AML1/ETO/DNMTs/HDACs complex alters the epigenetic status of PTEN genomic regions. (A) Relative qRT-PCR quantification of PTEN level in mononucleated cells (MNC) isolated from 81 AML FAB M2 subtype patients (supplemental Table 2; P = .0007). (B) Relative quantification of PTEN levels in SKNO-1, SKNO-1-siA/E, and U937 c treated or untreated with Zn²⁺. The results represent the average of 3 independent evaluations ± SD (*P = .003, #P = .007). (C) Schematic diagrams of the AML1 binding sites and of the CpG islands along the PTEN genes. Numbers are the nucleotides relative to PTEN (ATG). 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. (D) HEK293T cells were transiently cotransfected for 48 hours with luciferase reporter containing the sequence of the PTEN regulatory regions or its counterpart mutants (E), and increasing amounts (10, 50, and 100 ng) of pcDNA3.0 with AML1/ETO cDNA or without (293T-mock). The data are expressed as activity relative to that of the empty pGL3-LUC vector alone. pRL-TK was used as an internal control. The results shown are the average of 3 independent evaluations ± SD. (F) Genomic bisulfite sequencing assay was performed to detect the methylation status of the DNA sequences surrounding the AML1-binding site (− 2033) in PTEN promoter from CD34⁺ hematopoietic progenitors isolated from healthy donors PB (CD34+) and the indicated leukemia cell lines and leukemic blasts. 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 PTEN genes are indicated. (G) SKNO-1 cells (mock, and si-A/E) were treated or untreated with 2.5μM 5-Aza or/and SAHA 3.0μM for 40 hours. miR-193a and PTEN relative expression levels were evaluated by qRT-PCR. The results represent the average of 3 independent evaluations ± SD.