Myelomonocytic U-937, erythroid HEL and TF-1, megakaryocytic MEG-01 cells were grown in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS) (Gibco BRL, Life Technologies, Rockville, MD, USA); TF-1 cells also received 20 ng/ml GM-CSF (R&D Systems, MN, USA). The erythroid/megakaryocytic UT-7 cells were grown in MEM Alpha modification medium with L-Glutamine and Nucleosides (PAA Laboratories GmBh, Austria) supplemented with 20% FBS and 30 ng/ml GM-CSF. The acute myeloid leukemia Kasumi-1, Burkitt's lymphoma Raji, and promyelocytic HL-60 cells were supplemented with 20% FBS. Monkey kidney COS cells were grown in DMEM medium with 10% FBS supplemented with high glucose (4.5 g/L) and L-glutamine.
Identification of the transcription start site of the MTG16 gene
The 5'-end (transcription start) of the mRNA was identified by 5'-RACE carried out with mRNA prepared from HEL cells using Oligotex Direct mRNA mini Kit (Qiagen, Hilden, Germany), using the First choice RNA Ligase mediated (RLM)-RACE kit (Ambion Inc., TX, USA). Nested PCR of RACE reactions was performed with adapter primer 5'-CGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG-3' and nested gene specific primer 5'-GCCTTAGCTTTCCTGTCCACTGG-3'. RACE- products were cloned into pGEM-T Easy Vector system (Promega Corporation, WI, USA) and sequenced.
Amplification of MTG16 promoter region
A 1208 bp region upstream of the translation start (from -1264 to -57 bp) was amplified from human genomic DNA by PCR. The forward and reverse primers used were 5'-TGAGGCGGTACCACCTCCAGCCATGGCATC -3' and 5'-TGCCTAGATCTACCTCCTGCAGCCTTGAGG -3', respectively. Regions corresponding to -905 to -57 and -820 to -57 bp were amplified from the 1208 bp fragment by nested PCR with forward primers 5'- TATGGTACCGTGGGGGAGCCCTGCTGTCTCCACA -3' (KpnI restriction site underlined),5'- AGGCGGGAGGTACCTTGAGGACAGGTCAGG -3' (KpnI restriction site underlined), and a common reverse primer 5'-TGCCTAGATCTACCTCCTGCAGCCTTGAGG -3' (BglII restriction site underlined).
Sequential 5' deletions of the -820 to -57 bp promoter region were generated by PCR from the cloned genomic DNA as template to generate -668 to -57, the -512 to -57, the -359 to -57 bp, the -339 to -57, the -299 to -57 and the -219 to -57 regions. Forward primers were 5'-TAAGGTACCCTGGGGAAAGCTCGGCTGAC-3', 5'-TATGGTACCCCTGCCCTGGGGCCGAGG -3', 5'-CCAGGTACCTCCTGTCAGGGAAGTGGCGG-3', 5'-TATGGTACCGGGCGCAGCTGCAGGCC
-3', 5'- TATGGTACCTCACGGGGACACAGCTGGC-3' and 5'-TATGGTACCATGAGCCCCAGGGCTCCCACCC -3' with KpnI restricton sites underlined. The common reverse primer is the same as used for amplification of the -1208 to -57 bp region. All sequences were verified by sequencing.
Amplification of MTGR1 promoter region
A 5' flanking region of 1965 bp from -1913 to +52 bp was amplified from human genomic DNA by PCR. The forward and reverse primers used were 5'-TATTGGTACCGCAACACCATGTCTGGCTAA -3' (KpnI restriction site underlined) and 5'- CTACAGATCTGCATTCACGCCCCACTTAC -3' (BglII restriction site underlined), respectively. Regions corresponding to -989 to +52, -656 to +52 and -499 to +52 bp were amplified from the 1965 bp fragment by nested PCR with forward primers 5'-TTGCGGTACCCTTCAAACTCCTGACCTCGTGA -3' (KpnI restriction site underlined), 5'- TTGCGGTACCCCCGGCCAACAGTTATTAAA -3' (KpnI restriction site underlined), 5'- TTGCGGTACCTGTAATCCCTCCCCCTAGCACT -3' (KpnI restriction site underlined), and a common reverse primer 5'-CTACAGATCTGCATTCACGCCCCACTTAC -3' (BglII restriction site underlined).
