. ddPCR and MLPA). There is very close agreement between the copy number measurements made by the two assays, as demonstrated by the narrow limits of agreement (-0.170 to 0.138) either side of the observed average agreement (-0.016).</p

Summary of the childhood ALL-associated SNPs investigated and the corresponding tumor DNA allelic imbalance results.

<p>* Number of heterozygous samples (for each SNP) with available bone marrow (i.e. tumor) DNA.</p><p>‡ % of HeH ALL samples with gains of that chromosome, based on data from Paulsson <i>et al</i>. (2010) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143343#pone.0143343.ref021" target="_blank">21</a>] and Dastugue <i>et al</i>. (2013) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143343#pone.0143343.ref022" target="_blank">22</a>].</p><p>† High hyperdiploid samples only.</p><p>Significant p-values highlighted in bold.</p><p>Summary of the childhood ALL-associated SNPs investigated and the corresponding tumor DNA allelic imbalance results.</p

<i>CDKN2A</i> and <i>IKZF1</i> SNP allele proportions in tumor DNA relative to genomic control copy number.

<p>Stacked histograms showing tumor DNA copy number of (A) <i>CDKN2A</i> and (B) <i>IKZF1</i> SNPs relative to a genomic control locus (<i>SLC24A3</i>). Black and grey bars represent the proportions of normalized SNP copy number accounted for by the risk and protective alleles respectively. White bars represent the difference between <i>CDKN2A</i>/<i>IKZF1</i> SNP copy number and the genomic control gene copy number. SMART-ddPCR was used to measure copy number of SNP risk/protective alleles, as well as the genomic control locus, in 35 leukemia bone marrow (tumor) DNA samples for <i>CDKN2A</i> (SNP rs3731249) and 75 tumor DNA samples for <i>IKZF1</i> (SNP rs4132601). Samples are grouped into those with allelic imbalance (AI) and those without AI, and arranged in order of normalized gene copy number relative to the genomic control.</p