Characterisation of the GRAF gene promoter and its methylation in patients with acute myeloid leukaemia and myelodysplastic syndrome

Abstract

We report the isolation of the 5' flanking region of GRAF (GTPase regulator associated with the focal adhesion kinase), previously described as a putative tumour suppressor gene of acute myelogenous leukaemia and myelodysplastic syndrome, and demonstrate its promoter activity in reporter gene assays. Two putative protein-binding sites are identified of which one was sensitive to CpG methylation. The suppressed GRAF expression could be restored in leukaemia cell lines by treatment with a demethylating agent and an inhibitor of histone deacetylases. In contrast to normal tissues, which tested negative for GRAF promoter methylation, 11 of 29 (38%) bone marrow samples from patients with acute myeloid leukaemia or myelodysplastic syndrome were positive.

Figure 1

Overview (A) and deletion analyses (B) of the GRAF promoter. (A) The GRAF promoter is a GC-rich area with a dense concentration of CpG dinucleotides and a CpG island extending from −981 in the promoter to +350 in the downstream intron (numbering of nucleotides relative to A (=+1) in the START-ATG codon). Segment −986 to −1 was examined with reporter gene assays. (B) Left panel: The promoter segments (numbering relative to A (=+1) in the START-ATG codon). Right panel: Relative firefly luciferase activity normalised against the promoting activity of SV40 (=100%). K562 cells were transiently transfected with a pGL3 plasmid containing different segments of the GRAF promoter. Inversion (arrow) or methylation of the promoter completely abolishes any promoting activity. The data represent the mean±s.d. of three independent experiments.

Figure 2

Footprint 1 site (Fp1). (A) Sequence of the footprint 1 site. The ciphers I, II and III correspond to the footprinting positions from the DNase I protection assay. The DNase I-protected position is shown in bold and DNase I-hypersensitive positions are underlined. The recognition sequence of M.FnuDII is boxed. (B) DNase I protection assays. Methylation of the promoter fragment modifies binding of nuclear extract proteins to footprint 1 site. Compared to the promoter fragments partially digested by DNase I alone (lane 1), addition of nuclear extracts (90 μg) from K562 cells changed the digestion pattern. While one signal (I) is reduced, the intensity of two other bands is increased (II and III). M.SssI methylates every cytosine residue in a CpG dinucleotide, and this methylation abolishes binding of trans-acting factors (lanes 4–6 are essentially identical). M.FnuDII only methylates the lateral cytosin residues in a CpG tetranucleotide and apparently this hemimethylation does not inhibit protective binding of trans-acting factors (lanes 7–9 are essentially identical to lanes 1–3). The A+G ladder (lane 10) shows the reverse complementary sequence. Numbers refer to positions relative to A in the START-ATG (=+1); the M.FnuDII recognition sequence is boxed. (C) DNase I protection assays: The binding of the trans-acting factors is not cell specific. DNase I protection assay with increasing amounts (20, 50, 90 and 120 μg) of nuclear extracts. Band I loses intensity as the amount of nuclear extracts is increased, whereas bands II and III gain intensity. Thus, band I is protected by protein binding and bands II and III are hypersensitive to DNase digestion by protein binding, regardless of the source of the nuclear extract.

Figure 3

Footprint 2 site (Fp2). (A) Sequence of the footprint 2 site (Fp2). The sequence marked with bold is protected from DNase I digestion by binding of trans-acting factors. (B) DNase I protection assays: The segment −555 to −528 is protected from DNase I digestion (lanes 2 and 5: 40 ng; lanes 3 and 6: 80 ng DNase I) by binding of trans-acting factors from nuclear extract from K562 cells (90 μg). This binding is insensitive to M.SssI methylation of the DNA, as the DNA methylation does not alter the restriction pattern. The protected segment is marked by the black bar. The A+G ladder (lane 7) shows the reverse complementary sequence. Numbers refer to positions relative to A in the START-ATG (=+1). (C) The interaction of proteins with the Fp2 region of the GRAF promoter is not cell specific. Nuclear extracts from both RAW264 and K562 cells (90 μg) can protect unmethylated DNA from digestion with increasing amounts of DNase I (40, 80 and 180 μg). (D) Deletion of the footprint sites Fp1 and Fp2 reduces the promoter activity. Left panel: The promoter segments (numbering relative to A (=+1) in the START-ATG codon). Right panel: Relative luciferase activity normalised against the promoting activity of the empty pGL3 plasmid (=1). The data represent the mean±s.d. of three independent experiments. (E) Promoting repression by the M.FnuDII hemimethylation is partially reversible by incubation with the histone deacetylase inhibitor TSA, whereas CpG methylation with M.SssI causes a complete block of the promoting activity, which cannot be restored by incubation with TSA. The reporter activities are shown as folds of increase of luciferase activity compared to the empty pGL3 plasmid. The data represent the mean±s.d. of three independent experiments.

Figure 4

GRAF expression levels in cell lines after treatment with 5-azadC and TSA. (A) Expression levels of GRAF analysed by quantitative RT–PCR relative to a housekeeping gene (ABL) in cells treated with 5 μM 5-azadC for 72 h vs untreated controls. ^*P<0.05; ^**P<0.005 (t-test of untreated vs treated). The data represent the mean±s.d. of three independent experiments. (B) Expression levels of GRAF by quantitative RT–PCR relative to a housekeeping gene (ABL) in cells treated with 0–300 ng TSA per ml culture medium for 16 h. ^*P<0.05; ^**P<0.005 (t-test of untreated vs treated). The data represent the mean±s.d. of three independent experiments.

Figure 5

Methylation-sensitive PCR of the GRAF promoter in patients’ samples. (A) Methylation-sensitive PCR. A 126 bp fragment of the GRAF promoter (segment −475 to −350) was amplified by applying MS-PCR. An artificially M.SssI-methylated promoter construct served as a positive control. (B) Sequences of MS-PCR products. Alignment of six PCR products (−475 to −350) generated from patient samples showing complete methylation of CpG dinucleotides within the GRAF promoter. Numbering refers to patients in Table 2. GP=germline promoter sequence; UP=unmethylated promoter sequence after MS-PCR; MP=methylated promoter after MS-PCR. Bold cytosine and guanine residues indicate sequence variations and inverted thymidine residues indicate a lack of methylation. Primer sequences are framed.