Sequential 5' deletions of the -499 to +52 bp promoter region were generated by PCR from the cloned genomic DNA as template to generate -308 to +52, the -257 to +52, the -207 to +52 bp and the -145 to +52 regions. Forward primers were 5'-TTGCGGTACCGTGCTTAATGGGAGCTGGTCT.-3', 5'-TTGCGGTACCGCGGCGAGAGCGCGCGGCGGGGGCG -3', 5'-TTGCGGTACCGCCGGGGGCGGGGCGGGGCGCGGCC -3' and 5'-TTGCGGTACCGTGGTGGTGTCTGGTTAGCTC-3', with KpnI restricton sites underlined. The common reverse primer is the same as used for amplification of the -499 to +52 bp region. All sequences were verified by sequencing.
Site-directed mutagenesis of transcription factor-binding sites
Oligonucleotide primers including the desired mutations were synthesized and used in two-step spliced overhang extension PCR. The following potential MTG16 promoter transcription factor binding sites were mutated: ETS1/PU.1 sites at positions -491, 5'-AGGAAA-3' changed to 5'- ATTCAA-3'; SP1 sites at positions -425, 5'-GGGAGG-3' changed to 5'-GTTCGG-3'; ETS1/SPI1 sites at positions -419, 5'-AGGAAGT-3' changed to 5' - ATTCAGT-3' TFAP2A sites at positions -389, 5'-GCCCGAGGG-3' changed to 5'-GCCTTCGGG-3'; GATA/YY1 sites at positions -356, 5'-GATGGT-3' changed to 5'-GACTTT-3'; SP1 sites at positions -316, 5'-CTCCGGCCCC-3' changed to 5'-CTCCTTACCC-3'; GATA sites at positions -301, 5'-TATCA-3' changed to 5'-TGGTA-3' and MZF1 sites at positions -251 & -191, 5'-TCCCCA-3' changed to 5'-TAAACA-3'.
The following potential MTGR1 promoter transcription factor binding sites were mutated: ETS1 sites at positions -335, 5'-CTTCCG-3' changed to 5'-CGTATG-3'; YY1 sites at positions -301, 5'-CCCATT-3'changed to 5'-CTTCTT-3'; SP1 sites at positions -285, 5'-CCCGGCCCCA-3' changed to 5'- CACTGTCCCA -3'; ETS1 sites at positions -276, 5'-CTTCCG-3' changed to 5'- CG TATG -3' SP1 sites at positions -243, 5'-CCCCCGCCGC-3' changed to 5'- CCCTTGCAGC -3'; SP1 sites at positions -229, 5'-CCCCGCCCGC-3' changed to 5'- CCTTGCACGC -3'; SP1 sites at positions -225, 5'-CCCCGCCCCG-3' changed to 5'- CCTTGCACCG -3'; SP1 sites at positions -213, 5'-CCCCGCCCCC-3' changed to 5'- CCTTGCACCC -3'; SP1 sites at positions -206, 5'-CCCCGCCCCG-3' changed to 5'- CCTTGCACCG -3'; SP1 sites at positions -192, 5'-CGCCGCCGCC-3' changed to 5'- CGTTGCAGCC -3'; SP1 sites at positions -178, 5'-CGCCGCCGCC-3' changed to 5'- CGTTGCAGCC -3' and SP1 sites at positions -170, 5'-CGCCGCCGCC-3' changed to 5'- CGTTGCAGCC -3'. After subcloning into promoterless pGL3/Basic reporter plasmid, all mutations were verified by sequencing.
Luciferase reporter assays of MTG16 and MTGR1 promoter
promoter was cloned into promoterless pGL3/Basic reporter plasmid using firefly luciferase as reporter to generate pGL3 -905-57, pGL3 -820-57, pGL3 -668-57, pGL3 - 512-57, pGL3 -359-57, pGL3 -339-57, pGL3 -299-57 and pGL3 -219-57 reporter constructs. Mutants were cloned into the same reporter plasmid. Transient transfections of hematopoietic cell lines were performed by electroporation as previously described by [49
]. The pGL3/SV40-promoter vector served as positive control and promoterless pGL3/Basic vector as negative control. Renilla luciferase, pRL/SV40 vector (Promega Corporation, WI, USA) was used as internal control for transfection efficiency. Thirtyfive μg pGL3 DNA were used for HL-60 target cells and 15 μg for HEL, TF-1, U937, UT-7, MEG-01, Raji and Kasumi-1 target cells. COS cells were transfected with 1.5 μg of DNA by use of Polyfect (Qiagen, Hilden, Germany). Twentyfour hours after transfection, cells were lysed and subjected to luciferase dermination using the Dual luciferase reporter assay kit (Promega Corporation, WI, USA). Firefly and Renilla luminiscense were quantified (Run Promega Protocol, DLR-0-INJ) in a GLOMAX 20/20 Luminometer (Promega Corporation, WI, USA). Firefly was normalized to Renilla luciferase as internal control for transfection efficiency. Results are given as adjusted Relative Luciferase Units (AdjRLU) normalized to pGL3/promoter (set to 1). Three to five independent transfections were performed in each case and each sample was measured in triplicate.
