Eicosapentaenoic acid demethylates a single CpG that mediates expression of tumor suppressor CCAAT/enhancer-binding protein delta in U937 leukemia cells

Abstract

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, α-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/EBPβ and C/EBPδ), 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/EBPβ and C/EBPδ bind the same response element as a heterodimer. We focused on the enhanced expression of C/EBPδ, 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/EBPδ promoter region, we found a site-specific CpG demethylation that was a determinant for the binding activity of Sp1, an essential factor for C/EBPδ 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/EBPδ is controlled by the methylation state of a site-specific CpG dinucleotide.

FIGURE 1.

Effects of fatty acids on U937 cell viability and morphology. A, U937 cells were treated with 50, 100, or 200 μm fatty acids for 24 h. Cells were harvested, stained with PI, and analyzed for cell cycle distribution using flow cytometry. Open circles, SA; open triangles, OA; open squares, LA; solid squares, LNA; solid diamonds, AA; solid circles, EPA; solid triangles, DHA. The means ± S.D. (error bars) of four separate experiments are shown. Two-way analysis of variance was utilized to determine the impact of fatty acid type and concentration (*, p < 0.001, LNA, AA, EPA, and DHA versus U937 untreated cells; #, p < 0.001, LNA, AA, and DHA versus EPA). B, cells were treated with 100 μm fatty acids as described in A. The percent cell cycle distribution was evaluated. One representative of five experiments is shown. C, cell proliferation was determined by counting triplicate samples for trypan blue dye exclusion after 100 μm fatty acid treatment. The means ± S.D. (error bars) of five separate experiments are shown (*, p < 0.001 versus U937 untreated cells). D, cells were treated with 100 μm fatty acids and incubated for 4 h with [³H]thymidine, and the uptake was evaluated as described under “Experimental Procedures.” The means ± S.D. (error bars) of three separate experiments are shown (*, p < 0.001 versus U937 untreated cells). E, morphological changes after 100 μm fatty acid treatment. Flow cytometry analyses for size (forward scatter) and granularity (side scatter). One representative of five experiments is shown. Forward scatter and side scatter peak signals were collected from about 10,000 cells. Mean values of these scattering signals were calculated using SYSTEM II software. Values shown (x axis, forward scatter; y axis, side scatter) are mean ± S.D. of five experiments (*, p < 0.05; #, p < 0.01; and **, p < 0.001 relative to U937 untreated cells).

FIGURE 2.

Effect of fatty acids on myeloid lineage-specific transcription factors. A, U937 cells were treated with 100 μm fatty acids for 24 h. Total cell lysates (50 μg of protein) were subject to Western blotting with the indicated antibodies as described under “Experimental Procedures.” For each protein, one representative of four experiments is reported. Images of independent blots were acquired using the VersaDoc Imaging System, and signals were quantified using Quantity One software. The -fold change in each protein was compared with control U937 cells (C) and was calculated after correction for β-tubulin loading differences. Data are the mean ± S.D. of four separate experiments (*, p < 0.01 versus U937 untreated cells). B, mRNA content was evaluated after 1-, 3-, and 24-h treatment with 100 μm fatty acids using quantitative RT-PCR as described under “Experimental Procedures.” White bars, unsupplemented U937; gray bars, OA; black bars, EPA. Data are presented as relative expression by calculating 2^−ΔΔCt normalized to untreated U937 cells. The means ± S.D. (error bars) of three separate experiments are shown (*, p < 0.01; and **, p < 0.001 versus U937 untreated cells).

FIGURE 3.

Effect of fatty acids on M-CSF receptor expression. A, U937 cells were treated with 100 μm fatty acids for 1, 3, and 24 h. mRNA content was evaluated using quantitative RT-PCR as described under “Experimental Procedures.” White bars, unsupplemented U937; gray bars, OA; black bars, EPA. Data are presented as relative expression by calculating 2^−ΔΔCt normalized to untreated U937 cells. The means ± S.D. (error bars) of three separate experiments are shown (*, p < 0.001 versus U937 untreated cells). B, M-CSF receptor protein levels after 24 h of 100 μm fatty acid conditioning. Total cell lysates (50 μg of protein) were subject to Western blotting as described under “Experimental Procedures.” One representative of four experiments is shown. C, cells were treated with 100 μm fatty acids for 24 h. Chromatin immunoprecipitations were performed in untreated (white bars), OA-treated (gray bars), and EPA-treated (black bars) U937 cells. Antibodies against C/EBPα, C/EBPβ, C/EBPδ, and PU.1 were used. Real time PCR was performed using M-CSF receptor promoter-specific primers. The results shown are the mean ± S.D. (error bars) of three independent experiments.

FIGURE 4.

Effect of EPA on C/EBPδ protein expression and methylation status in U937 cells. A, Western blot analysis of C/EBPδ in U937 untreated cells (lane 1), cells after 2- (lane 2) and 5-day (lane 3) treatment with 1 μm 5-aza 2′-deoxycytidine, and cells after 24-h 100 μm EPA treatment (lane 4). Cell lysates (50 μg of protein) were loaded. One representative of three experiments is shown. Images of independent blots were acquired using the VersaDoc Imaging System, and signals were quantified using Quantity One software. The -fold change of C/EBPδ protein was compared with control U937 cells and was calculated after correction for β-tubulin loading differences. Data are the mean ± S.D. of three separate experiments. B, cells were treated with 100 μm fatty acids for 24 h, and the methylated DNA levels of C/EBPδ CpG islands were quantified using the Methyl-Profiler qPCR Primer Assay as described under “Experimental Procedures.” The means ± S.D. (error bars) of three separate experiments are shown (*, p < 0.001 versus OA-treated or U937 untreated cells). C, C/EBPδ gene proximal promoter DNA sequence contains a CpG island. The underlined sequences indicate the forward and reverse nested primers utilized for cloning and sequencing after bisulfite reaction. The cloned fragment (350 bp) contains 32 CpGs (in bold). The arrow indicates the CpG demethylated nucleotide after EPA treatment. D, sequencing of the individual clones generated by PCR after bisulfite reaction. Black and white circles represent methylated and unmethylated CpGs, respectively.

FIGURE 5.

Influence of demethylated CpG on Sp1 binding to C/EBPδ gene promoter. For the electrophoretic mobility shift assay, end-labeled double-stranded unmethylated and methylated oligonucleotides were incubated with U937 nuclear extracts. Sequences in bold italics represent the Sp1 consensus sequence. U, unmethylated oligonucleotides; M, methylated oligonucleotides; +Sp1 Ab, unmethylated oligonucleotides plus Sp1 antibody.