Pim1 is a direct ERG target activated by a +10 enhancer site. (A) Pim1 levels by quantitative RT-PCR in TgERG leukemia and mouse WT whole or lineage-negative bone marrow, in human CMK (low ERG) or CMY (high ERG) cells, and in CMY cells transfected with either nontargeting shRNA (shNT) or ERG (shERG) shRNA. (B, left panel) ERG and Pim1 protein levels in the bone marrow of TgERG leukemic mice and WT littermates; (right panel) ERG and PIM1 protein levels in CMK and CMY human AML cells. (C) Density plots showing ERG binding to Pim1 loci in 2 TgERG leukemias. (D) Density plots showing occupancy of the PIM1 loci in human CD34⁺ cells by a heptad of transcription factors and by open chromatin marks. (E) Luciferase reporter assay. The Pim1 +10 candidate regulatory region was cloned into a luciferase reporter plasmid and stably transfected to L8057 cells. Cells expressing the Pim1 +10 region showed a four to sixfold increase in promoter activity compared with the empty vector. Mutations in the 2 ETS motifs in the Pim1 +10 region nearly abolished its enhancing effect. (F) Transgenic reporter mice: The Pim1 +10 region was cloned into a lacZ reporter vector and used to generate transgenic F0 embryos. Typical images showing Xgal staining in fetal liver of E10.5 embryo in either whole embryo (left) or in fetal liver sections (right).