BMC Cancer 2006, 6:132 http://www.biomedcentral.com/1471-2407/6/132 FPGS minimal promoter does not play a role in the observed lineage-specific differences in FPGS expression in ALL cell lines. Role of putative NFY-box and E-box binding sites on FPGS expression To identify specific regulatory elements involved in FPGS gene transcription, we analyzed the nucleotide sequence of the minimal FPGS gene promoter for presence of known transcription factor recognition motifs using the MatInspector program (Genomatix, release 7.3.1). As shown in Figure 2, two Sp1 (GGGCGG; -10, -15), one reverse Sp1 (+5), one inverted NFY-box (CCAAT; -20), and one E-box (CANNTG; +61) transcription factor bind- ing sites were identified within the FPGS minimal pro- moter (minP). We examined the role of putative NFY and E-box DNA binding sites by generating mutants at each site with substitutions reported to reduce or abolish gene promoter activity [22,23]. Mutant constructs for NFY (NFY-868; CCAAT → CTTTT) and E-box (Ebox-952; CAC- CTG → CATTCG) were co-transfected with pCMVβ in both CCRF-CEM and NALM6 cells and assayed for luci- ferase and β-galactosidase activities. As shown in Figure 4B, mutation in the NFY site (NFY-868) reduced the level of FPGS transcription by 45% in NALM6 (p < 0.001) and 40% in CCRF-CEM cells (p < 0.005) when compared to the wild type construct (pGL2628-ATGm/c). Normalized luciferase activities were 1.9 × 10-2 ± 2.0 × 10-3 (pGL2628- ATGm/c) and 1.0 × 10-2 ± 1.2 × 10-3 (pGL868NFY-ATGm/ c) in NALM6, and 2.4 × 10-1 ± 2.4 × 10-2 (pGL2628- ATGm/c) and 1.5 × 10-1 ± 1.4 × 10-2 (pGL868NFY-ATGm/ c) in CCRF-CEM cells. These data suggest that the NFY binding site is required to activate FPGS gene transcrip- tion in lymphoid cells. In contrast, no significant differ- ences were observed in either cell line with the mutant Ebox-952 construct, suggesting a negligible role in FPGS expression (Figure 4B). In NALM6 and CCRF-CEM cells, the normalized level of luciferase activity was 1.6 × 10-2 ± 8.4 × 10-4 (pGL952Ebox-ATGm/c) and 2.2 × 10-1 ± 1.8 × 10-2 (pGL952Ebox-ATGm/c), respectively. To establish NFY protein interaction with the NFY-box element (CCAAT) at position -20 upstream the FPGS gene promoter, we performed electrophoretic mobility shift assays (EMSAs) using an oligonucleotide probe contain- ing FPGS gene sequence from -32 to -14. The sequences of double-stranded biotinylated oligonucleotide probes and competitors were incubated with nuclear extracts pre- pared from CCRF-CEM or NALM6 cells. The EMSA revealed the formation of two complexes with the NFY oligonucleotide probe (Figure 4C, C1 and C2). To further ascertain the specificity of binding, we demonstrated that the formation of complexes C1 and C2 were inhibited by excess amounts of unlabeled NFY oligonucleotides (Fig- ure 4C, lanes 3 and 8) but not with unlabeled non-specific Epstein-Barr nuclear antigen (EBNA) oligonucleotides (Figure 4C, lane 6). To directly evaluate the proposed par- ticipation of NFY in the shifted complexes, anti-NFY pep- tide (CBF-A and CBF-B subunits) antibodies were incubated with EMSA binding reactions before electro- phoresis. Addition of anti NFY-A (CBF-A) antibody to these reactions resulted in strongly retarded supershift complex (SSC) (Figure 4C, lanes 4 and 9). In contrast, addition of NFY-B (CBF-B) antibody abolished the forma- tion of the complex C2 (Figure 4C, lanes 5 and 10) with- out formation of a supershifted band. These data clearly demonstrate that NFY transcription factor is present in the complex that binds to the putative NFY binding site (CCAAT) at position -20. Discussion Folate antimetabolites such as MTX are essential chemo- therapeutic drugs in the treatment of children with acute lymphoblastic leukemia (ALL). MTX is retained within the cell by cellular metabolism catalyzed by the enzyme FPGS. The human FPGS promoter has been previously characterized using CCRF-CEM cells and it encompasses a GC-rich region without a typical TATA sequence, usually a characteristic of housekeeping genes and proto-onco- genes. The minimal portion of the promoter required to drive transcription in CCRF-CEM cells consists of a region starting -43 to +150 of the main transcription start site including part of exon 1 [17]. When 5'-flanking sequences upstream of exon 1 were analyzed for promoter and/or enhancer activity in Bp- vs. T-cells, we found significantly lower luciferase activity in the Bp-ALL cell line NALM6 compared to the T-ALL cell line CCRF-CEM. These results are opposite of what one would predict based on the known lineage differences resulting in two- to three-fold higher FPGS expression Bp- vs. T-ALL [9,11]. These low levels of FPGS-luciferase activity observed with our con- structs in NALM6 vs. CCRF-CEM cells, lead us to conclude that the minimal promoter region is not sufficient to effectively drive FPGS transcription in Bp-cells (NALM6). Consequently, we hypothesize that additional regulatory elements are required to drive FPGS expression in Bp-ALL. Our analysis failed to demonstrate promoter and/or enhancer activity in the immediate 5'-flanking region of exon A1b, between exons A1b and 1, and in the 3'-UTR. We propose that Bp-cell specific enhancer(s) are required for FPGS gene expression in Bp-ALL cells and that distant yet unidentified regulatory loci exist. The human FPGS transcription start sites have been mapped previously in human CCRF-CEM and HepG2 (hepatoma) cells and shown that FPGS transcription was initiated from multiple start sites spread over 80 bp clus- tered in two major regions differing by the presence of the mitochondrial vs. cytosolic initiation codons [13,14,17]. The first transcription initiation site generates a long tran- Page 9 of 12 (page number not for citation purposes)