Eicosapentaenoic Acid Demethylates a Single CpG That Mediates Expression of Tumor Suppressor CCAAT/Enhancer-binding Protein δ in U937 Leukemia Cells

Polyunsaturated fatty acids (PUFAs) inhibit proliferation and induce differentiation in leukemia cells. To investigate the molecular mechanisms whereby fatty acids affect these processes, U937 leukemia cells were conditioned with stearic, oleic, linolenic, alpha-linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acids. PUFAs affected proliferation; eicosapentaenoic acid (EPA) was the most potent on cell cycle progression. EPA enhanced the expression of the myeloid lineage-specific transcription factors CCAAT/enhancer-binding proteins (C/EBP beta and C/EBP delta), PU.1, and c-Jun, resulting in increased expression of the monocyte lineage-specific target gene, the macrophage colony-stimulating factor receptor. Indeed, it is known that PU.1 and C/EBPs interact with their consensus sequences on a small DNA fragment of macrophage colony-stimulating factor receptor promoter, which is a determinant for expression. We demonstrated that C/EBP beta and C/EBP delta bind the same response element as a heterodimer. We focused on the enhanced expression of C/EBP delta, which has been reported to be a tumor suppressor gene silenced by promoter hypermethylation in U937 cells. After U937 conditioning with EPA and bisulfite sequencing of the -370/-20 CpG island on the C/EBP delta promoter region, we found a site-specific CpG demethylation that was a determinant for the binding activity of Sp1, an essential factor for C/EBP delta gene basal expression. Our results provide evidence for a new role of PUFAs in the regulation of gene expression. Moreover, we demonstrated for the first time that re-expression of the tumor suppressor C/EBP delta is controlled by the methylation state of a site-specific CpG dinucleotide.