Mechanisms underlying loss of RARA expression differ between AML cell lines and patient samples. (A) RARα2 promoter is methylated in AML cell lines but not patient samples. Following bisulphite modification, specifically amplified PCR products were sequenced using primers (gray arrows) mapping to regulatory regions of (i) RARα1 or (ii) RARα2. CpG islands are indicated by heavy gray lines; all positions are shown relative to the transcription initiation sites (black broken arrows). Samples from patients, cell lines, and controls are indicated. nm indicates nonmethylated plasmid control; m, methylated plasmid control. Methylated CpG dinucleotides are represented by ●, and unmethylated CpGs are represented by ○. Sequencing revealed the RARα2 promoters of individual clones to be completely demethylated. All unmethylated samples were sequenced on both the sense and antisense strands, and no CpG hemimethylation was found. Methylation levels for RARα2 CpG island 1 from 3 bisulphite-modified APL patient samples were quantified by pyrosequencing,¹⁶  and a mean value of 3.7% methylated cytosine per CpG dinucleotide was observed. Bisulphite modification of genomic DNA was performed as previously described,¹⁷  and PCR primers were selected using the MethPrimer program¹⁸ : BS-RARA1-Fwd, 5′-GTTTTGGGTTTGAGGGAGGGAT-3′ and BS-RARA1-Rev 5′-AACTTTACCCRAAACCCCAAACTAA-3′; BS-RARA2#1-Fwd, 5′-GGTYGGAGTTATATATGATGT-3′ and BS-RARA2#1-Rev 5′-AATAATCCCRATATCCTCCCCTTAA-3′; BS-RARA2#2-Fwd, 5′-GTAGAGTTGGGGTGGGGG-3′ and BS-RARA2#2-Rev, 5′-CAAAATACAACRACTCCCCAAATCC-3′. 1 and 2 refer to RARα2 CpG islands 1 (promoter) and 2 (corresponds to 5′ UTR of RARα2), respectively. PCR was performed using the FastStart system (Roche, Indianapolis, IN) supplemented with GC-rich solution. PCR products were separated by agarose gel electrophoresis, recovered, and sequenced directly using the previously mentioned PCR primers. For NB4 and U937 cell line samples, recovered PCR products were cloned into the pDrive vector (Qiagen) and sequenced using M13 forward and reverse primers. (B) Chromatin associated with the RARα2 promoter in primary AML cells is characterized by reduced transcriptional competence. Chromatin immunoprecipitation assays with antibodies directed against acetyl-histone H3 or dimethyl-histone H3 lysine 4 (K4) were performed on CD33⁺ CB cells and samples from AML patients (n = 2; consisting of AML FAB subtypes M4 and M5). DNA obtained from the input or from immunoprecipitated DNA was analyzed by real-time PCR using primers mapping to the RARα2 promoter region. Results were quantified as a percentage of immunoprecipitated material relative to input DNA, and levels of H3 acetylation (H3^Ac) and H3K4 dimethylation (H3K4^Me2) are shown relative to CD33⁺ CB cells. As expected, an irrelevant antibody-negative control did not immunoprecipitate the target DNA sequence (data not shown). The following PCR primers were used: ChIP-RARα2-Fwd, 5′-GAGCTGCACAATGTCACACC-3′ and ChIP-RARα2-Rev, 5′-GGCTGAACTCTCGCTGAACT-3′. Error bars are SD.