C/EBPα expression is repressed by methylation rather than by acetylation. (A) NB4 (left panel) or U937PR9 (right panel) treated (■) or not (□) every 24 hours with 100μM zinc (Zn) were treated for 72 hours with 1μM (NB4) or 5μM, respectively, 5-Aza or for 24 hours with 300nM TSA. C/EBPα mRNA expression was detected by quantitative real-time PCR and normalized to GAPDH expression. This graph is representative of 3 independent experiments. Methanol (MetOH) and dimethyl sulfoxide are vehicle controls for TSA and 5-Aza, respectively. (B) C/EBPα protein was assessed in NB4 cells treated with TSA or 5-Aza by Western blot. Equal loading of the protein was assessed with β-tubulin. (C) Western blot of C/EBPα protein in U937MT PML-RARα AHT mutant cells either untreated or treated for 16 hours with 100μM Zn. Equal loading was assessed with an antibody against β-tubulin. (D) In vitro methylation represses C/EBPα promoter activity. (Di) A vector expressing luciferase driving by C/EBPα promoter sequence was treated in vitro with the CpG methyltransferase MSssI. Efficiency of the methylation was assessed by digesting the non–MSssI-treated and the MSssI-treated vector with a methylation-insensitive enzyme MspI (M) and a methylation-sensitive enzyme, HpaII (H). (Dii) U937 cells were electroporated with the methylated (MSssI) or untreated vector (nontreated) driving luciferase activity. Shown are relative luciferase units (RLU) representing the ratio between the firefly activity driven by the C/EBPα promoter before and after methylase treatment normalized to the CMV Renilla activity driven by a control vector.²⁶  When the C/EBPα promoter was methylated, relative luciferase activity was decreased by 85% compared with the activity obtained with the unmethylated vector (P < .001).