Electrophoretic mobility shift assay (EMSA). 32
P-labeled DNA probes were incubated for binding with 1 to 4 μg of nuclear extracts for 20 min at 15°C in a buffer containing 5% glycerol, 50 mM NaCl, 20 mM Tris, pH 7.9, 0.5 mM EDTA, 5 mM MgCl, 1 mM dithiothreitol (DTT), 100 ng/μl poly(dI-dC), and 50 ng/μl bovine serum albumin (BSA) in a 15-μl final reaction mixture. The reaction mixture was then loaded onto a 5% polyacrylamide gel (29:1, acrylamide/bisacrylamide ratio) and run at 4°C at 150 V for 3 h. Nuclear extracts were prepared according to standard protocols (21
Competition experiments were performed by incubating 50 to 100 ng of unlabeled oligonucleotides in the reaction mixture for 10 min, prior to the addition of 32
P-labeled probes. The anti-CP2 and the anti-NF-E4 antibodies are described in references 14
; the anti GATA-1 antibody N6 was obtained from Santa Cruz. The experiments with bacterial recombinant proteins (see below) were performed using 2 ng/μl of poly(dI-dC); EMSAs with proteins produced by in vitro coupled transcription-translation reactions (TNT kit; Promega) were carried out in the presence of 25 ng/μl of poly(dI-dC).
GATA-1 and EKLF probes were made by PCR using either the forward (FW) or the reverse (REV) 32P end-labeled primer (see below for sequences). All other probes used in this work were generated by labeling the “sense” strand oligonucleotide with 32P and T4 polynucleotide kinase and annealing it with the complementary oligonucleotide; in this case, only the sequence corresponding to the sense strand is reported below. Labeled probes were purified by polyacrylamide gel electrophoresis.
Primers used for probes generated by PCR (mutations are underlined) were as follows: GATA-1 WT FW, 5′-CGAGTCCATCTGATAAGACTTATC-3′; GATA-1 GATAmut5′ FW, 5′-CGAGTCCATCGTCTAAGACTTATC-3′; GATA-1 GATAmut3′ FW, 5′-CGAGTCCATCTGATAAGACTTACA-3′; GATA-1 REV, 5′-ATAAAGCCTGGATCCTGGGGCTTACGC-3′; EKLF FW, 5′-CCCCTACCTGATAGCGG-3′; and EKLF REV, 5′-CCTTTCAGGCATTATCAGACAC ACC-3′.
Short GATA-1 CP2 consensus oligonucleotides (nucleotides [nt] −695 to −660) were as follows: WT, 5′-CTGCTGCCCCAGAGCAGGCCAGAGCTGGCGTAAGC-3′; MUT 1/3, 5′-CTGCTGCCACAGAGCAGGTAAGAGCTGGCGTAAGC-3′; MUT 2/4, 5′-CTGCTGCCCCAGATTGAGCCAGATTGAGCGTAAGC-3′; and MUT 1-4, 5′-CTGCTGCCACAGATTGAGTAAGATTGAGCGTAAGC-3′.
p45 NF-E2 oligonucleotides (nt 450 to 493) were as follows: WT, 5′-CCTGCAGCTGATAAACCCCTTATCTGGCCCAGGCAGGGGAACCT-3′; MUT 1-3, 5′-CCTGCAGCTGATAAACCCCTTATCTGTCCTAGGTAGGGGAACCT-3′; and MUT 2/3, 5′-CCTGCAGCTGATAAACCCCTTATCTGGCCTAGGTAGGGGAACCT-3′.
EKLF oligonucleotides (nt −683 to −634) were as follows: WT, 5′-CCTACCTGATAGCGGCCTGAAACATCTGGTGTGTCTGATAATGCCTGAAA-3′; and MUT, 5′-CCTACCTGATAGCGGCCTGAAACATTTGGTGTGTTTGATAATGCCTGAAA-3′.
The following constructs were also used: α-globin CP2 site, 5′-TAGAGCAAGCACAAACCAGGCCAA-3′ (22
); γ-globin SSE site, 5′-TCCAGTGAGGCCAGGGGCCGGCGGCTGGCTAGGGATGA-3′ (14
); α-globin GATA site, 5′-GGCAACTGATAAGGATTCCCA-3′ (24
); and γ-globin CCAAT box, 5′-GCCTTGCCTTGACCAATAGCCTTGACA-3′ (24
Recombinant protein expression and purification.
For the expression of the GST-GATA-1 fusion protein, the murine GATA-1 cDNA was cloned in the
polylinker region of the pMGSTev expression vector (10
), in frame with the GST moiety. GST-CP2 fusion protein was produced from the pGEX vector as described previously (14
). The Escherichia coli
BL21 strain cells were transformed with the above plasmids, cultures were grown at mid-logarithmic phase (0.6 A600
), and protein expression was induced with 0.1 mM IPTG (isopropyl-β-d
-thiogalactopyranoside) for 3 h at 37°C. GST-CP2 and GST-GATA-1 proteins present in the soluble fraction were bound to GST-Sepharose 4B (Amersham Bioscience) and purified according to the manufacturer's instructions.
