Identification of ERG Specific Target Genes by Genome-Wide Screening in T-Lymphoblastic Leukemia

The Ets related gene, ERG, encodes a transcription factor with a vital role in hematopoiesis. Recent findings have shown that ERG knockout mice require a minimum of one functional allele to ensure embryonic blood development and adult stem cell maintenance. Moreover, it was earlier reported that enforced expression of ERG induced oncogenic transformation in 3T3 cells. Overexpression of ERG, observed in a subset of acute T-lymphoblastic and acute myeloid leukemia patients, was associated with an inferior outcome. However, the impact of ERG contributing to this unfavourable phenotype has yet to be determined, as downstream targets of ERG in leukemia remain unknown. Herein, we conducted a genome-wide analysis of ERG target genes in T-lymphoblastic leukemia. Chromatin immunoprecipitation-on-chip array (ChIP-on-chip) analyses were performed using two ERG specific antibodies for the enrichment of ERG-bound DNA templates in T-lymphoblastic leukemia cells (Jurkat) with input DNA or IgG precipitated DNA as controls. Enriched DNA templates and control DNA were differentially labelled and co-hybridized to high resolution promoter chip arrays with 50–75mer probes (770,000) representing 29,000 annotated human transcripts (NimbleGen). Based on two independent ChIP-on-chip assays, bioinformatic analysis (ACME) yielded statistically significant enriched peaks (using a sliding window of 1000 bp, and a P-value < 0.0001) identifying promoter regions of 365 potential ERG target genes. From these genes, clustering by functional annotation was performed using the DAVID database and subsequently genes related to leukemia were further selected for quantitative PCR validation. The design of promoter primers included the highly conserved ETS GGAA DNA binding site. Genes with greater than two-fold enrichment (ERG ChIP versus control) included WNT2 (17-fold), OLIG2 (14-fold), WNT11 (7-fold), CCND1 (5-fold), WNT9A (4-fold), CD7 (3-fold), EPO (3-fold), ERBB4 (3-fold), RPBJL (3-fold), TRADD (3-fold), PIWIL1 (2-fold), TNFRSF25 (2-fold), TWIST1 (2-fold), and HDAC4 (2-fold). Interestingly, enriched target genes involved in developmental processes (WNT2, WNT9A, WNT11, TWIST1, PIWIL1, ERBB4, and OLIG2) have shown oncogenic potential when mutated or overexpressed. Thus, we hypothesize that overexpression of ERG may contribute to T-cell leukemogenesis by the deregulation of these oncogenic targets. Further disclosure of ERG directed downstream pathways may contribute to the design of specific treatment strategies (such as WNT inhibitors) with particular effectiveness in ERG deregulated leukemia.