C/EBPδ promoter activity is suppressed by methylation. (A) Luciferase assays showing C/EBPδ promoter repression in response to methylation. Methylated (SssI) or mock-methylated C/EBPδ promoter reporter constructs were transfected into 32D cells. The fold repression was calculated as 1/activity (normalized to Renilla values) with the control set as 1. The results are shown as the mean ± SD of 3 independent experiments. (B) Luciferase assay in S2 Drosophila cells, indicating that C/EBPδ promoter repression is dependent on MeCP2 activity. Methylated or mock-methylated reporter constructs were transfected into S2 Drosophila cells along with an Sp1 expression plasmid and either an empty vector or a MeCP2 expression vector. Fold repression was calculated as 1/relative activity, with the control set as 1. The results are shown as the mean ± SD of 3 independent experiments. (C) Luciferase assay showing fold repression in promoter activity of C/EBPδ, C/EBPα, cyclin E, and c-myc promoters after SssI methylase treatment. Methylated (SssI) or mock-methylated C/EBPδ promoter reporter constructs were transfected into 32D cells. The results are shown as the mean ± SD of 3 independent experiments. (D) Chromatin modification of histone H3-K4 methylation and histone H3 acetylation. Chromatin immunoprecipitations were performed with Aza-exposed U937 cells. Antibodies against histone H3-methylated K4 (K4-Me), acetylated histone H3, or IgG isotype control were used. Real-time PCR was performed using C/EBPδ promoter–specific primers and probe. The results shown are mean ± SD of 2 independent experiments.