ERG +85 stem cell enhancer is regulated by HSC TFs in AML cells. (A) H3K9/14Ac ChIP-chip profiles across the ERG locus are shown for 2 high (ME-1 and KG-1) and 1 low (K562) ERG-expressing AML cell lines. qRT-PCR confirmation of enrichments (from an independent ChIP experiment) is shown to the right of each profile. (B) Abundance of ERG transcripts originating from either the distal or proximal promoter is shown relative to total ERG expression in each AML cell line. (C) The +85 enhancer has a number of highly conserved TF binding sites including E-box (EB, CANNTG, yellow), Ets (E, GGAW, blue), Myb (M, YAACNG, purple), Gata (G, GATA, red), and Gfi (AAATCA, cyan). (D) Stable (KG-1 and K562) and transient (ME-1) transfection assays show activity of the +85 enhancer in conjunction with a heterologous SV40 promoter. (E) Mutation of conserved TF binding sites in conjunction with transient (ME-1) and stable (KG-1) transfection assays in ERG-expressing AML cells shows the dependence of the +85 enhancer on specific binding sites for its activity in leukemic cells. (F) Activity of the endogenous ERG promoters in AML cells either alone or in conjunction with the +85 enhancer measured by transient (ME-1) and stable (KG-1) transfection assays. (G) ChIP-qPCR enrichment of HSC TFs at the +85 enhancer and promoters of ERG in ME-1 and KG-1 AML cells. (H) ChIP-qPCR enrichment of TFs at the ERG +85 stem cell enhancer and promoters in AML patient 1, a patient with H3K9/14Ac enrichment at both promoters and enhancer. *P < .05; **P < .01. NS, not significant.