Oncogene (2011) 30, 2044–2056 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Genome-wide screen reveals WNT11, a non-canonical WNT gene, as a direct target of ETS transcription factor ERG LH Mochmann1, J Bock1, J Ortiz-Ta´nchez1, C Schlee1, A Bohne1, K Neumann2, WK Hofmann3, E Thiel1 and CD Baldus1 1Department of Hematology and Oncology, Charite´, Campus Benjamin Franklin, Berlin, Germany; 2Institute for Biometrics and Clinical Epidemiology, Charite´, Campus Mitte, Berlin, Germany and 3Department of Hematology and Oncology, University Hospital Mannheim, Mannheim, Germany E26 transforming sequence-related gene (ERG) is a current therapies in acute leukemia patients with poor transcription factor involved in normal hematopoiesis prognosis characterized by high ERG mRNA expression. and is dysregulated in leukemia. ERG mRNA over- Oncogene (2011) 30, 2044–2056; doi:10.1038/onc.2010.582; expression was associated with poor prognosis in a subset published online 17 January 2011 of patients with T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Herein, a Keywords: ETS-related gene (ERG); WNT11; acute genome-wide screen of ERG target genes was conducted leukemia; ERG target genes; 6-bromoindirubin-3-oxime by chromatin immunoprecipitation-on-chip (ChIP-chip) in (BIO); morphological transformation Jurkat cells. In this screen, 342 significant annotated genes were derived from this global approach. Notably, ERG-enriched targets included WNT signaling genes: WNT11, WNT2, WNT9A, CCND1 and FZD7. Further- more, chromatin immunoprecipitation (ChIP) of normal Introduction and primary leukemia bone marrow material also confirmed WNT11 as a target of ERG in six of seven The erythroblastosis virus E26 transforming sequence patient samples. A larger sampling of patient diagnostic (ETS) encodes the ETS-related gene (ERG) that has an material revealed that ERG and WNT11 mRNA were co- important physiological role in hematopoiesis (Loughran expressed in 80% of AML (n ¼ 30) and 40% in T-ALL et al., 2008), angiogenesis (Birdsey et al., 2008), vascular (n ¼ 30) bone marrow samples. Small interfering RNA (Ellett et al., 2009) and bone development (Iwamoto (siRNA)-mediated knockdown of ERG confirmed down- et al., 2007). ERG belongs to the highly conserved ETS transcription factor family that is defined by the ETS regulation of WNT11 transcripts. Conversely, in a tet-on 0 0 ERG-inducible assay, WNT11 transcripts were co- DNA-binding motif 5 -GGA(A/T)-3 (Sharrocks, 2001; stimulated. A WNT pathway agonist, 6-bromoindirubin- Wei et al., 2010). In hematopoietic development, ERG 3-oxime (BIO), was used to determine the effect of cell has long been postulated to function during early stages growth on the ERG-inducible cells. The addition of BIO of T-cell development as its mRNA abundance peaks resulted in an ERG-dependent proliferative growth and diminishes as cells undergo T-lineage commitment advantage over ERG-uninduced cells. Finally, ERG (Anderson et al., 1999). In addition, ectopic ERG induction prompted morphological transformation where- expression was shown to induce megakaryocytic differ- by round unpolarized K562 cells developed elongated entiation in human K562 cells (Rainis et al., 2005) and protrusions and became polarized. This morphological in hematopoietic progenitors ERG promotes expansion transformation could effectively be inhibited with BIO of megakaryocytes (Stankiewicz and Crispino, 2009). and with siRNA knockdown of WNT11. In conclusion, ERG function was further characterized by Loughran ERG transcriptional networks in leukemia converge on et al. in heterozygous mice harboring a missense muta- WNT signaling targets. Specifically, WNT11 emerged as tion that phenotypically displayed mild cytopenia in a direct target of ERG. Potent ERG induction promoted the B-cell compartment and notably had a reduction morphological transformation through WNT11 signals. of progenitor cells by 50%. Furthermore, homozygosity The findings in this study unravel new ERG-directed for the same mutation failed to establish definitive hemato- molecular signals that may contribute to the resistance of poiesis at the embryonic stage. Thus, normal ERG function was necessary to establish and maintain hematopoiesis. ERG dysregulation has been reported in solid tumors and hematological malignancies. Three fusion proteins Correspondence: Dr CD Baldus, Department of Hematology and composed of ERG with TMPRSS2 (Tomlins et al., Oncology, Charite´, Campus Benjamin Franklin, Hindenburgdamm 30, 2005; Klezovitch et al., 2008), EWS (Sorensen et al., 12203 Berlin, Germany. E-mail: [email protected] 1994) or TLS (Kong et al., 1997) create oncogenic Received 14 July 2010; revised 2 November 2010; accepted 23 November proteins. The