Figure 4

Methylation status and analysis of NFY and E-box transcription binding sites on FPGS expression. (A) Genomic DNA from CCRF-CEM and NALM6 cells was treated with sodium bisulfite and DNA region of the FPGS gene promoter was PCR-amplified using primer sets specific to methylated (M) and unmethylated (U) forms (Table 1). Expected MSP products were M, 139 bp and U, 142 bp. L, 100 bp ladder molecular weight marker. (B) The putative NFY and E-box transcriptional binding sites were mutagenized as described in Materials and Methods. Wild type pGL2628-ATGm/c (grey bars) and mutated pGL868NFY-ATGm/c (black) and pGL952Ebox-ATGm/c (white) plasmids were transfected by nucleofection in CCRF-CEM and NALM6 cells. Level of luciferase activity (RLU) was initially subtracted from the level of luciferase activity detected with pGL3 (empty vector), standardized with respect to the level of β-galactosidase activity, and normalized to wild type pGL2628-ATGm/c level. Each experiment was performed at least two times in triplicate. Data are expressed as mean ± S.E.M. (C) EMSA of biotinylated double stranded oligonucleotides containing the FPGS NFY binding site (region -32/-14) incubated with nuclear extracts prepared from CCRF-CEM (lanes 2–6) or NALM6 (lanes 7–10) cells. For competition experiments, 0.4 μM of unlabeled NFY (lanes 3 and 8) or EBNA oligonucleotides (lane 6) were included in the reaction mixture. NFY antibody CBF-A (lanes 4 and 9) or CBF-B (lanes 5 and 10) were added to the reaction mixtures. Specific DNA-protein complexes C1 and C2, and the supershifted complex (SSC) are indicated. Lane 1 represents negative control with no nuclear extract.