The ERG +85 stem cell enhancer is active in T-ALL cells, relies on E-Box and Ets binding motifs, and is bound by oncogenic transcription factors in vivo. (A) Nucleotide sequence alignment of the Erg +85 enhancer region. Alignment of human (Hs), mouse (Mm), dog (cf), and opossum (Md) sequences extracted from the UCSC genome browser with conserved Ets (blue), Gata (red), E-Box (yellow) motifs colored for clarity. Black boxes indicate 100% cross-species sequence conservation, and gray boxes show less conserved sequences. (B) Enhancer activity of the hERG +85 region. Results of stable transfection assays in MOLT4 T-ALL, 416B hematopoietic precursors, and REH B-ALL cells showing luciferase activity of the +85 enhancer/pGL2P construct relative to pGL2P alone. The +85 fragment was active in MOLT4 and 416B, but not REH cells. (C) Mutational analysis of +85 enhancer activity in MOLT4 cells. Schematic on the left shows TF binding motifs of WT and mutant enhancers as detailed in panel A. (D) ChIP-PCR analysis of TF binding. The enhancer has active chromatin marks (AcH3) in 416B blood progenitors as well as in T-ALL cells. There is strong LMO2 binding in all cell types, whereas SCL and LYL1 enrichments are more pronounced in T-ALL cells. The Ets factors, FLI1 and ERG, are also enriched in T-ALL cells. Consistent with their expression profiles, Gata2 is enriched at the +85 enhancer in 416B progenitors, whereas GATA3 is enriched in T-ALL cells.