For EMSA experiments, the GST moiety of CP2 was cleaved with thrombin (Amersham Biosciences). Briefly, the eluted GST-CP2 protein was dialyzed against a buffer containing 20 mM HEPES, pH 7.9, 100 mM KCl, 20% glycerol, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and then incubated overnight with thrombin at room temperature. The cleaved GST moiety was removed by binding to GST-Sepharose 4B resin at room temperature. The unbound fraction containing the CP2 cleaved protein was recovered and stored in aliquots at −80°C.
Plasmids. (i) GATA-1 reporter plasmids.
The GATA-1 mouse promoter region from nucleotide −856 to nucleotide −655 flanked by BamHI sites was obtained from the previously described pSVo GATA-1 plasmid (28
) and cloned into the compatible BglII site present in the polylinker of the pGL2 Basic luciferase reporter vector (Promega). The mouse GATA-1 minimal promoter from the same pSVo GATA plasmid (HindIII fragment from nucleotides −330 to −31) was transferred to the HindIII site of the pGL2 polylinker, immediately downstream to BglII.
To generate mutations in the CP2 boxes, a two-step PCR approach was used: in the first series of PCRs, mutations were introduced by amplifying the wild-type template sequence with REV oligonucleotides, carrying the desired mutations, corresponding to the 3′ end of the enhancer sequences, in conjunction with a common FW primer carrying an external BamHI site for subsequent cloning. The amplified fragments were gel purified and used as a template for the second round of PCRs with the same FW primer and a common REV primer, external to mutations, to introduce a 3′ BamHI site. The resulting fragments were then BamHI digested and cloned in the compatible BglII site of pGL2 Basic vector upstream to the GATA-1 minimal promoter.
Primers for GATA-1 constructs were as follows: FW-856 BamHI, 5′-CATAAAGCTTGGATCCACTCTGGGTGTCACCTC-3′; REV-655mut 1/3, 5′-CTGGGGCTTACGCCAGCTCTTACCTGCTCTGTGGCA-3′; REV-655mut 2/4, 5′-CTGGGGCTTACGCTCAATCTGGCTCAATCTGGGGCA-3′; REV-655mut 1-4, 5′-CTGGGGCTTACGCCAACTCTTACCTACTCTGTGGCAGCAGATAAGTCTTATGACATGGACTCG-3′; and REV-655BamHI, 5′-ATAAAGCCTGGATCCTGGGGCTTACGC-3′.
(ii) p45 NF-E2 reporter plasmids.
The p45 NF-E2 pGL2 plasmids (wild type and mutated in GATA-1 sites) have been described previously (27
). To generate the two mutations in CP2 boxes, we used a two-step PCR approach, using a pGEM-T (Promega) plasmid containing the p45 NF-E2 mouse fetal promoter (nucleotides 233 to 605) as a template (provided by B. Giglioni). In the first series of PCRs, partially overlapping primers carrying the desired mutations were used in conjunction with external primers on the pGEM-T flanking sequences to obtain the 5′ and 3′ portions of the fragment of interest. The amplified fragments were then gel purified and used as a template for a second round of PCRs with the same external pGEM-T primers. The resulting fragments were then cut with MboI and cloned in the compatible BglII site of pGL2 Basic vector.
Primers used for p45 mutants were as follows: pGEM-T FW, 5′-TAATACGACTCACTATA-3′; pGEM-T REV, 5′-ATTGGTGACACTATAGAA-3′; mut 2/3 sense, 5′-GATAAACCCCTTATCTGGCCTAGGTAGGGG-3′; and mut 1-3 sense, 5′-GATAAACCCCTTATCTGTCCTAGGTAGGGG-3′.
(iii) EKLF reporter plasmids.
The EKLF HS1 fragment was obtained by digestion with ApaI and NcoI of a PCR fragment from MEL cell DNA (nucleotides −938 to −24), amplified with oligonucleotides FW (5′-ACGTCTCGAGAACGGCATACTAGCTGCAGCTC-3′) and REV (5′-ACGTGGATCCGGCTCCTGTCTGCCCACATC-3′).
The fragment was blunted and cloned into the SmaI site of the pGL2 Basic vector containing the GATA-1 minimal promoter. Again, the mutations in CP2 boxes 2 to 3 were obtained by subsequent PCRs: overlapping sense and antisense oligonucleotides, together with the above external primers, were used to generate templates for the second round of PCR, carried out with the same external primers. The EKLFmut sense primer was 5′-GCGGCCTGAAACATTTGGTGTGTTTGATAATG-3′. The resulting mutated fragment was ApaI/NcoI digested, blunted, and cloned into the SmaI site of the pGL2 Basic vector containing the GATA-1 minimal promoter. All constructs obtained by PCR were sequenced to exclude the presence of undesired mutations.
