AML1/ETO/DNMTs/HDACs complex acts on the AML1 dna-binding site present on the upstream sequence of pre-miR-193a gene and alters its epigenetic status. (A) Schematic diagrams of the AML1 binding sites and of the CpG islands along the miR-193a genes. Numbers are the nucleotides relative to pre-miR-193a (+1). Vertical arrows indicate AML1 binding sites, horizontal arrows indicate the location of the primers used in the ChIP assay. Vertical lines indicate CpG dinucleotides, horizontal bars below the CpG sites show the regions analyzed by bisulfite sequencing. (B) Human 293T cells were transiently cotransfected for 48 hours with luciferase reporter containing the WT sequence of the miR-193a regulatory regions or its counterpart mutants, and increasing amounts (10, 50, and 100 ng) of pcDNA3.0 with AML1/ETO cDNA or without (293T-mock). (C) U937 (mock and −A/E-HA) cells were transiently transfected with luciferase reporter and treated with Zn²⁺ (100μM for 16 hours). (D) Chromatin was immunoprecipitated using the indicated antibodies or IgG. PCR was performed using oligo 1 primers designed for the amplification of a distal region on miR-193a gene containing the predicted AML1 binding site to evaluate the specificity of protein binding. Oligo 2 primers were designed to amplify DNA sequences surrounding the proximal AML1-binding site. PTEN primers were designed to amplify DNA sequences surrounding the AML1-binding site (− 2033) and the CpGs in the PTEN gene regulatory region. Input shows the amplification from sonicated chromatin. Amplification of GAPDH was a control for nonspecific precipitated sequences. (E) Genomic bisulfite sequencing was performed to detect the methylation status of the DNA sequences surrounding the AML1-binding site (− 280) in the pre-miR-193a gene upstream region from CD34⁺ hematopoietic progenitors isolated from PB of healthy donors (CD34+) and the indicated leukemia cell lines and leukemic blasts. Each row of circles represents the sequence of an individual clone. Black circles and empty circles represent methylated and unmethylated CpG dinucleotides, respectively. Cells were either untreated or treated with 2.5μM 5-Aza for 40 hours. For each sample, the percentages of global methylation level of these regions on the miR-193a gene are indicated.