Similarly, MTGR1 pGL3 -1913 + 52, pGL3 -989 + 52, pGL3 -656 + 52, pGL3 -499 + 52, pGL3 -308 + 52, pGL3 -257 + 52, pGL3 -207 + 52 and pGL3 -145 + 52 reporters were generated, transfected into cells and assayed as described above.
Transfection of GATA-1 small interfering RNA
Twentyfour-well Falcon plates (Becton Dickinson, New Jersey 07417, USA) were coated with 1 mg/ml retronectin (Takara Bio inc, Verviers, Belgium) in PBS for two h at room temperature. Excess retronectine was removed and wells were blocked with 2% BSA in PBS for 30 min. After removal of the supernatant, 50,000 HEL cells were added per well in 600 μl RPMI medium containing 10% FBS and 20 nM phorbol 12-myristate 13-acetate (PMA) to generate adherent cells by incubation at 37°C for 24 h. After washing with PBS transfection was carried out with 5 μM siRNA using the N-TER transfection reagent (Sigma-Aldrich Corp. St. Louis, MO USA) in RPMI without FBS. After 4 h, 300 μl of RPMI with 20% FBS was gently added and cells were incubated for 48 h. Eventually, gently rinsed with PBS, tripsinized and examined by Western blotting. The GATA-1 siRNA sequence was 5'-AGUUGAGGCAGGGUAGAGC-3' (Oligo SASI_Hs02_00333089, Sigma).
Inhibition of GATA-1 transcriptional activity by HERP2
The transcription factor HERP2 represses transcriptional activation by GATA-1 [29
] by physical interaction. To get support for a role of GATA-1 in gene activation of MTG16
, HEL, TF-1, UT-7, and MEG-01cells were co-transfected with 10 μg MTG16 pGL3 -668 luciferase reporter and 10 μg HERP2 plasmid (kindly provided by Dr N Goldfarb, Charlottesville, Virginia) and the reporter signal was measured after 24 h as described above for luciferase reporter assays. The results were normalized to the luciferase activity of pGL3 -668 luciferase reporter.
Electrophoretic Mobility Shift Assay (EMSA) of MTG16 promoter
EMSA was performed as described previously [25
]. The potential GATA-1 sites at -301 was examined. The probe sequence was biotin-5-CCCGGCATTATCACGGGGACAC -3'. Nuclear extracts from HEL, TF-1, UT-7 and MEG-01 cells were prepared as described by Andrews and Faller [50
]. One to two μl polyclonal anti-GATA-1 (Active Motif, Carlsbad, CA, USA), monoclonal anti-GATA-2 (Santa Cruz Biotechnology Inc., CA, USA, sc-9008) or polyclonal anti-CD63 (sc-7080, Santa Cruz Biotechnology Inc., CA, USA) antibodies were added to the reaction mixtures.
Chromatin Immunoprecipitation (ChIP) assay of MTG16 promoter
ChIP was performed as described previously [25
]. For IP, 4 μl of polyclonal anti-GATA-1 (Active Motif, Carlsbad, CA, USA) or 0.8 μg monoclonal anti-GATA-2 antibodies (R&D Systems, MN, USA) were used. Forward and reverse primers for GATA sites were 5'-CAGATGGTTCCTGTCAGGGAAGTGGCG-3' and 5'-AGGTCTCCCTGCAGCCTGCGGGTGAG-3'. Control forward and reverse primers were: 5'-TAACACAGAGTACCCAGCCACTGTGC-3 ' and 5'-TGGTTGCACGGACAGAAGCCCCT-3'. Three different chromatin preparations were used for each IP.
Quantitative real-time PCR
Real-time PCR was performed as described previously [18
]. Transcript levels were calculated from a standard curve based on the Ct values of the samples,. Relative quantification based on the ΔCt method [51
] was used. Normalization: ΔCt = Ct (sample) - Ct (HEL corresponding DNA concentration). Relative quantification = 2 - ΔCt
Immunoprecipitation (IP) and Western blotting
IP and Western blotting were performed as described previously [52
]. The following antibodies were used: polyclonal anti-GATA-1 (Active Motif, Carlsbad, CA, USA) and polyclonal anti-GATA-2 (R&D Systems, MN, USA).
The statistical significance between two samples was determined by unpaired t-test in Prism Programme. Triple and single asterisk represents P < 0.0001 and P < 0.05, respectively.