AML1/ETO induces epigenetic modification of the RARβ2 gene in t(8;21)-positive cells. (A) Schematic representation of the distribution of the CpGs (black circles) along the promoter and exon 1 (nt 370 to +238) of the RARβ2 gene. The black arrows indicate the DNA sequences amplified by different sets of primers sensitive to the methylation status of CpGs (P3 and P4). White arrows indicate the regions of annealing of the PCR primers used in ChIP assays. (B) ChIP analysis was performed in mock, A/E-HA, and SKNO-1 cell lines using an antibody specific for 5-methylcytosine (α-5MetC) or with no antibody (no-Ab) as negative control. Immunoprecipitated chromatins were analyzed by PCR primers specific for the amplification of a genomic region containing the βRARE binding site and the 5′-UTR exon 1 of RARβ2 gene. Samples representing 0.02% of sonicated chromatin (input) were included in the PCR analysis. The amplification of a GAPDH coding region was performed as a control of nonspecific precipitated sequences. (C) Bisulfite sequencing assay was performed to detect the methylation status of each CpG along the RARβ2 promoter/exon 1 sequence on genomic DNA isolated from mock, A/E-HA, and SKNO-1 cells. Black (▪) and empty (□) squares represent methylated and unmethylated CpGs, respectively. For each sample, the percentages of global methylation level in the promoter and in the 5′-UTR exon 1 regions are indicated. (D) MSP PCR assay was performed on bisulfite-treated DNA isolated from mock, A/E-HA, and SKNO-1 cell lines to analyze the methylation status of RARβ2 promoter/exon 1 regions. U and M indicate the unmethylated and methylated forms amplified by MSP primers, respectively. (E) ChIP assay was performed on the SKNO-1 cell line using antibodies specific for the acetyl-H4, acetyl-H3, acetyl-H3-Lys9, and methyl-H3-Lys9 forms or without antibody (no-Ab) as a negative control.