Multiple hematopoietic TFs bind the +6-kb enhancer. (A) ChIP-seq tracks for TFs occupying the +6-kb enhancer region in a hematopoietic progenitor cell line (HPC-7).32 (B) ChIP-seq peaks for CEBPA, FLI1, ERG, and SPI1 at the +6-kb enhancer in sorted GMP cells from mice expressing MLL-AF9 in hematopoietic cells.28 (C) ChIP-seq for CEBPA in different murine hematopoietic cells (region encompassing the +6 kb region has been highlighted). Lp30 GMP, GMPs isolated from mice transplanted with fetal liver from Lp30 mice.34-36 (D) ChIP-qPCR shows enrichment of CEBPA binding within the +6-kb enhancer using 3 primer pairs (P1-P3). (E) Luciferase reporter assay. The +6-kb enhancer sequence was amplified into 3 fragments (peak 1: 0-570 bp, peak 2: 571-1240 bp, and peaks 1+2: 0-1240 bp) and cloned into pGL4–basic vector. Results represent fold induction of relative luciferase activity after normalization to Renilla control in 2 independent experiments, each done in triplicates. (F) EMSA was performed with oligonucleotide (oligo) sequences located within the +6-kb enhancer region. Biotin-labeled target and mutant oligonucleotides were mixed with protein extracts from 293T cells transfected with an empty vector or an expression vector for CEBPA. The reaction mixtures were resolved on native 10% polyacrylamide–TBE gel. Cold competition was carried out with 100-fold molar excess of unlabeled oligo. *P < .05.