Protein/DNA interactions on the NF-E2 1A promoter. (A- B) EMSA of the putative AML1 binding sites in the NF-E2 promoter. Nuclear extracts from HEL cells were incubated with a 32P-labeled oligonucleotide containing either a consensus AML1 binding site (A) or an oligonucleotide spanning bp −3394 to −3360 of the NF-E2 1A promoter, which contains a predicted AML1 binding site (B). (A-B) In the indicated lanes, a 100× excess of the indicated, nonradioactive oligonucleotide was added. Numbers indicate the position of the oligonucleotides within the NF-E2 promoter; “cons” indicates the consensus sequence; “mut” indicates oligonucleotides containing 3-bp mutations in the AML1 binding sites (see Figure 3). Alternatively, an antibody to AML1 (B, lane 6) or a control antibody (B, lane 7) was added. A filled arrowhead indicates the specific AML1/DNA complex. The open circle shows nonspecific binding to the DNA probe, and the open arrowhead denotes unbound oligonucleotide. A filled circle shows the supershifted AML1-antibody complex. (C-D) ChIP analysis of AML1 binding sites on the NF-E2 1A promoter. (C) HEL cell lysates were chromatin immunoprecipitated (ChIPed) either with an antibody to AML1 or with an unrelated IgG control, as indicated. ChIPed DNA was amplified by PCR by the use of either primers covering the AML1 binding sites in the NF-E2-1A promoter or control primers from a distal region in the NF-E2 1A promoter or from the GAPDH promoter, as indicated. In lane 1, a 1:50 dilution of the input DNA was used; lane 4 shows control PCRs without DNA. (D) Lysates from purified peripheral blood granulocytes of 4 PV patients and 3 healthy controls were used for ChIP with an antibody to AML1 (top) or with an unrelated IgG control (middle). The ChIPed DNA was amplified by the use of primers spanning the AML1 binding sites between bp −3466 and −3192 in the NF-E2 1A promoter. In the bottom panel, a 1:50 dilution of the input DNA was used.
