DNMT1 promoter activity was down-regulated by pre-miR-29b through repression of Sp1. (A) EMSA of DNMT1 promoter DNA and nuclear extracts prepared from K562 cells transfected with miR-29b construct (miR-29b) or scrambled control (sc). The arrow represents the DNMT1-protein complex. (B) EMSA of DNMT1 promoter DNA with human recombinant Sp1 protein (0.5 mg, rhSp1, Promega, catalog no. E6391) or no protein extracts. These data further indicate that Sp1 is binding to the DNMT1 promoter DNA. (C) EMSA of DNMT1 promoter DNA using K562 nuclear extracts with unlabeled DNA competitors (Sp1-binding elements and DNMT1 promoter DNA, WT). As a control, we used no DNA competitor (−) and a nonspecific unlabeled probe (TFIID) that contains the TATA box sequence 5′-GCAGAGCATATAAGGTGAGGTAGG A-3′. This sequence is not related to the DNMT1 promoter DNA. The fact that this oligo did not compete shows specificity of DNA competition by DNMT1 DNA and by SP1 DNA. (D) EMSA of DNMT1 promoter DNA using K562 nuclear extracts and unlabeled wild-type (WT) or mutants (M) excess (20 times) DNA competitors. The first 2 lanes (from the left) are without competitors. The sequences of the DNMT1 promoter DNA and various DNMT1 mutants with linker scanned mutations (shown in bold-type) are shown below (M1-M5).