PU.1 binds to the pDP4 promoter in vitro and in vivo. (A) Isolation of the complete 5′ end of the pDP4 transcript by RACE PCR. Using a gene-specific antisense primer, which is located within exon 2 at bp +210, and a 5′ capping primer (for sequence see manufacturer's protocol), a single PCR fragment was amplified from bone marrow (BM) but not spleen (SP) cDNA. Sequencing of the subcloned PCR product revealed a 25-bp-long 5′ untranslated region. (B) Sequence comparison of the murine and human (CELERA database GA_x5J8B7W6JWY and NCBI accession NT_039606) pDP4 proximal promoter region showing a highly conserved PU.1-binding site (boxed), the presence of a TATA box (overlined), the transcription start site (+1 followed by an arrow), and the ATG marking the start of the coding region (underlined). The adenosine residue in the initial methionine codon corresponds to bp 26 196 078 of mouse chromosome 14 (NCBI accession NT_039606). (C) Electromobility shift (EMSA) experiment using reticulocyte lysate synthesized in vitro PU.1 (left panel) or 10 μg nuclear extract from WEHI-3 cells (right panel). The probe spanned base pairs -64 to -92 of the murine pDP4 promoter. A 50-fold excess of the cold probe with (comp.^*) or without (comp.) an introduced mutation (TTCC to GCGA) was used to test the specificity of the DNA-protein interactions. ss indicates the PU.1 complex supershifted with specific antisera. (D) Chromatin immunoprecipitation (ChIP) assay to analyze PU.1 binding to the pDP4 promoter in WEHI-3 cells. The upper panel shows a PCR-amplified 0.4-kb pDP4 promoter fragment that was specifically immunoprecipitated by the anti-PU.1 antibody. The lower panel shows a control experiment demonstrating that the anti-PU.1 antibody did not pull down a 0.6-kb thymidine kinase (TK) promoter fragment from the same extracts. The input lane represents a 1:20 dilution of the supernatant from the IgG immunoprecipitation.