most frequent chromosomal fusion in 2010; published online 17 January 2011 prostate cancer consists of the 50-untranslated region of ERG genome-wide screen in acute leukemia LH Mochmann et al 2045 TMPRSS2 fused with 30-end ERG. This fusion has ChIP (C20) and with double (C20 and C17) precipitat- multiple variants, of which two of the most common ing antibodies for increased accessibility to multiple variants are associated with poor outcome (Narod et al., ERG epitopes (refer to Materials and methods for a 2008). In leukemia, ERG overexpression is also believed detailed description). Each ChIP with single and double to contribute to the molecular pathogenesis in a sub- precipitating antibodies was carried out in duplicate. set of T-cell acute lymphoblastic leukemia (T-ALL) and The in vivo assay allowed for the enrichment and acute myeloid leukemia (AML) patients. High ERG identification of ERG-bound DNA sequences that were expressers were associated with an inferior clinical subsequently hybridized to a high-resolution human outcome (Marcucci et al., 2005; Baldus et al., 2006). promoter chip. Duplicate ChIP-chips 1 and 3 (single- The pathogenesis of ERG was also observed in antibody ChIP) resulted in 13 070 and 4405 signifi- sublethally irradiated mice transplanted with ERG cant peaks, respectively. Duplicate ChIP-chips 2 and 4 transduced progenitor cells, whereby megakaryoblastic (double-antibody ChIP) resulted in 11 227 and 6630 leukemia developed (Salek-Ardakani et al., 2009). Thus, significant peaks, respectively. As expected, many several clinical and experimental studies indicate that significant peaks were detected at individual gene ERG contributes to the pathogenesis in cancer and promoters, which yielded 1683, 3066, 1304 and 973 leukemia; however, the underlying biological mechan- single genes, in ChIP-chips 1–4, respectively. Pooled isms are not yet fully understood. gene sets from ChIP-chips 1 and 3 are denoted as ChIP- To unravel the molecular function of ERG in acute chip I and pooled gene sets from ChIP-chips 2 and 4 are leukemia, we have conducted a genome-wide screen of denoted as ChIP-chip II. Finally, only overlapping genes ERG target genes. Chromatin immunoprecipitation- from ChIP-chips I and II were combined to condense on-chip (ChIP-chip) analyses of ERG candidate target the significant candidate gene pool to 342 gene annota- genes revealed that ERG may participate in a broader tions (Table 1). DAVID Functional Annotation Tool spectrum of biological signaling than previously des- (Laboratory of Immunopathogenesis and Bioinfor- cribed. ERG loss and gain of function experiments matics, Frederick, MD, USA) was used to characterize directly affected WNT11, a non-canonical WNT path- biological themes (Table 2). Statistically enriched gene way gene. Moreover, a proliferative growth advantage ontology categories suggest a broad functional role for was observed when ERG-induced cells were treated ERG that included developmental processes, multi- with WNT agonist 6-bromoindirubin-3-oxime (BIO) cellular processes, biological adhesion and biological and a glycogen synthase-3b (GSK-3b) inhibitor. Finally, regulation (P-value o0.05). Furthermore, ERG candi- co-expression of ERG and WNT11 stimulated morpho- date genes subjected to Ingenuity Pathway Analyses logical transformation of round hematopoietic cells to revealed an overlap of enriched WNT target genes in polarized cells with protrusions upon ERG induction. In several key developmental pathways (Table 3). addition, the elongation process of ERG-induced cells was effectively inhibited by the addition of BIO and with small interfering RNA (siRNA)-mediated knockdown Validation of enriched promoter regions and selection of WNT11. These findings show that in human leukemia of ERG target genes WNT11 is a direct target of ERG and highlight a role Based on the gene’s described relevance to hemato- for ERG in the WNT signaling pathway. poiesis and leukemia in the scientific literature (versus a random calculated approach), 24 of the 342 enriched promoter regions were selected for further examination. MATCH algorithm was used to determine the signifi- Results cance and location of conserved ETS-binding motif (5-GGAA/T-3) in each of the 24 putative promoter Genome-wide screen of ERG candidate genes regions up to 2 kb from the transcription start site (TSS) ERG transcriptional networks in leukemia are un- (Table 4 and Supplementary Table 1). In all, 17 of 24 known. Thus, in order to construct ERG-related selected targets were confirmed by quantitative PCR, networks, a genome-wide screen by ChIP-chip was with at least twofold enrichment relative to total chro- conducted in a human T-cell leukemia line, Jurkat. ChIP matin