Myeloid-specific PU.1 autoregulation at the −12-kb element. (A) Sequence alignment of the −12-kb DHS of mouse, human, and dog. Identical residues are shown in black. The PU.1 binding motifs as identified by rVista software and the dimethylsulfate footprinting pattern obtained with macrophages (see supplemental Figure 6) are indicated. (B) Electromobility shift assay showing PU.1 binding to the −12-kb DHS. Nuclear extracts from RAW264.7 macrophages (left) and NIH-3T3 fibroblasts (right) were incubated with a ³²P-labeled probe containing the potential PU.1 binding site of the −12-kb DHS, as well as an antibody to PU.1 and unlabeled competitor oligonucleotides, as indicated. Base pair exchanges from gaggaagc to gagcgcgc in the PU.1 motif are indicated (mut). The position of PU.1 and a supershift complex (ss) are shown. (C) ChIP-qPCR showing strong PU.1 in vivo binding to the −12-kb DHS in RAW264.7 macrophages but not in 3T3-NIH fibroblasts. Binding to the −14-kb element and the proximal promoter are shown as controls. The values were normalized to a ChIP with the use of an IgG-control antibody. (D) Reporter assay data with RAW264.7 cells stably carrying a −12-kb DHS-driven luciferase construct. The cells were transiently transfected with constructs expressing shRNAs against either PU.1 or the LacZ gene, as a control, along with a GFP marker to allow flow cytometric sorting of the transfected cells. The values are normalized to luciferase activities driven by PU.1 promoter alone. (E) ChIP-qPCR with RAW264.7 cells that were untreated (mock) or were stably transfected with a shRNA against PU.1 showing reduced H3K9ac at the −12-kb region after PU.1 knockdown. The values are normalized to a ChIP with an IgG-control antibody. (F) UCSC Genome browser representation of PU.1 occupancy at the PU.1 locus in mouse peritoneal macrophages and splenic CD19⁺ B cells as shown by ChIP-seq tag counts (log₂).