K562 human erythroleukemic cells were grown in RPMI 1640 medium supplemented with l-glutamine and 5% fetal bovine serum. Exponentially growing K562 cells (1 × 107 to 2 × 107) were electroporated at 400 V and 960 μF with a Bio-Rad apparatus in 800 μl of phosphate-buffered saline with 20 μg of plasmid. To normalize experiments for transfection efficiency, 2 μg of pRL-TK plasmid carrying the Renilla luciferase gene under the control of the ubiquitous TK promoter was cotransfected in each sample. After 48 h, total cellular extracts were prepared and the double luciferase activity was measured according to the Promega Dual-Luciferase reporter system protocol. All experiments were repeated in triplicate with at least three independent plasmid preparations.
ChIP assays were performed as described previously (15
). Isolated DNA fragments were purified with a QIAquick spin kit (QIAGEN), and 2 μl from a 40-μl DNA extraction was amplified quantitatively by real-time PCR with the GATA-1, NF-E2, or EKLF promoter-specific primer or MyoD primer as a negative control (37
). For ChIP/chromatin reimmunoprecipitation (Re-ChIP) experiments, we immunoprecipitated the soluble chromatin fraction with antibody to CP2, washed it, and released the bound immune DNA complexes in 20 mM DTT solution for 30 min at 37°C. We resuspended the precipitates in 1 volume of the immunoprecipitated dilution buffer and performed a Re-ChIP with antibody to GATA-1. We also carried out control ChIP to test antibody specificity with either a control (no antibody) or normal rabbit serum.
The primers used were as follows: GATA-1 (forward), 5′-CTTTCCTACCCTATCCCACTCCTCG-3′; GATA-1 (reverse), 5′-TGCTGGATTTGAACTAGAGCCTGTG-3′; P45 NF-E2 (forward), 5′-GTTAAGGTATGGCCCAAATGACCCT-3′; P45 NF-E2 (reverse), 5′-AAGTTGTGGAAAGAGGCAAGCAGAC-3′; EKLF (forward), 5′-ATTGAACGCCAGGCTAATTTGAAGA-3′; EKLF (reverse), 5′-GAAAAGGCAGAAAGGGTATTCTGGG-3′; MyoD (forward), 5′-TGCAAGGCGTGCAAGCGCAAGAC-3′; and MyoD (reverse), 5′-CTCGATATAGCGGATGGCGTTG-3′.
In vitro protein-protein interaction assay.
The plasmid used to produce GATA-1 in vitro in the reticulocyte cell-free coupled transcription-translation system (TNT kit, Promega) was derived from the above-mentioned pMGSTev plasmid by removing the GST coding sequence by a double SpeI-NcoI digestion. The resulting ends were then blunted with Vent polymerase and religated. The CP2 cDNA was inserted into a pBluescript plasmid under the T7 promoter. 35S-labeled proteins were synthesized in the presence of [35S]methionine (Amersham). We incubated 1 μg of purified GST or GST fusion proteins with the above 35S-labeled proteins for 2 h at 4°C, adjusting the reaction mixture to a final volume of 40 μl with a buffer containing 50 mM Tris HCl, pH 7.5, 100 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and protein inhibitor cocktail (Roche). After incubation, 60 μl of 50% glutathione-Sepharose beads was added together with 500 μl of binding buffer (150 mM NaCl, 10 mM Tris HCl, pH 8, 0.3% NP-40, 1 mM DTT, PMSF, protein inhibitor cocktail, 0.25% BSA). Incubation was continued for 90 min at 4°C. Beads were collected by centrifugation and washed three times with 1 ml of IPP250 buffer (20 mM Tris HCl, pH 7.9, 250 mM NaCl, 0.05% NP-40, 1 mM DTT, PMSF, protein inhibitor cocktail). Proteins bound to the resin were eluted by boiling in 20 μl of Laemmli buffer, loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and visualized by autoradiography after drying of the gel.
In vivo protein-protein interaction assay.
K562 nuclear extract (50 μg) was preincubated with 7.5 μl of protein G agarose beads (Kirkegaard & Perry Laboratories) and 100 μl of NDB buffer (100 mM KCl, 20 mM Tris HCl, pH 7.8, 0.5 mM EDTA, 1 μg/μl BSA, 0.1% NP-40, 5 mM β-mercaptoethanol, 20% glycerol) for 1 h at 4°C. Precleared nuclear extracts were then incubated with 2 μg of anti-GATA-1 antibody (N6; Santa Cruz) or with 2 μg of anti-T antigen antibody (Santa Cruz) as a negative control for 4 h at 4°C. One hundred microliters of 50% protein G agarose beads was added to the reaction for 1 h at 4°C and then washed three times with 150 μl of NDB buffer. Bound proteins were eluted by boiling in 20 μl of Laemmli buffer, loaded onto a 10% SDS-PAGE gel, blotted, and probed for the presence of CP2.